Extended-spectrum β-lactamase and their molecular mechanism in Enterobacteriaceae were analyzed in 126 fish samples of 9 various wild species, living in the lagoon of Bizerte in Tunisia. Fifty-nine (59) Gram-negative strains were isolated and identified as Escherichia coli (n=24), Klebsiella pneumonia (n=21), Citrobacter freundii (n=8), and Shigella boydii (n=6). Forty-seven ESBL producers were identified using the synergic test. β-Lactamase genes detected were blaCTX-M-1 (E. coli/15; K. pneumonia/8; C. freundii/1; Sh. boydii/1), blaCTX-M-1+ blaOXA-1 (E. coli/4; K. pneumonia/3), blaCTX-M-1+ blaTEM-1-a (K. pneumonia/2), blaCTX-M-15+ blaTEM-1-a (K. pneumonia/1; Sh. boydii/1), blaCTX-M-15+ blaOXA-1 (K. pneumonia/1), blaCTX-M-15 (E. coli/3; K. pneumonia/1; Sh. boydii/3), and blaCTX-M-9 (C. freundii/3). Most strains (84.7%) showed a multiresistant phenotype. qnrA and qnrB genes were identified in six E. coli and in ten E. coli+one K. pneumonia isolates, respectively. The resistance to tetracycline and sulfonamide was conferred by the tet and sul genes. Characterization of phylogenic groups in E. coli isolates revealed phylogroups D (n=20 strains), B2 (n=2), and A (n=2). The studied virulence factor showed prevalence of fimA genes in 9 E. coli isolates (37.5%). Similarly, no strain revealed the three other virulence factors tested (eae, aer, and cnf1). Our findings confirmed that the lagoons of Bizerte may be a reservoir of multidrug resistance/ESBL-producing Enterobacteriaceae. This could lead to indisputable impacts on human and animal health, through the food chain.
1. Introduction
The increasing rates of land-based anthropogenic pollution in marine ecosystems have become an important factor that promotes the emergence of multidrug-resistant (MDR) bacteria in aquatic animals [1–3]. The rapid dissemination of extended-spectrum β-lactamase-producing Enterobacteriaceae in marine coastal ecosystems is worrisome because enterobacteria species (mostly Escherichia coli) are commensal bacteria in the gut microbiota of fishes [4, 5].
Fish living in the natural environment harbored pathogenic Enterobacteriaceae [6–8]. Therefore, fish are considered as a potential vehicle of foodborne bacterial infections, which may present a threat for the human public health. Contamination of fish with MDR bacteria could demonstrate the risk of the persistence of these bacteria in the fish gut flora and explain possible human gut contamination [9]. Besides, significant antibiotics are excreted unaltered or as metabolites (up to 75%), which present a major source of antibiotic input into the aquatic environment. It is estimated that 49% of marine ecosystems worldwide are strongly affected by some anthropogenic factors of stress with significant and serious economic implications [10, 11]. Many of these compounds can now be detected easily in water resources.
Human survival and well-being depend on different services of the marine ecosystem (such as fishing) and, therefore, on the conservation and the best management of the ecosystems. Research on marine or lagoon ecosystem services has grown exponentially during the last decade, for better marine risk assessments. This pollution from a variety of sources (urban, agricultural, and industrial) contributed to altering the communities of different living beings that are prevailing in the marine and lagoon environment and indirectly presenting risks for human health [9, 12].
If mismanaged, multiple anthropogenic impacts on marine ecosystems might also affect coastal fisheries and aquaculture. The wide use of antimicrobial agents in clinical settings, veterinary medicine, livestock industries, and aquaculture has led to a large-scale dissemination of antibiotic-resistant bacteria in many environments [7, 8, 13, 14].
For enterobacteria, the problem of antibiotic resistance is essentially related to the broad-spectrum family of cephalosporin. Such resistance is usually due to the production of extended-spectrum β-lactamases (ESBLs) or cephalosporinase plasmid (pAmpC). The producing ESBL or pAmpC strains are largely isolated in hospital settings, animals, and food products and from different aquatic environments (such as lakes, rivers, and urban runoff). The CTX-M gene is the most common ESBL gene detected [15].
Resistance genes may be transferred between bacteria via mobile genetic elements [16]. One such mobile element called integrons that mediate the integration of resistance genes may also be involved, resulting in the development of multidrug-resistant bacteria [17].
Thus, the lagoon of Bizerte could be a site of choice allowing studies of the important triangular thematic aspect of “pollution-aquatic ecosystems-biotic component,” by the national and international scientific communities [18–20]. The lagoon of Bizerte receives various discharges through urban and industrial wastewater and agricultural runoff [21, 22].
The main aim of this work was to detect, isolate, and identify species multiresistant to antibiotics and producing ESBL enterobacteria (ESBL-Eb), from the intestines and gills of some species of wild fish, trapped in the lagoon of Bizerte. In the first part, we have studied the mechanisms of resistance in these enterobacteria isolates phenotypically and genetically. In the second part, we investigated important virulence factors involved in these isolates.
