1. INTRODUCTION
Shrimp culture has become an important industry which is accounting for approximately 18% of the total world’s aquaculture economy. Asian and Latin America are major production areas whose economic value is estimated at USD 28 billion per year [1]. However, this high-value industry has faced serious disease outbreaks, particularly caused by Vibrio bacteria, which have caused widespread losses to farmers. A certain Vibrio species can cause high mortality to shrimp such as V. parahaemolyticus, V. alginolyticus, V. harveyi, V. mimicus, or V. vulnificus. Vibrio bacteria are the major agent responsible for acute hepatopancreatic necrosis disease, shell disease, white gut disease, luminescent disease, red disease, and loose shell [2,3].
Antibiotics were widely applied in aquaculture to control pathogenic bacterial infections including Vibrio species. The overuse of antibiotics is recently being a serious problem to the water ecosystem by spreading antibiotic-resistant bacteria and antimicrobial resistance genes to the environment. Moreover, opportunistic Vibrio bacteria have been reported carrying out multiple virulence factors and multiple resistomes. The diversity of resistomes found higher in shrimp farms than in coastal waters and depended on the farming stage [4-6]. The overuse of antibiotics in shrimp farming in Vietnam has recently escalated, according to the National Agro-Forestry-Fisheries Quality Assurance Department (Nafiqad). Antibiotics including oxytetracycline, chloramphenicol, ciprofloxacin, ormetoprim, and enrofloxacin are typically to blame for violations of shrimp batches [7]. As a result, many antibiotics are now listed as banned substances including ciprofloxacin, chloramphenicol, erythromycin, oxytetracycline, and sulfonamides [2].
Thua Thien Hue province has the advantage of a long coastline, many dunes along the coast, which are favorable for shrimp farming. Besides, this place has the Tam Giang-Cau Hai coastal lagoon system which is the largest lagoon of Southeast Asia, which has many favorable conditions for the development of shrimp culture [8]. Over the past years, white leg shrimp (Litopenaeus vannamei) farming becomes more popular due to great economic benefits to farmers. However, enormous difficulties and challenges have been rising for the shrimp farming markets such as environmental pollution and epidemics outbreaks caused by Vibrio species[8,9]. According to a preliminary investigation, for instance, a 2000 m2 pond stocked with 50,000 shrimp in Phong Dien district (Thua Thien Hue province) would require spending 50–60 million VND on mineral salts, digestive enzymes, and antibiotics (tetracycline, berberine, etc.) over the course of 2.5 months [10]. Moreover, the data on antibiotic-resistant Vibrio related to the shrimp farming industry in this area are not well documented. This study alerts the occurrence of antibiotic resistance Vibrio species in intensive shrimp farming systems, contributing to the approaches to minimize the antibiotic resistance Vibrio in aquaculture.
2. METHODS AND MATERIALS
2.1. Medium and Antibiotics
Thiosulfate Citrate Bile Sucrose agar (TCBS) agar medium was used to isolate Vibrio [11]. Mueller-Hinton broth was used for the antimicrobial susceptibility test. Antibiotics including ciprofloxacin, gentamycin, kanamycin, oxytetracycline hydrochloride, doxycycline, and streptomycin from Nam Thai Veterinary Medicine Co. (Vietnam) were used to evaluate the antibiotic resistance ability of Vibrio isolates.
2.2. Determination of Antibiotic-resistant Vibrio Isolates
Five hundred μL of water from each shrimp pond in four districts of Thua Thien Hue province (Quang Dien, Phu Vang, Phu Loc, and Phong Dien) was collected monthly for the study, with three ponds in each district. Water was diluted between 10-1 and 10-4 in a sterile 0.9% saline solution. One hundred μL of the diluted solution was then spread on TCBS agar plate supplemented with various antibiotics including ciprofloxacin (50 μg/mL), gentamycin (10 μg/mL), kanamycin (30 μg/mL), oxytetracycline hydrochloride (30 μg/mL), doxycycline (30 μg/mL), and streptomycin (10 μg/mL). A TCBS agar plate without antibiotics was used as a control. The cultures were kept at 37°C for 24 h. Bacterial concentrations were determined based on the number of Vibrio colonies growing on TCBS agar with or without antibiotics. The prevalence of antibiotic-resistant Vibrio bacteria (PARV) was calculated as follows:
 |
Where: CARV: antibiotic-resistant Vibrio cells, CTV: total Vibrio cells
2.3. Determination of Minimum Bacterial Inhibitory Concentration (MIC)
MIC50 and MIC90 were determined as described by Ataee et al. [12]. Antibiotic concentrations were prepared from 30 to 300 μg/mL (in increment of 30) for each, except for ciprofloxacin was from 5 to 50 μg/mL (in increment of 5).
