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Introduction
According to the data from the Food and Agriculture Organization, aquaculture has provided nearly half of the fishery products for human consumption, which has fulfilled the huge requirement for edible proteins and nutrients around the world (Liu et al. 2019). China is the largest aquaculture products provider in the world and yielded more than 52 million tons of farmed food aquatic products till 2020 (Bureau of Fisheries 2021). However, accompanied by the growing intensive production mode, the aquaculture system also suffers from frequent outbreaks of infectious diseases and high mortality (Reverter et al. 2020). To deal with bacterial infection, enormous amounts of antibiotics were applied in the aquaculture industry for the purpose of combating or preventing bacterial infection. In 2017, China took the largest share of global antibiotic consumption in aquaculture at 58% and was projected to remain the largest antibiotic consumer up to 2030 (Schar et al. 2020).
There is no doubt that the overuse and misuse of antibiotics in aquaculture could not only stimulate a rapid emergence of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) but also lead to a widespread of ARGs in the aquatic environment and reduce the antibiotics’ therapeutic potential against human and animal pathogens (Jia et al. 2020; Preena et al. 2020). The antibiotics and their corresponding ARGs were prevalent in major mariculture sites in China, and the number and proportion of antibiotic-resistant bacteria were significant, which may pose a grave threat to the marine environment and human health. (Gao et al. 2018). In Dongshan Bay marine aquaculture, 175 multi-drug-resistant isolates possessed at least three ARG subtypes, and ten percent of multiple-drug-resistant microorganisms belonged to potentially pathogenic genera or species (Cui et al. 2022). Freshwater aquaculture is an important source of ARGs too. Polymerase chain reaction results revealed that the sul1 and sul2 genes were the most reported ARGs, and tetracycline resistance genes had the most often reported subtype in Chinese freshwater aquaculture ponds (Wang et al. 2021). The high prevalence of ARB and ARGs in the aquaculture environment highlights the roles of aquatic products as potential sources of ARB and ARGs. It raises the need for controlling their transmission to humans through the food chain.
Liu et al. (2017), summarizing the antibiotics applied in the Chinese aquaculture industry from 2003 to 2016, found that quinolones were the most frequently detected class in aquatic products, followed by sulfonamides and macrolides. Sulfonamides, macrolides, fluoroquinolones, and chloramphenicol also were detected in the mariculture ponds (Zhang et al. 2021). Furthermore, fluoroquinolones and tetracyclines have gradually become the dominant antibiotics in aquaculture since 2006 (Dang et al. 2007; He et al. 2016; Wang et al. 2021). Based on the previous reports and our previous research works concerning the aquaculture environment in Zhejiang (Yuan et al. 2017; Ye et al. 2019), six frequently detected antibiotic compounds, including sulfonamides, tetracyclines, enrofoxin, cephalexin, gentamincin, and florfenicol, were selected for their related ARB and ARGs investigated in this study. The objective of this study was to assess the status of distribution and diversity of the ARBs and ARGs among the popular aquatic products in Zhejiang, China. Furthermore, the phenotypic and genotypic antibiotic resistance properties were investigated among the ARB isolates. A deep insight into the ARB and ARGs associated with aquatic products is beneficial in understanding their risks to aquatic organisms and humans.
Experimental
Materials and Methods
Samples. Aquaculture products, including crucian carp (Carassius auratus, 400–500 g), black carp (Mylopharyngodon piceus, 3,000–4,000 g), yellow catfish (Pelteobagrus fulvidraco, 150–200 g), loach (Misgurnus anguillicaudatus, 50–60 g), and giant river prawn (Macrobrachium rosenbergii, 35–40 g) were collected from different aquaculture farms in the Linghu, Huzhou (120.03°–120.14°E, 30.39°–30.47°N) in the north of the Zhejiang province, China. Large yellow croaker (Larimichthys crocea, 300–350 g) was collected from Dachen Dao, Wenzhou (121.90°E, 28.49°N), southeast of the Zhejiang province. Five samples were collected at random for each species. All the samples were transported to the lab in an icebox within 6 h after collection.
Isolation of bacteria. Initially, epidermal mucus, gills, and gut contents were collected. All the tissue samples were pooled and homogenized together by a sterile homogenizer (Yetuo, YT-PJ-400, Shanghai) immediately. Then, 100 μl of 10-fold serial dilutions up to 10–7 were plated onto nutrient agar (2216E Agar for large yellow croaker; Qingdao Hope Bio-Technology Co., Ltd., China) containing sulfadiazine (30 μg/ml), gentamicin (15 μg/ml), enrofloxacin (5 μg/ml), cephalexin (30 μg/ml), tetracycline (30 μg/ml), and chloramphenicol (30 μg/ml). The concentration of antibiotics added to the media was selected based on the CLSI (2018) breakpoints and modified slightly according to our previous studies (Yuan et al. 2017). After the cultures were incubated at 30°C for 48 h, at least five colonies from suitable dilution plates of each sample were randomly picked up and purified by streak plating twice. The purified culture was preserved in 30% glycerol at –80°C.