2. Materials and Methods2.1. Sites of Fish Sampling
The isolation of enterobacteria strains was performed by analyzing 126 samples of 9 wild species of fish trapped during the second half of the year 2016, in the surroundings of Menzel Abdurrahman port (north coast of Bizerte lagoon) (Table 1, Figure 1). The lagoon of Bizerte is an isodiametric saltwater body, located between latitude 37° 8′ and 37° 16′ N and longitude 9° 46′ and 9° 56′ E, in the north of Tunisia. It covers approximately 150 km2 and has an average depth of 8 m. It communicates with the Mediterranean Sea by a channel of 8.5 km in length and is connected to the Lake of Ichkeul by the river of Tinja. It represents an economically important body of water due to a variety of fishing and aquaculture activities. These fish samples were transported aseptically to the laboratory in a refrigerated cooler at +4°C and analyzed within the 24 h following their collection. The samples of fish tissues were primarily experimented for their intestinal tract and gills.
Distribution of positive bacterial strains resistant to cefotaxime according to the different species of fish sampled.
Family
Common name
Scientific name
Number of samples
Distribution isolates
Gills
Stomach contents
Moronidae
Loup
Dicentrarchus labrax
14
4
2
Carangidae
Chinchard commun
Trachurus trachurus
14
6
6
Mugilidae
Muges
Mugil cephalus
14
2
0
Mullet
Chelon labrosus
14
5
3
Mullidae
Rouget de roche
Mullus surmuletus
14
5
3
Pomatomidae
Tassergal
Pomatomus saltatrix
14
0
0
Soleidae
Sole commune
Solea solea
14
0
0
Sparidae
Marbré
Lithognathus mormyrus
14
1
0
Saupe
Sarpa salpa
14
12
10
Total
126
35
24
Map of Bizerte lagoon (Northern Tunisia). The black full star in the northern part of the lagoon shows the sampling site location.
2.2. Bacteria Isolation
After dissecting the sampled fish, the gills and the stomach contents were separately inoculated into 225 mL of buffered peptone water, pH=7. After incubation under shaking for 24 h at 37°C, dilutions in sterilized physiological water were performed, and a volume of 1 mL was seeded on MacConkey agar growth medium supplemented with 2 μg/mL of cefotaxime (CTX) for enterobacteria recovery. All the plates were incubated at 37°C for 24 h.
From each inoculate sample and based on colony size and morphology, a maximum of five suspected Enterobacteriaceae colonies were selected for E. coli isolate identification by biochemical tests and specific PCR amplification of the uidA gene [23]. Identification of other Enterobacteriaceae was realized by PCR amplification and sequencing of 16S rRNA genes [24]. E. coli strain (ATCC 25,922) was used as the control.
2.3. Antibiotic Susceptibility Testing
Susceptibility to 16 antimicrobial agents was performed using the disk diffusion method, following the Clinical and Laboratory Standard Institute (CLSI) recommendations [25]. The following antimicrobial agents were tested (concentration in μg): ampicillin (10 μg), cefotaxime (30 μg), cefoxitin (30 μg), ceftazidime (30 μg), chloramphenicol (30 μg), ciprofloxacin (5 μg), gentamycin (10 μg), imipenem (10 μg), nalidixic acid (30 μg), streptomycin (10 μg), sulfamethoxazole (300 μg), tetracycline (30 μg), ticarcillin (75 μg), tobramycin (10 μg), trimethoprim (5 μg), and trimethoprim-sulfamethoxazole (1.25+23.75μg). A screening test for the detection of ESBLs was carried out by the double-disc synergy test (DDST), according to the CLSI criteria [25].
2.4. Antibiotic Resistance Gene Characterization
The genes encoding TEM, SHV, OXA, and CTX-M β-lactamases were analyzed by PCR and sequencing in all ESBL-Eb [23]. The nucleotides and their amino acid sequences were compared with those deposited in the GenBank database and those reported in the website http://www.lahey.org/Studies, to confirm the specific β-lactamase genes. Genes encoding resistance to tetracycline (tet A and tet B), quinolone (aac[6′]-Ib, qnrA and qnrB), and sulfonamide (sul 1 and sul 2) were analyzed by PCR (and sequencing sometimes) [26].
2.5. Phylogenetic Groups and Virulence Factors
Identification of the major phylogenetic groups of E. coli isolates was determined by PCR, using a combination of three genes (chuA, yjaA, and tSpeA.C2) [27].
E. coli strains were screened for the following virulence factors: eae that codes for intimin, aer for aerobactin, cnf1 for cytotoxic necrotizing factors, and fimA for fimbriae of type I genes [28].
3. Results3.1. Identification of Isolates
Enterobacteria (n=59) were isolated from 126 wild fish sampled from the Bizerte lagoon. All recovered isolates were cefotaxime resistant, and the number of isolates from the gills (n=35) was slightly higher when compared to those from the viscera and the stomach contents (n=24) (Table 1). Biochemical and molecular identification showed that the 24 isolates were assigned to E. coli species harboring the specific gene iudA. Analysis of the sequences of the 16S rRNA genes of the 36 remaining isolates allowed the characterization of three enterobacteria species: Klebsiella pneumonia [K. pneumonia] (n=21), Citrobacter freundii [C. freundii] (n=8), and Shigella boydii [Sh. boydii] (n=6).