Vibrio isolates were cultured overnight in Mueller-Hinton broth at 37°C with 180 rpm of shaking, then diluted to an OD600 of 1 (approximately 1.5 × 108 CFU/mL). The diluted bacterial solution was mixed with 152.5 μL of Mueller-Hinton broth and antibiotic solution to a final concentration of 105 CFU/mL. The mixture was incubated at 37°C with 150 rpm of shaking for 18 h. The optical density of the mixture before and after incubation was measured at 600 nm using a UV-VIS spectrophotometer. The antibiotic susceptibility ratio (RAS, %) was determined as follows [13]:
 |
Where, Aa and Ad are the initial optical density of sample and control (without antibiotics); Ab and Ac are the final optical density of sample and control (without antibiotics). MIC50 and MIC90 were defined as the antibiotic concentration that inhibits approximately 50% and 90% of Vibrio cells, respectively.
2.4. Molecular Identification of Vibrio Isolates
Total DNA from Vibrio isolates was extracted by the cetyltrimethylammonium bromide method as described by Sambrook [14]. 16S rRNA genes were amplified by PCR with universal primers (forward: 5’-AGAGTTTGATCMTGGCTCAG-3’ and reverse: 5’-CTGCTGCSYCCCGTAG-3’) and sequenced by the Sanger method. Their sequences were then searched homology against the GenBank database to identify species. The phylogenetic tree was generated using MEGA 11 software using the maximal likelihood method with a bootstrap of 1000 replicates [15].
2.5. Data Analysis
The data were processed with Minitab software version 16.2.0 and Microsoft Excel 2013 to calculate mean values. A one-way ANOVA with a significance level of 0.05 was used to compare the differences in means between treatments.
3. RESULTS
3.1. Determination of MIC
Based on inhibition rate data for the 10 antibiotics used, Vibrio isolates grown on ciprofloxacin-TCBS plates were selected to determine antibiotic resistance ability. The observation found five isolates exhibited a broad antibiotic resistance against gentamycin (10 μg/mL), kanamycin (30 μg/mL), oxytetracycline hydrochloride (30 μg/mL), doxycycline (30 μg/mL), and streptomycin (10 μg/mL). These isolates named as Vibrio KS01 (NCBI, accession number: OK560361), Vibrio KS03 (OK560362), Vibrio KS04 (OK560363), Vibrio KS06 (OK560364), and Vibrio KS07 (OK560365). The minimum inhibitory concentration of 10 antibiotics used to eliminate these five isolates is shown in Table 1.
Table 1: The minimum inhibitory concentration of antibiotics against Vibrio KS01, KS03, KS04, KS06, and KS07.
| Antibiotics | KS01 | KS03 | KS04 | KS06 | KS07 | |||||
|---|---|---|---|---|---|---|---|---|---|---|
| MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | MIC50 | MIC90 | |
| 1. Aminoglycoside | ||||||||||
| Gentamycin | 90 (51.5%) | 180 (94.1%) | 30 (56.4%) | 150 (95.6%) | 270 (54.9%) | - | 240 (80.1%) | - | 60 (51.6%) | - |
| Kanamycin | 60 (55.3%) | 150 (99.5%) | 30 (85.8%) | 150 (95.4%) | 120 (69.3%) | 150 (91.5%) | 240 (57.9%) | - | 120 (70.5%) | 150 (96.6%) |
| Streptomycin | - | - | - | - | - | - | - | - | - | - |
| 2. Tetracycline | ||||||||||
| Oxytetracycline hydrochloride | 180 (50.6%) | - | - | - | 150 (54.7%) | - | - | - | - | - |
| Doxycycline | 30 (84.2%) | - | 300 (52.3%) | - | 120 (62%) | - | 30 (72.3%) | - | - | - |
| 3. Quinolone | ||||||||||
| Ciprofloxacin | 50 (58.68%) | - | 25 (50.47%) | - | - | - | - | - | - | - |
No inhibition
The results showed that kanamycin, one of the four aminoglycoside antibiotics investigated, had the strongest inhibitory effect on Vibrio. Vibrio KS03 expressed high sensitivity to this antibiotic group than other isolates whose MIC50 was at 30 μg/mL. Meanwhile, Vibrio KS06 was strongly resistant to these antibiotics with MIC50 found to be above 120 μg/mL. MIC90 of all Vibrio isolates was ≥150 μg/mL. In contrast, streptomycin did not significantly affect bacterial growth.