Taxonomy of the isolates based on the 16S rDNA sequencing. The isolates were identified by sequencing their the 16S rRNA gene. The genomic DNA of the isolates was extracted using a Bacterial DNA Extraction Kit (Tiangen Biotech Co. Ltd., China). The 16S rDNA sequences were amplified using primers 27F and 1492R with the protocol described by Yuan et al. (2017). All the amplicons were sent directly to the Shanghai Sangon Biotechnology Co. Ltd. for sequencing. The DNA sequences were analyzed using the sequence alignment tool BLAST (Bacteria 16S ribosomal RNA sequences database of GenBank), available on the National Centre for Biotechnology Information website (http://www.ncbi.nlm.nih.gov). The BLAST results with E-values of 0.0, good query coverage (98–100%), and 98–100% identity could be confirmed to a species level.
Testing of antibiotic susceptibility. Tests for susceptibility to six antibiotics were carried out using the disk diffusion method (CLSI 2018) at the following concentrations: 25 μg sulfamethoxazole (1.25/23.75, TMP/SMZ), 10 μg gentamicin, 4 μg enrofloxacin, 30 μg cephalexin, 30 μg tetracycline, 30 μg chloramphenicol (Hangzhou Microbial Reagent Co., Ltd., China). After incubation at 35°C for 24 h, the zones of inhibition surrounding the disks were measured, and the strains were recognized as susceptible or resistant based on interpretative criteria according to the CLSI guidelines (CLSI 2018). Escherichia coli ATCC® 25922™ was used as the quality control strain.
Analysis of antibiotic-resistance genes. The antibiotic-resistance genes from all the isolates were amplified. Templates for PCR were prepared by boiling as follows: about 1–2 colonies from a fresh culture were picked up and suspended in 50 μl deionized sterile water. After boiling for 5 min, the suspension was immediately incubated on ice for 2 min. Then, the suspension was centrifuged at 5,000 × g for 1 min. About 2 μl of the supernatant was extracted and used as a PCR template. PCR assays were performed in 50 μl reaction mixture containing 1.5 μl of 10 × LA Buffer (Mg2+), 2.0 μl of 2.5 mmol/l dNTP Mix, 1 μl of each primer (10 μmol/l), 0.1 μl of 5 U/μl TaKaRa LA Taq™ DNA Polymerase (TaKaRa, Japan), and 2 μl of DNA template. The primers for the target genes sul2, aac(6’)-Ib, qnrS, blaPSE, tetA, cmlA, and floR are described in Table SI. The target genes were amplified using the following protocol: 94°C for 5 min, followed by 35 cycles of 94°C for 60 s, appropriate annealing temperatures for each primer pair for 40 s, and 72°C for 60 s, followed by a final extension step of 72°C for 10 min. All the amplicons were resolved using electrophoresis in a 1.5% agarose gel and checked by illumination under UV light after staining with ethidium bromide. The PCR products were purified using the Gel Mini Purification Kit (Zomanbio, China) and submitted for sequencing to the Shanghai Personalbio Biotechnology Co. Ltd. The sequencing data were analyzed using the BLAST tool in the NCBI database.
Statistical analysis. All the analyses were performed in triplicate, and statistical analysis was performed using the programs Origin 2019 (Origin Lab Corporation, USA) and AutoscriptProlog (IBM® SPSS® Statistics 21.0). The data were expressed as mean values ± standard deviation.
Results and Discussion
Identification of antibiotic-resistant bacteria. Colonies were obtained in the nutrient agar plates without antibiotics for each sample, and the distribution of antibiotic-resistant bacteria varied among different samples according to the number of colonies exhibited in Table SII. In total, 136 cultivable bacteria resistant to the tested drugs were picked up and purified. All the isolates could be classified into 22 genera and 49 species (Table SIII) according to 16S rDNA sequencing data. The corresponding GenBank accession numbers were as follows, MN216250-MN216297, MN220510-MN220522, MN220538-MN220542, and MN220557-MN220626. The predominant microbiota was represented by 106 isolates within six genera, including 41 Aeromonas spp., 19 Shewanella spp., 14 Acinetobacter spp., 13 Myroides spp., 10 Pseudomonas spp., and 9 Citrobacter spp., accounting for 80% of the total isolates.