The rate of enterobacteria strain isolation showed a high ratio of distribution over the fish species in the Bizerte lagoon. However, we noticed a low ratio of segregation for the fish species of Lithognathus mormyrus, Pomatomus saltatrix, and Solea solea, with a ratio close to zero (Table 1).
3.2. Antimicrobial Resistance Phenotypes
The highest resistance frequency among the 59 cefotaxime-resistant strains was registered for the family of β-lactam antibiotics. The respective antibiotic resistance percentages were of 100% for ampicillin, 96.3% for ticarcillin, 11.9% for amoxicillin/clavulanic acid, 40.7% for ceftazidime, 24% for cefoxitin, 74.6% for ertapenem, and 5.1% for imipenem.
This highest resistance frequency to β-lactam was followed by an important resistance frequency of 71.2% for the sulfonamides. For non-β-lactam antibiotics, such isolate resistance frequencies were as follows for tobramycin (47.5%), gentamicin (40.7%), nalidixic acid (37.3%), tetracycline (37.3%), ciprofloxacin (3.4%), and chloramphenicol (1.7%). None of the strains was resistant to trimethoprim/sulfamethoxazole. Fifty (84.7%) out of the 59 isolates showed multiresistant phenotypes, including resistance to at least three families of antimicrobial agents. A higher multiresistance was noticed especially for the isolates from the gills than from the viscera and the stomach (58% and 42% of the isolates, respectively). Detection of ESBL isolates by the double-disc synergy test (DDST) revealed that 47 cefotaxime-resistant strains were ESBL-Eb-producing ones and belonged further to the following four species: E. coli (n=22), Klebsiella pneumonia (n=16), Citrobacter freundii (n=4), and Shigella boydii (n=5).
3.3. Characterization of ESBL Genes
The sequence analysis of the ESBL genes (blaCTXM, blaTEM, and blaOXA) of the isolated strains from different fish species showed the dominance of blaCTX-M-1 in the Enterobacteriaceae isolates. The ESBL genes were (species/number of isolates) blaCTX-M-1 for E. coli/15, K. pneumonia/8, C. freundii/1, and Sh. boydii/1; blaCTX-M-1+ blaOXA-1 for E. coli/4 and K. pneumonia/3; blaCTX-M-1+ blaTEM-1-a for K. pneumonia/2; blaCTX-M-15+ blaTEM-1-a for K. pneumonia/1 and Sh. boydii/1; blaCTX-M-15+ blaOXA-1 for K. pneumonia/1; blaCTX-M-15 for E. coli/3, K. pneumonia/1, and Sh. boydii/3; and blaCTX-M-9 for C. freundii/3.
3.4. Resistance Mechanism of Non-β-Lactam Antimicrobial Agents
Resistance to the sulfonamide of ESBL-Eb isolates (n=43) was shown to be conferred by either sul 1 genes for 15 isolates (E. coli [n=7], K. pneumonia [n=4], C. freundii [n=3], and Sh. boydii [n=1]) or sul 2 genes for 13 other strains (E. coli [n=10], K. pneumonia [n=2], and C. freundii [n=1]), with no sul gene being detected for the last 15 strains. For the resistance to the tetracycline, the tet A gene appeared in all 22 resistant isolates of E. coli (n=21) and K. pneumonia (n=1). Besides, for quinolone family resistance, only six out of the 22 strains harbored the aac(6′)-Ib-cr gene for E. coli (n=5) and K. pneumonia (n=1). Analysis of qnr plasmids by PCR allowed the detection of the qnrA gene in E. coli isolates (n=10) and the qnrB gene in 10 strains [E. coli (n=9) and K. pneumonia (n=1)].
3.5. Phylogenetic Groups of E. coli Isolates and Virulence Factors
Amplification of the three genes ChuA, YjaA, and TSPE4C2and their presence or absence in 24 ESBL-producing E. coli strains has revealed that 20 strains (83.3%) appeared to belong to the group D and 2 strains (8.3%) to the group B2, while the two other strains (8.3%) belonged to the group A. Characterization of the virulence factor demonstrated the prevalence of the fimA gene in nine E. coli isolates (37.5%), but there was no strain revealing the three other virulence factors considered (eae, aer, and cnf1) (Tables 2 and 3).
Characteristic of ESBL-Eb strains detected in fish from lagoon of Bizerte.
