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. 2021 Mar 20;18(6):3218.
doi: 10.3390/ijerph18063218.

Tetracycline-Resistant Bacteria Selected from Water and Zebrafish after Antibiotic Exposure

Ana Rita Almeida  1 , Marta Tacão  1 , Joana Soares  1 , Inês Domingues  1 , Isabel Henriques  2
Affiliations

Tetracycline-Resistant Bacteria Selected from Water and Zebrafish after Antibiotic Exposure

Ana Rita Almeida et al. Int J Environ Res Public Health. .

Abstract

The emergence of antibiotic-resistant pathogens due to worldwide antibiotic use is raising concern in several settings, including aquaculture. In this work, the selection of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) was evaluated after exposure of zebrafish to oxytetracycline (OTC) for two months, followed by a recovery period. The selection of ARB in water and fish was determined using selective media. The abundance of tetA genes was estimated through qPCR. Higher prevalence of ARB was measured in all samples exposed to the antibiotic when compared to control samples, although statistical significance was only achieved five days after exposure. Isolates recovered from samples exposed to the antibiotic were affiliated with Pseudomonas and Stenotrophomonas. Various antibiotic susceptibility profiles were detected and 37% of the isolates displayed multidrug resistance (MDR). The selection of the tetA gene was confirmed by qPCR at the highest OTC concentration tested. Two MDR isolates, tested using zebrafish embryos, caused significant mortality, indicating a potential impact on fish health and survival. Overall, our work highlights the potential impact of antibiotic contamination in the selection of potential pathogenic ARB and ARGS.

Keywords: Danio rerio; microcosm; multidrug resistance; pathogenicity test; qPCR.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Total counts (Log (CFU/mL)) of antibiotic-resistant bacteria from nonexposed (0 µg/L) and oxytetracycline (OTC)-exposed (10 µg/L) samples at each sampling time (5 days of exposure: 5 dE; 2 months of exposure: 2 mE and 5 days of postexposure: 5 dPE). Counts were determined in selective media (Glutamate Starch Phenol Red Agar (GSP), Membrane Fecal Coliform agar (mFC) and Plate Count Agar (PCA)) supplemented with tetracycline (TET) at 16 µg/mL. Asterisks (*) represent statistically significant differences (p ≤ 0.05) towards the respective control (nonexposed water and fish skin).
Figure 2
Figure 2
Resistance pattern (susceptible; susceptible, increased exposure; and resistant) of isolated bacteria according to the genera (Stenotrophomonas and Pseudomonas) and type of sample (water or fish skin). Aztreonam: ATM; cefepime: FEP; ceftazidime: CAZ; chloramphenicol: C; ciprofloxacin: CIP; gentamicin: CN; imipenem: IMI; ticarcillin: TIC; ticarcillin/clavulanic acid: TIM; tigecycline: TGC; and trimethoprim/sulfamethoxazole: STX.
Figure 3
Figure 3
Survival percentage (%) of zebrafish embryos exposed to multidrug-resistant bacteria isolated from water and zebrafish skin. Strains were obtained from samples exposed to 10 µg/mL of OTC, after a 5-day postexposure period (5 dPE) period. Asterisks (*) represent statistically significant differences (p ≤ 0.05) towards the control.
Figure 4
Figure 4
Relative abundance of tet(A) and absolute abundance of 16S rRNA gene (Log gene copy number/mL or fish gut), determined by qPCR in (a) zebrafish gut and (b) water exposed to OTC (0, 0.01 and 10 µg/mL) at three sampling times (5 days of exposure: 5 dE; 2 months of exposure: 2 mE; and 5 days postexposure: 5dPE). Asterisks (*) represent statistically significant differences (p ≤ 0.05) towards the respective control (0 µg/mL).
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