Aeromonas spp., the major pathogens causing zoonotic diseases and was distributed naturally in diverse aquatic ecosystems and aquaculture products, was the predominantly isolated genera (31%). The antibiotic-resistant Aeromonas spp. could be frequently isolated from both healthy and disease-out broken aquaculture environments or farmed products (Zdanowicz et al. 2020; Dien et al. 2023). In this study, ten antibiotic-resistant Aeromonas species were isolated. Among them, Aeromonas veronii took the highest ratio (28 strains, 21%), and most isolates were recovered from crucian carp (17 strains). A. veronii is a widely distributed novel pathogen that can cause sepsis in fish, leading to high mortality and severe economic losses to aquaculture (Sun et al. 2017). Generally, Aeromonas spp. is regarded as an indicator of antibiotic resistance in the aquatic ecosystem since considerable antibiotic resistance was frequently found in Aeromonas infections (Patil et al. 2016). In this work, presumptive drug-resistant Aeromonas spp. were detected in all samples, which suggested that an excess of the tested antibiotics might be applied or exist in the aquaculture environment.
Shewanella spp. was the dominant ARB genus in large yellow croaker (14 strains, 52%), and the most frequently recovered species was Shewanella baltica (Table SIII). S. baltica and Shewanella putrefaciens are specific spoilage organisms of several chilled marine fish (Gu et al. 2013). Antibiotic-resistant Shewanellaceae, mainly the species Shewanella algae, were often recovered together with Vibrio spp. from the aquatic environments (Zago et al. 2020), and sometimes those species were closely related to specific diseases (Cao et al. 2018). The antibiotic-resistant Shewanella spp. in the farmed large yellow croaker added more risk to food safety.
Apart from the two genera mentioned above, other commensal and pathogen bacterial including Acinetobacter spp., Myroides spp., Pseudomonas spp., and Citrobacter spp. also accounted for a large proportion of all the isolates, while the remaining ARB recovered in this study belonged to 16 other genera. The diversity of the ARB also revealed that the ARGs might horizontally transfer between different genera and species (Fu et al. 2022).
Phenotypic characterization of antibiotic resistance. Phenotypic antibiotic resistance was checked accordingly to the CLSI (2018) (Fig. S1). A high percentage of resistance to tetracycline and sulfadiazine was shown for 38% and 37% of the total isolates (Table SIV). It was consistent with the results obtained in previous research works (Gao et al. 2012; Huang et al. 2017). Similar resistance rates to chloramphenicol (38 isolates, 28%), cephalexin (37 isolates, 27%), and enrofloxacin (35 isolates, 26%) were observed in this study. Furthermore, 24 (18%) isolates exhibited resistance to gentamicin.
Multiple drug-resistant bacteria were widely distributed in the samples of this study, which confirmed the widespread multi-drug resistance (Deng et al. 2020; Wang et al. 2021). In total, 109 isolates (80%) exhibited resistance to at least one antibiotic, whereas 27 isolates were sensitive to all six tested drugs (Table SIV). Twenty-seven isolates exhibited resistant to only one tested drug, but the numbers of double resistant isolates and triple resistant isolates had reached to 46 and 25 respectively, which in total account for 52% of the isolates. Furthermore, eight isolates were resistant to four different drugs, while two isolates were even resistant to five. These results were consistent with previous findings of Sun et al. (2017), who reported that the multi-drug resistance rate of digestive tract bacteria in cultivated abalone was as high as 66%. The heavy or alternate use of multiple antibiotic complexes during aquaculture production might confer high multi-drug resistance to bacteria (Gao et al. 2018).
The distribution of antibiotic resistance among different samples showed great diversities (Fig. 1), which might be related to the disease outbreak frequency and administration of the antibiotic drugs. Even in the same pond, the number of ARB varied largely (Wu et al. 2019). Here in this study, cultivable bacteria from the giant river prawn displayed high resistance to sulfadiazine (59%), enrofloxacin (47%), cephalexin (59%), tetracycline (47%), and chloramphenicol (41%). The isolates from the large yellow croaker displayed high tolerance to sulfadiazine (52%), chloramphenicol (56%), and tetracycline (44%). Bacteria isolated from crucian carp also showed high resistance to sulfadiazine (45%), enrofloxacin (52%), and tetracycline (72%). Isolates from yellow catfish only had high resistance to cephalexin (60%). The resistance rates of the bacteria from black carp to the tested antibiotics were relatively low but ubiquitous, ranging between 25% and 36%. Bacteria from loach were sensitive to the tested antibiotics and showed no resistance to enrofloxacin, tetracycline, and chloramphenicol.
Fig. 1.
The resistance rates of the isolates originated from different aquatic products to the six tested antimicrobials.