Isolates
Sampling
Fish species
ESBL
Resistance to non-β-lactam
Resistance genes
Phylogenetic group
Virulence factor
EC 138
SC
S. salpa
CTX-M-1+OXA-1
TET, NAL, SUL
tet A, sul 2, qnrB
D
—
EC 155
G
T. trachurus
CTX-M-1+OXA-1
TET, NAL, SUL
tet A, sul 1, qnrB
D
—
EC 159
SC
T. trachurus
CTX-M-1+OXA-1
TET, NAL, SUL
tet A, sul 2, qnrB
D
—
EC 163
G
T. trachurus
CTX-M-1+OXA-1
TET, NAL, SUL
tet A, sul 2, qnrB
D
—
EC 71
SC
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, qnrB
B2
fimA
EC 80
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 1, aac(6′)ib-cr
D
fimA
EC 87
SC
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, qnrB
D
fimA
EC 94
SC
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, aac(6′)ib-cr
D
—
EC 98
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 1, qnrA
B2
fimA
EC 101
SC
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, qnrA
D
fimA
EC 105
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 1, aac(6′)ib-cr
D
fimA
EC 109
SC
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, qnrA
D
—
EC 113
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, qnrA
D
—
EC 119
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 1, qnrA
D
—
EC 129
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 2, aac(6′)ib-cr, qnrB
D
—
EC 131
G
S. salpa
CTX-M-1
TET, NAL, SUL
tet A, sul 1, qnrB
D
—
EC 182
G
P. saltatrix
CTX-M-1
TET, STR
tet A
D
—
EC 319
SC
M. surmuletus
CTX-M-1
CN, TOB, SUL
—
A
—
EC 61
SC
S. salpa
CTX-M-15
TET, NAL, SUL
tet A, qnrA
D
fimA
EC 62
G
S. salpa
CTX-M-15
TET, NAL, SUL
tet A, sul 2, aac(6′)ib-cr
D
fimA
EC 68
G
S. salpa
CTX-M-15
TET, NAL, SUL
tet A, sul 1, qnrB
D
fimA
K. p 286
SC
T. trachurus
CTX-M-1+TEM-1-a
CN, TOB, SUL
—
K. p 291
G
T. trachurus
CTX-M-1+TEM-1-a
CN, TOB, SUL
—
K. p 282
G
T. trachurus
CTX-M-15+TEM-1-a
CN, TOB, SUL, STR
—
K. p 135
SC
S. salpa
CTX-M-1+OXA-1
TET, NAL, SUL
tet A, sul 1, aac(6′)ib
K. p 206
G
D. labrax
CTX-M-1+OXA-1
CN, TOB
—
K. p 246
SC
Ch. labrosus
CTX-M-1+OXA-1
CN, TOB
—
K. p 216
G
D. labrax
CTX-M-15+OXA-1
CN, TOB
—
K. p 211
SC
D. labrax
CTX-M-1
CN, TOB
—
K. p 222
SC
D. labrax
CTX-M-1
CN, TOB, SUL
—
K. p 234
G
Ch. labrosus
CTX-M-1
C, CN, TOB
—
K. p 243
G
Ch. labrosus
CTX-M-1
CN, TOB, SUL
sul 2
K. p 249
G
Ch. labrosus
CTX-M-1
CN, TOB, SUL
sul 2
K. p 254
SC
Ch. labrosus
CTX-M-1
CN, TOB, SUL
sul 1
K. p 257
G
Ch. labrosus
CTX-M-1
CN, TOB, SUL
—
K. p 263
SC
T. trachurus
CTX-M-1
CN, TOB, SUL
—
K. p 219
G
D. labrax
CTX-M-15
CN, TOB
—
C342
G
T. trachurus
CTX-M-1
CN, TOB, SUL
sul 2
C66
SC
S. salpa
CTX-M-9
—
—
C79
SC
S. salpa
CTX-M-9
—
—
C225
G
D. labrax
CTX-M-9
SUL
sul 1
SH278
SC
T. trachurus
CTX-M-15+TEM-1-a
CN, TOB, SUL
—
SH260
SC
Ch. labrosus
CTX-M-1
CN, TOB, SUL
—
SH273
G
T. trachurus
CTX-M-15
CN, TOB, SUL
—
SH307
G
M. surmuletus
CTX-M-15
CN, TOB, SUL
—
SH315
G
M. surmuletus
CTX-M-15
CN, TOB
—
SC: stomach content analysis; G: gills; EC: Escherichia coli; K. p: Klebsiella pneumoniae; CF: Citrobacter freundii; (SH): Shigella boydii; T: Thermococcus; S. salpa: Sarpa salpa; T. trachurus: Trachurus trachurus; P. saltatrix: Pomatomus saltatrix; M. surmuletus: Mullus surmuletus; D. labrax: Dicentrarchus labrax; Ch. labrosus: Chelon labrosus; TET: tetracycline; Nal: nalidixic acid; TOB: tobramycin; SUL: sulfonamide; CN: gentamicin; STR; streptomycin; C: chloramphenicol.
Characteristic of non ESBL-Eb strains detected in fish from lagoon of Bizerte.