Oxytetracycline hydrochloride did not show susceptibility to Vibrio strains KS03, KS06, and KS07, while the MIC50 of KS01 and KS04 was 180 μg/mL and 150 μg/mL, respectively. Although, the MIC50 of doxycycline ranges from 30 to 300 μg/mL. This antibiotic did not completely inhibit the growth of Vibrio.
Our study revealed that ciprofloxacin was not significantly effective against Vibrio isolates compared with other tested antibiotics. It only partially inhibited the growth of strains KS01 and KS03, but not strains KS04, KS06, and KS07 [Table 1]. This result indicated that ciprofloxacin-resistant ability of Vibrio was highly stable.
In general, Vibrio KS01 isolate was completely resistant to streptomycin, while KS03 isolate resists streptomycin and oxytetracycline hydrochloride. Streptomycin and ciprofloxacin inhibited KS04 growth. Meanwhile, streptomycin, oxytetracycline hydrochloride, and ciprofloxacin had no inhibitory effect against KS06. KS07 isolate still grew well in the presence of streptomycin, doxycycline, oxytetracycline hydrochloride, and ciprofloxacin.
3.2. Antibiotic Resistance Status of Vibrio
As shown in Table 2, the water contained Vibrio with concentrations ranging from 102.5 to 103.8 CFU/mL. Vibrio growth was slightly increased in the 1st month of culture but then more rapidly from the second to the 2nd month, reaching about 105 CFU/mL and 106 CFU/mL, respectively.
Table 2: Vibrio in shrimp culture ponds in Thua Thien Hue.
| Sample source | Culture time (month) | Vibrio concentration (CFU/mL) | |||
|---|---|---|---|---|---|
| Quang Dien | Phu Vang | Phu Loc | Phong Dien | ||
| Water | Supply water | 102.9 | 102.5 | 103.2 | 103.8 |
| 1 | 102.1 | 104.2 | 103.7 | 104.1 | |
| 2 | 105.3 | 104.4 | 103.8 | 105.5 | |
| 3 | 106.4 | 105.4 | 104 | 106.6 | |
| Vibrio isolates | KS03, KS01, KS04, KS07 | KS03, KS01, KS06 | KS03, KS01, KS06, KS07 | KS03, KS01, KS07 | |
The percentage of antibiotic resistance varied and was quite different among the water sources and culture time [Table 3]. Water in Quang Dien was clean without any antibiotic resistance Vibrio except for doxycycline. On the contrary, antibiotic resistance Vibrio was found in water obtained from Phu Vang, Phu Loc, and Phong Dien. Vibrio’s high resistance ratio with gentamycin and streptomycin was found in supply water at one shrimp pond in Phu Vang. Meanwhile, there was no ciprofloxacin resistance Vibrio in all investigated areas. The percentage of antibiotic resistance Vibrio dramatically increased over time during the cultivation of the shrimp.