Genotypic characterization of antibiotic resistance. The existence of specific ARGs distributed on the conjugative plasmids, including qnrS, tetA, floR, sul2, aac(6’)-Ib, blaPSE, and cmlA in the 109 isolates, was checked by PCR amplification. The percentages of the amplicons were presented in Fig. 2. The prevalence of ARGs of the corresponding ARBs was presented in Table I. The genes qnrS, tetA, floR, and cmlA were principally found in isolates of phenotypically resistant characteristics, whereas sul2, aac(6’)-Ib, and blaPSE were less frequently detected, even in the isolates of the resistant phenotype.
Fig. 2.
Amplicons of partial antimicrobial resistance genes.
The coincidence of the resistant phenotype and genotype among the isolates.
Antimicrobial
No. of ARB isolates
ARGs
No. of the ARGs carrier
Coincidence rate (%)
Sulfadiazine
50
sul2
10
20%
Gentamicin
24
aac(6’)-Ib
10
42%
Enrofloxacin
35
qnrS
26
74%
Cephalexin
37
blaPSE
12
32%
Tetracycline
52
tetA
35
67%
Chloramphenicol
38
floR
30
79%
cmlA
30
79%
The chloramphenicol resistance-related genes floR and cmlA had the highest detection rate in ARBs (79%). Although chloramphenicol has been prohibited in aquaculture since 1999 in China, chloramphenicol-resistant pathogens or environmental bacteria remain prevalent. The application of the chloramphenicol derivative florfenicol in aquaculture productions might be a direct cause. Moreover, the chloramphenicol-resistant genes left over from history also could transfer between different microorganisms in aquatic environments without a high selective pressure (Yoo et al. 2003; Wang et al. 2011). The presence of the qnrS gene among the ARB was over 74%, which to some degree corroborated with the fact that the quinolone-related ARGs such as qnrA, qnrB, qnrS were frequently discovered in the farming water, sediments, aquatic products, and pathogens causing disease in fish (Wang et al. 2021), as a result of the widespread and frequent application of quinolone and fluoroquinolone drugs (Chenia et al. 2016).
In this work, tetA was distributed among 67% of tetracycline-resistant ARBs. Many studies revealed that among different tetracycline-resistance tet genes, tetA was the most frequently detected in samples, including water and sediment in fish farms, fresh vegetables, and fish (Xiong et al. 2019). The tetA gene was also suggested to be a potential indicator of the abundance of tetracycline resistance genes in specific aquaculture modes in the Pearl River Delta (Huang et al. 2017). The gentamicin-related gene aac(6’)-Ib, β-lactam-resistance gene blaPSE, and the sulfonamide-resistance gene sul2 showed a lower detection rates of 42%, 32%, and 20%, respectively.
The inconsistency between the phenotypic and genotypic resistant data could owe to the following reasons. Firstly, the mechanism of drug resistance of bacteria was complicated, and diverse antibiotic resistance mechanisms against the particular antibiotic exist. For example, the plasmid-mediated quinolone resistance (PMQR) genes included qnr, qepA, and aac(6’)-Ib genes (Suzuki, 2012). However, in this study, only the qnrS was selected to reflect fluoroquinolone-related gene pollution, which might be the main reason for the inconsistency between the phenotypic and genotypic resistant ratio. Secondly, the expression of some ARGs could be impressed by other factors during the dissemination (Wardenburg et al. 2019). Last but not least, limited samples in this study may not fully reveal the objective rules.
Overall, the ARB from different aquatic products showed varying resistance to the tested antibiotics, which might be related to the differential application of antibiotics during the farming process. The results also confirm that high rates of multi-drug resistance were prevalent within the isolates from all the tested samples.
The prevalence of drug-resistant opportunistic pathogens such as A. veronii and Aeromonas hydrophila indicates a potential safety hazard. The previous studies indicated that most ARGs come from plasmids that originate from Vibrio and Aeromonas (Liu et al. 2019). The abuse of antibiotics and the horizontal transfer of ARGs have increased the prevalence of multi-drug-resistant bacteria in the aquatic environment. All the tested ARGs responding to the six tested drugs could be detected among the ARBs. The widespread ARGs may increase the antibiotic resistance of pathogenic bacteria and reduce antibiotic efficacy.
Conclusions
This work showed a great diversity of ARB and ARGs in popular farmed aquatic products in Zhejiang, China. Since the potential pathogen Aeromonas spp. and spoilage bacteria Shewanella spp. were the predominant ARB in the collected samples, more concerns need to be put on the antibiotic administration. Multi-drugs resistant isolates consisted of more than half of the isolates and were distributed widely among all the collected samples. The ARGs corresponding to the common antibiotics applied in aquaculture were detected easily, though the inconsistency between the phenotypic and genotypic antibiotic resistance was detected. The data obtained in this work could improve understanding of the risks to aquatic organisms and human beings.