Isolates
Sampling
Fish species
Resistance to non-β-lactam
Resistance genes
Phylogenetic group
Virulence factor
EC 3
G
S. salpa
CIP, TET, NAL
tet A, qnrB
D
—
EC 24 (1)
G
L. mormyrus
—
—
D
—
K. p 25
G
S. salpa
NAL
qnrB
K. p 75
G
S. salpa
—
—
K. p 296
SC
M. surmuletus
TOB, SUL
sul 1
K. p 311
SC
M. surmuletus
TOB, SUL
sul 1
K. p348
G
M. surmuletus
STR
—
C170
G
P. saltatrix
—
—
C145
SC
T. trachurus
—
—
C242
G
Ch. labrosus
CN, TOB, SUL
sul 1
C270
SC
T. trachurus
CIP, TOB, SUL
sul 1
SH300
G
M. surmuletus
TOB, SUL
sul 1
SC: stomach content analysis; G: gills; EC: Escherichia coli; K. p: Klebsiella pneumoniae; CF: Citrobacter freundii; SH: Shigella boydii; S. salpa: Sarpa salpa; T. trachurus: Trachurus trachurus; P. saltatrix: Pomatomus saltatrix; M. surmuletus: Mullus surmuletus; Ch. labrosus: Chelon labrosus; L. mormyrus: Lithognathus mormyrus; TET: tetracycline; Nal: nalidixic acid; TOB: tobramycin; SUL: sulfonamide; CN: gentamicin; STR; streptomycin; C: chloramphenicol.
4. Discussion
In the present study, 59 Gram-negative and cefotaxime resistant isolates, belonging to the Enterobacteriaceae family, were recovered out of 126 samples of different wild fish species, collected from the lagoon of Bizerte. Molecular identification showed the dominance of E. coli isolates (40%) along with K. pneumoniae (n=21), C. freundii (n=8), and Sh. boydii (n=6). The high frequency of E. coli and K. pneumoniae isolation of 40.7 and 35.6%, respectively, registered in this study is well reported in several similar studies worldwide and different environments [29–32].
In addition, the detection of CTX-resistant enterobacteria strains in the gills and the stomach contents of the different fish species was realized. Thus, the gill content appeared to show higher frequency isolation of bacteria resistant to cefotaxime as compared to those from the stomach content. This would infer that the gills may be a very favorable reservoir to host resistant flora as compared to the stomach. Resistant Enterobacteriaceae in the gills seems to be caused by the ingestion of water contaminated with fecal bacteria and antibiotic residues in the lagoon of Bizerte. Some studies reported that the high frequency of concentrations of antibiotics reported in the marine environment and their potential impacts on the aquatic ecosystems explain the widespread of antibiotic resistance largely reported in fish, marine mammals, and seabirds living in coastal waters [33].
In addition, the most important resistance frequencies, registered in the different fish species tested, were shown for Sarpa salpa, followed by the species of Trachurus trachurus, Chelon labrosus, Mullus surmuletus, and, finally, Dicentrarchus labrax; such frequencies showed ratios fluctuating between 1.6 and 0.4. However, reports on bacteria isolation were low variable and being closer to zero for Mugil cephalus, Lithognathus mormyrus, Pomatomus saltatrix, and Solea solea. These reports infer a great chance of fish exposure to bacteria resistant to cefotaxime and other antibiotics, and such exposure seems to be primarily related to the biological behavior of the fish as well as their wide geographical distribution in the marine environment and lagoons [34–37].
But, we have detected 47 ESBL-Eb-producing isolates, especially in E. coli (n=22), K. pneumoniae (n=16), Sh. Boydii (n=5), and C. freundii (n=4). This agrees with the recent report of Ben Said et al. [38] that has described ESBL Enterobacteriaceae isolates in sewage water, in Tunisia. Singh et al. [39] have also reported a study of multiple antibiotic-resistant, ESBL-producing enterobacteria in fresh seafood such as Escherichia coli the predominant species followed by Klebsiella oxytoca and K. pneumonia. It was shown that the ESBL-positive phenotype is detected in 169 (78.60%) tested isolates, with E. coli being the predominant species (53), followed by Klebsiella oxytoca (27) and K. pneumoniae (23).
Characterization of the ESBL genetic profile by amplification and sequencing of different CTX-M groups showed the dominance of blaCTX-M-1 among 35 isolates of E. coli (n=19), K. pneumonia (n=13), Sh. Boydii (n=6), and C. freundii (n=8). A similar study conducted, in Tunisia, by Ben Said et al. [40] revealed the detection of enterobacteria-producing ESBL species in agronomic soil, irrigation water, and various vegetables. Besides the prevalence of CTX-M-1 enzymes, CTX-M-15 (n=10) and CTX-M-9 (n=3) were also detected in the study. Among these strains, we have noticed the existence of an association between blaCTX-M-1/blaCTX-M-15 and blaOXA-1 and blaTEM-1a. Indeed, the association between genes encoding cefotaximases and those of other beta-lactamases (TEM and OXA types) has been frequently identified among our isolates and also previously reported in others [41].
All these results confirmed the high circulation of these resistance genes in the environment and the lagoon of Bizerte. In addition, the gene blaCTX-M-15, known as an ESBL gene usually associated with strains found in the environment, and the gene blaCTX-M-1, known as the most detected ESBL gene in food products, in vegetation, and in different natural environments, explained the origin of the high transmission and dissemination of these variants of resistant bacteria through the residual releases into the lagoon of Bizerte.