Table 3: Percentage of antibiotic resistance Vibrio in shrimp pond.
| Antibiotic group | Antibiotic | Collection time | Location | |||
|---|---|---|---|---|---|---|
| QD | PV | PL | PD | |||
| Aminoglycoside | Gentamycin | Supply water | 0 | 21.21 | 43.00 | 8.29 |
| 1st month | 58.33 | 25.86 | 41.82 | 35.71 | ||
| 2nd month | 53.02 | 39.19 | 47.85 | 51.08 | ||
| 3rd month | 78.57 | 39.13 | 52.07 | 60 | ||
| Kanamycin | Supply water | 0 | 13.13 | 30 | 2.86 | |
| 1st month | 0 | 15.45 | 4.55 | 11.9 | ||
| 2nd month | 18.06 | 28.90 | 11.69 | 25.12 | ||
| 3rd month | 33.33 | 36.31 | 14.6 | 35.42 | ||
| Streptomycin | Supply water | 8.75 | 16.16 | 42.67 | 7.14 | |
| 1st month | 16.67 | 18.09 | 48.18 | 36.43 | ||
| 2nd month | 69.04 | 55.49 | 57.69 | 37.05 | ||
| 3rd month | 67.26 | 50.81 | 62.07 | 97.91 | ||
| Tetracycline | Oxytetracycline hydrochloride | Supply water | 0 | 0.00 | 23.67 | 5.57 |
| 1st month | 0 | 26.07 | 16.36 | 37.86 | ||
| 2nd month | 59.07 | 55.32 | 33.23 | 78.72 | ||
| 3rd month | 98.10 | 85.07 | 41.47 | 83.64 | ||
| Doxycycline | Supply water | 7.5 | 24.24 | 0 | 3.71 | |
| 1st month | 41.67 | 75.97 | 18.79 | 42.14 | ||
| 2nd month | 43.02 | 75.96 | 27.69 | 61.96 | ||
| 3rd month | 49.11 | 80.13 | 23.73 | 65.92 | ||
| Quinolone | Ciprofloxacin | Supply water | 0 | 0 | 0 | 0 |
| 1st month | 0 | 0.06 | 0 | 0.71 | ||
| 2nd month | 5.60 | 0.19 | 0 | 1.41 | ||
| 3rd month | 12.91 | 0.26 | 0 | 3.80 | ||
QD: Quang Dien, PV: Phu Vang, PL: Phu Loc, PD: Phong Dien
Study results have shown that more than 90% of Vibrio present in water was resistant to antibiotics, especially to streptomycin and oxytetracycline hydrochloride [Table 3]. Among the investigated areas, the shrimp farm in Phong Dien had the highest rate of antibiotic-resistant Vibrio bacteria, 97.91% after 3 months of culture. In contrast, quinolone antibiotics such as ciprofloxacin inhibited most Vibrio isolates.
3.3. Identification Antibiotic Resistance Vibrio
Species identification of the five Vibrio isolates was carried out by sequencing their 16S rRNA gene. Comparison of 16S rRNA nucleotide sequences with the database on the GenBank showed that Vibrio KS03 has high similarity with V. parahaemolyticus (100%); Vibrio KS04, KS06, and KS07 like V. alginolyticus (100, 100, and 99%, respectively); while Vibrio KS01 was identified with Vibrio sp. (100%). Therefore, the three isolates of Vibrio KS04, KS06, and KS07 were named V. alginolyticus KS04, V. alginolyticus KS06, and V. alginolyticus KS07. The Vibrio KS03 isolate was named V. parahaemolyticus KS03. The phylogenetic tree of these isolates was constructed using the maximum likelihood method and the Tamura-Nei model, as shown in Figure 1. The nucleotide sequences of Vibrio isolates KS01, KS03, KS04, KS06, and KS07 have been deposited on NCBI with the accession numbers OK560361, OK560362, OK560363, OK560364, and OK560365, respectively.