It is well confirmed that the environment could play a very important role in the diffusion of resistance. Antibiotics used in agriculture and fish farming or arboriculture are frequently found as metabolites in the soils and the waters where they exert and maintain high genetic selection pressure [40].
Most of our ESBL-Eb have shown a multiresistance phenotype and carried a different type of resistance genes (tet, sul) encoding for tetracycline and sulfonamide resistance, respectively, with this character being typical of ESBL-producing bacteria in other studies [40, 42].
For quinolone resistance, it was conferred by aac(6′)-ib-cr, qnrA, and qnrB genes. These antibiotic genetic determinants are specifically related to pathogenic clinical strains and were recently detected in the environment [40, 43, 44].
Detection of quinolone plasmid QNR confirmed the circulation of these resistance genes by mobile genetic elements. In parallel, determining the phylogenetic groups and analyzing the different virulence factors of E. coli isolates resistant to cefotaxime showed mainly the dominance of the phylogenetic group D with 81.9% (18/22 isolates). This group is known to be principally composed of pathogenic strains called “extraintestinal” pathogens. Among other antibiotic-resistant isolates, two strains (9%) belonged to the phylogenetic group B2. This group B2 is known to be the most virulent strain group [41]. Finally, only two strains belonged to the phylogenetic group A as known as “commensal” phylogenetic group strains. All these results are similar to those reported by Ben Said et al. [38]. The investigation on the 4 known virulence genes studied in all E. coli isolates has revealed the dominance of the fimA gene that belongs to the phylogenetic groups D and B2. The study of Jouini et al. [45] has confirmed these findings and has found that all strains isolated from many varieties of food presented the fimA gene of virulence. Therefore, no other isolates have revealed at least one of the four genes investigated in the present work; these strains could host other virulence determinants not yet investigated.
5. Conclusion
Our study appeared to be the first work done in Tunisia concerning the lagoon of Bizerte and showed a high occurrence of ESBL-Enterobacteriaceae as well as the CTX-M-1 group, in some tested wild fish species. It allowed determination of tet, sul, aac(6′)-ib-cr, and qnr resistance genes that confer resistance to tetracycline, sulfonamide, and quinolone, respectively. These findings demonstrated the role of the Bizerte lagoon as hotspot collectors of ESBL-Enterobacteriaceae with high likelihood of dissemination and spread to humans and animals throughout the food chain.
Data Availability
All data used to support the findings of the study are approved and included in the article.
Conflicts of Interest
The authors declare that they have no conflicts of interest.
Authors’ Contributions
Bilel Hassen and Ahlem Jouini contributed equally to this work.
Acknowledgments
The sampling was carried out in collaboration with the National Institute of Science and Technology of the Sea, 2025 Salammbô, Tunisia, University of Carthage. We thank Pr. Abdeljalil Ghram for the critical reading of the manuscript.
FotiM.GiacopelloC.BottariT.FisichellaV.RinaldoD.MamminaC.Antibiotic resistance of gram negatives isolates from loggerhead sea turtles (Caretta caretta) in the central Mediterranean Sea20095891363136610.1016/j.marpolbul.2009.04.0202-s2.0-6894918032719473669AhasanM. S.PicardJ.ElliottL.KinobeR.OwensL.ArielE.Evidence of antibiotic resistance in Enterobacteriales isolated from green sea turtles, Chelonia mydas on the Great Barrier Reef20171201–2182710.1016/j.marpolbul.2017.04.0462-s2.0-8501893943628476351FernandesP.MartensE.Antibiotics in late clinical development201713315216310.1016/j.bcp.2016.09.0252-s2.0-85002202416BrahmiS.TouatiA.Dunyach-RemyC.SottoA.PantelA.LavigneJ. P.High prevalence of extended-spectrum β-lactamase-producing Enterobacteriaceae in wild fish from the Mediterranean Sea in Algeria201824329029810.1089/mdr.2017.01492-s2.0-8504137621428805537GerzovaL.VidenskaP.FaldynovaM.SedlarK.ProvaznikI.CizekA.RychlikI.Characterization of microbiota composition and presence of selected antibiotic resistance genes in carriage water of ornamental fish201498, article e10386510.1371/journal.pone.01038652-s2.0-8490546033425084116KolářM.UrbánekK.LátalT.Antibiotic