 | Figure 1: Phylogenetic tree among Vibrio sp. KS01, Vibrio sp. KS03, Vibrio sp. KS04, Vibrio sp. KS06, Vibrio sp. KS07, and other Vibrio sp. from GenBank. The tree was built using the maximum likelihood method. The tree was generated with branch lengths measured in the number of substitutions per site. [Click here to view] |
4. DISCUSSION
A previous study by Chikwendu (2004) showed that the rate of Vibrio resistant to mezlocillin, doxycycline, carbenicillin, and ampicillin ranged from 81.3 to 97.8%, while the rate of resistance to kanamycin was only 40.9% [16]. The present study reported that Vibrio can resist other strong antibiotics including streptomycin, gentamycin, and ciprofloxacin which are often used to treat pathogenic bacteria for humans. Data in Table 3 indicated that ciprofloxacin is the last barrier to eliminating antibiotic resistance Vibrio. Ciprofloxacin has stronger antibacterial activity than antibiotics of other groups such as amino acids, b-lactams, tetracyclines, and penicillins [17,18]. Ciprofloxacin inhibited bacteria growth through inactivation of the DNA gyrase, which further prevents chromosome replication [17]. The supply water did not contain any Vibrio which can resist ciprofloxacin. However, it was found in pond-reared water after 2 months of cultivation. Similar studies also recorded ciprofloxacin resistance Vibrio from fish [19] and shrimp [20]. Gentamycin resistance of Vibrio has been reported as dominant as in their community in Nigeria and Iran, accounting for 93% and 83.3%, respectively [21,22]. A high prevalence of streptomycin-resistant Vibrio has also been noted here [21]. A study on antibiotic resistance of V. parahaemolyticus appeared in Korea and found that 27.7% of isolates were resistant to tetracycline [23]. Meanwhile, more than 90% of V. parahaemolyticus isolates from seafood samples in Malaysia were sensitive to tetracycline [24,25].
Kang et al. [26] identified that 15/16 Vibrio isolates from oysters had resistance profiles against 16 antibiotics in which five V. alginolyticus isolates showed multiple resistance to at least three antimicrobials. V. parahaemolyticus isolates also alerted high resistance to ampicillin, cefazolin, streptomycin, trimethoprim, kanamycin, and trimethoprim [27]. A report by Manjusha et al. [21] showed that 16.8% of Vibrio isolates collected from brackish water and coastal areas in India resisted all types of tested antibiotics. Vibrio isolated from aquaculture water in Nigeria resisted gentamycin, nitrofurantoin, tetracycline, augmentin, chloramphenicol, amoxicillin, ofloxacin, cotrimoxazole, ceftriaxone, and ciprofloxacin [28]. These results combine with our similar findings that demonstrated antibiotic resistance Vibrio present in brackish water, especially in aquaculture. This shows that strict adherence to safe farming guidelines as well as regular assessment of bacterial susceptibility to antibiotics is essential.
5. CONCLUSION
Vibrio presents in brackish water with the number from 102.5 to 103.8 CFU/mL and dramatically increasing during shrimp culture, reaching 104 to 106.6 CFU/mL after 3 months. Antibiotic-resistant Vibrio also occurred in brackish water and depends on farming location. Gentamycin, kanamycin, streptomycin, oxytetracycline hydrochloride, and doxycycline did not completely control the Vibrio growth. After 3 months of culture, pond water consists of up to 98.87% antibiotic resistance Vibrio. Ciprofloxacin exhibited the strongest inhibitory against Vibrio. MIC evaluation results in high resistance ability to other antibiotics of ciprofloxacin-resistant Vibrio. According to the present study, antibiotic-resistant Vibrio bacteria can be controlled by routinely using ciprofloxacin in shrimp farming.
6. ACKNOWLEDGMENTS
The authors would like to thank Prof Loc’s Core Research Group from Hue University for supporting the study.
7. AUTHORS’ CONTRIBUTION
Investigation: Tuan LC, Khanh NV, Tien HTNB, Phuong PT, and Thanh LTH; data analysis: Loc NH; writing-original draft preparation: Tuan LC; and writing-review and editing: Loc NH.
8. FUNDING SOURCES
This study was supported in part by grant from Hue University (Code: HH-2020-01-176) and VLIR - IUC Programme - Hue University (Project 3 - Coastal Ecosystem and Natural Resources Management).
9. CONFLICTS OF INTEREST STATEMENT
The authors report no financial or any other conflicts of interest in this work.
10. ETHICAL APPROVAL
This article does not contain any studies with human participants or animals performed by any of the authors.
11. DATA AVAILABILITY
All data generated and analyzed are included within this research article.
12. PUBLISHER’S NOTE
This journal remains neutral with regard to jurisdictional claims in published institutional affiliation.
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