selective pressure and development of bacterial resistance200117535736310.1016/S0924-8579(01)00317-X2-s2.0-003503332311337221KümmererK.Antibiotics in the aquatic environment – a review – part I200975441743410.1016/j.chemosphere.2008.11.0862-s2.0-63449084512KümmererK.Antibiotics in the aquatic environment – a review – part II200975443544110.1016/j.chemosphere.2008.12.0062-s2.0-6344908959519178931BoeningD. W.An evaluation of bivalves as biomonitors of heavy metals pollution in marine waters199955345947010.1023/A:10059952179012-s2.0-0032896975AlexanderM.Aging, bioavailability, and overestimation of risk from environmental pollutants200034204259426510.1021/es001069+2-s2.0-0034666852MartínezM. L.IntralawanA.VázquezG.Pérez-MaqueoO.SuttonP.LandgraveR.The coasts of our world: ecological, economic and social importance2007632–325427210.1016/j.ecolecon.2006.10.0222-s2.0-34250193587WarwickR.Taxonomic distinctness as an indicator of stress in the marine macrobenthos2005R Warwick 20051011YangJ.WangC.ShuC.LiuL.GengJ.HuS.FengJ.Marine sediment bacteria harbor antibiotic resistance genes highly similar to those found in human pathogens201365497598110.1007/s00248-013-0187-22-s2.0-84876940241MölstadS.LundborgC. S.KarlssonA. K.CarsO.Antibiotic prescription rates vary markedly between 13 European countries200934536637110.1080/003655401100800342-s2.0-0035987169RossoliniG. M.D'AndreaM. M.MugnaioliC.The spread of CTX-M-type extended-spectrum β-lactamases200814334110.1111/j.1469-0691.2007.01867.x2-s2.0-3724907228118154526DrögeM.PühlerA.SelbitschkaW.Horizontal gene transfer as a biosafety issue: a natural phenomenon of public concern1998641759010.1016/S0168-1656(98)00105-92-s2.0-03447660699823660PartridgeS. R.TsafnatG.CoieraE.IredellJ. R.Gene cassettes and cassette arrays in mobile resistance integrons200933475778410.1111/j.1574-6976.2009.00175.x2-s2.0-6674910206419416365BarhoumiB.ElbarhoumiA.ClérandeauC.Al-RawabdehA. M.AtyaouiA.TouilS.DrissM. R.CachotJ.Using an integrated approach to assess the sediment quality of an Mediterranean lagoon, the Bizerte lagoon (Tunisia)20162561082110410.1007/s10646-016-1664-42-s2.0-8496635630327146821MartinsM. V. A.ZaaboubN.AleyaL.FrontaliniF.PereiraE.MirandaP.ManeM.RochaF.LautL.El BourM.Environmental quality assessment of Bizerte lagoon (Tunisia) using living foraminifera assemblages and a multiproxy approach2015109, article e013725010.1371/journal.pone.01372502-s2.0-8494593086726372655SaidO. B.LouatiH.SoltaniA.Preud’hommeH.Cravo-LaureauC.GotP.PringaultO.AissaP.DuranR.Changes of benthic bacteria and meiofauna assemblages during bio-treatments of anthracene-contaminated sediments from Bizerta lagoon (Tunisia)20152220153191533110.1007/s11356-015-4105-72-s2.0-8494548245925618309SaidO. B.Goñi-UrrizaM.El BourM.AissaP.DuranR.Bacterial community structure of sediments of the Bizerte lagoon (Tunisia), a southern Mediterranean coastal anthropized lagoon201059344545610.1007/s00248-009-9585-x2-s2.0-7795248712519789910BarhoumiB.MenachK. L.ClérandeauC.AmeurW. B.BudzinskiH.DrissM. R.CachotJ.Assessment of pollution in the Bizerte lagoon (Tunisia) by the combined use of chemical and biochemical markers in mussels, Mytilus galloprovincialis2014841-237939010.1016/j.marpolbul.2014.05.0022-s2.0-8490277092924913071JouiniA.VinuéL.SlamaK. B.SaenzY.KlibiN.HammamiS.BoudabousA.TorresC.Characterization of CTX-M and SHV extended-spectrum β-lactamases and associated resistance genes in Escherichia coli strains of food samples in Tunisia20076051137114110.1093/jac/dkm3162-s2.0-3544895694117855726WeisburgW. G.BarnsS. M.PelletierD. A.LaneD. J.16S ribosomal DNA amplification for phylogenetic study1991173269770310.1128/jb.173.2.697-703.19912-s2.0-00260677971987160Clinical and Laboratory Standards InstitutePerformance standards for antimicrobial susceptibility testing201727thWayne, PA, USAthe Clinical and Laboratory Standards Institute 2017SáenzY.BrinasL.DomínguezE.RuizJ.ZarazagaM.VilaJ.TorresC.Mechanisms of resistance in multiple-antibiotic-resistant Escherichia coli strains of human, animal, and food origins200448103996400110.1128/AAC.48.10.3996-4001.20042-s2.0-464433271715388464ClermontO.BonacorsiS.́.BingenE.Rapid and simple determination of the Escherichia coli phylogenetic group200066104555455810.1128/aem.66.10.4555-4558.20002-s2.0-003377563311010916RuizJ.SimonK.HorcajadaJ. P.VelascoM.BarrancoM.RoigG.Moreno-MartinezA.MartinezJ. A.Jimenez de AntaT.MensaJ.VilaJ.Differences in virulence factors among clinical isolates of Escherichia coli causing cystitis and pyelonephritis in women and prostatitis in men200240124445444910.1128/jcm.40.12.4445-4449.20022-s2.0-1874436547912454134ChouchaniC.MarrakchiR.HenriquesI.CorreiaA.Occurrence of IMP-8, IMP-10, and IMP-13 metallo-β-lactamases located on class 1 integrons and other extended-spectrum β-lactamases in bacterial isolates from Tunisian rivers20124529510310.3109/00365548.2012.7177122-s2.0-8487233687622992193ZurfluhK.HächlerH.Nüesch-InderbinenM.StephanR.Characteristics of extended-spectrum β-lactamase-and carbapenemase-producing Enterobacteriaceae isolates from rivers and lakes in Switzerland20137993021302610.1128/AEM.00054-132-s2.0-8487633629123455339KittingerC.LippM.FolliB.KirschnerA.BaumertR.GallerH.GrisoldA. J.LuxnerJ.WeissenbacherM.FarnleitnerA. H.ZarfelG.Enterobacteriaceae isolated from the river Danube: antibiotic resistances, with a focus on the presence of ESBL and carbapenemases20161111, article e016582010.1371/journal.pone.01658202-s2.0-8499435754327812159TafouktR.TouatiA.LeangapichartT.BakourS.RolainJ. M.Characterization of OXA-48-like-producing Enterobacteriaceae isolated from river water in Algeria201712018518910.1016/j.watres.2017.04.0732-s2.0-8501825701328486169GawS.ThomasK. V.HutchinsonT. H.Sources, impacts and trends of pharmaceuticals in the marine and coastal environment20143691656, article 2013057210.1098/rstb.2013.05722-s2.0-8490783536325405962AustinB.The bacterial microflora of fish, revised2006694510.1100/tsw.2006.1812-s2.0-3374742466216906326HansenD. L.ClarkJ. J.IshiiS.SadowskyM. J.HicksR. E.Sources and sinks of Escherichia coli in benthic and pelagic fish200834222823410.3394/0380-1330(2008)34[228:SASOEC]2.0.CO;22-s2.0-46849099627OzaktasT.TaskinB.GozenA. G.High level multiple antibiotic resistance among fish surface associated bacterial populations in non-aquaculture freshwater environment201246196382639010.1016/j.watres.2012.09.0102-s2.0-8486830254923039919ZhangM.SunY.ChenL.CaiC.QiaoF.DuZ.LiE.Symbiotic bacteria in gills and guts of Chinese mitten crab (Eriocheir sinensis) differ from the free-living bacteria in water2016111, article e014813510.1371/journal.pone.01481352-s2.0-8495819104426820139SaidL. B.JouiniA.AlonsoC. A.KlibiN.DziriR.BoudabousA.SlamaK. B.TorresC.Characteristics of extended-spectrum β-lactamase (ESBL)- and pAmpC beta-lactamase-producing Enterobacteriaceae of water samples in Tunisia20165501103110910.1016/j.scitotenv.2016.01.0422-s2.0-8495753560026871556SinghA. S.LekshmiM.PrakasanS.NayakB.KumarS.Multiple antibiotic-resistant, extended-spectrum-β-lactamase (ESBL)-producing enterobacteria in fresh seafood2017535310.3390/microorganisms503005328867789SaidL. B.JouiniA.KlibiN.DziriR.AlonsoC. A.BoudabousA.SlamaK. B.TorresC.Detection of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in vegetables, soil and water of the farm environment in Tunisia2015203869210.1016/j.ijfoodmicro.2015.02.0232-s2.0-8492497630325791254MendonçaN.LeitãoJ.ManageiroV.FerreiraE.CaniçaM.Spread of extended-spectrum β-lactamase CTX-M-producing Escherichia coli clinical isolates in community and nosocomial environments in Portugal20075161946195510.1128/AAC.01412-062-s2.0-3425016981017371815MuzslayM.MooreG.AlhussainiN.WilsonA. P. R.ESBL-producing Gram-negative organisms in the healthcare environment as a source of genetic material for resistance in human infections2017951596410.1016/j.jhin.2016.09.0092-s2.0-8500887762427771149ConteD.PalmeiroJ. K.da Silva NogueiraK.de LimaT. M. R.CardosoM. A.PontaroloR.Degaut PontesF. L.Dalla-CostaL. M.Characterization of CTX-M enzymes, quinolone resistance determinants, and antimicrobial residues from hospital sewage, wastewater treatment plant, and river water2017136626910.1016/j.ecoenv.2016.10.0312-s2.0-8499431140927816836XuY.GuoC.LuoY.LvJ.ZhangY.LinH.WangL.XuJ.Occurrence and distribution of antibiotics, antibiotic resistance genes in the urban rivers in Beijing, China201621383384010.1016/j.envpol.2016.03.0542-s2.0-8496202231027038570JouiniA.Ben SlamaK.KlibiN.Ben SallemR.EstepaV.VinuéL.SáenzY.Ruiz-LarreaF.BoudabousA.TorresC.Lineages and virulence gene content among extended-spectrum β-lactamase—producing Escherichia coli strains of food origin in Tunisia201376232332710.4315/0362-028X.JFP-12-2512-s2.0-8487407667823433382