EVALUATION OF ANTIBIOTIC RESISTANCE OF GRAM NEGATIVE BACTERIA ISOLATED FROM DONG NAI RIVER

78 ABSTRACT EVALUATION OF ANTIBIOTIC RESISTANCE OF GRAM NEGATIVE BACTERIA ISOLATED FROM DONG NAI RIVER Vo Thi Thu Thao, Pham Thi Hoa International University - Vietnam National University, Ho Chi Minh city Email: pthoa@hcmiu.edu.com This study assessed the current status of antibiotic resistance of Gram-negative bacteria on Dong Nai Saigon river. Water samples were collected from 4 different areas on the river including: Nam Cat Tien forest, La Nga district (Dong Nai province), Sai Gon river (HCMC) and Can Gio district (Ho Chi Minh city). Sampling sites are close to large aquaculture areas, ranging from upstream to downstream of the river. The results showed that 20 strains of Gram negative bacteria, which belong to five bacterial species, were identified: Vibrio sp, Serratia sp Enterobacteriaceae sp, Pseudomonas sp and Escherichia coli. Sensitivity to antibiotics was determined with four common antibiotics used in aquaculture including Ampicillin, Amoxicillin, Chloramphenicol (CHL), Tetracycline (TET) and Kanamycin (KAN). AMP antimicrobial resistance was highest (55%), and lowest was TET (25%). Most isolated strains (80%) were resistant to at least one antibiotic, of which 3 were resistant to all antibiotics. Antimicrobial resistance rates in the five-isolated species have shown greater concern needed for the protection of the environment and human health, particularly the antibiotics used in agriculture and aquaculture activities. Keywords: Dong Nai river, Antibiotic resistance, Gram-negative bacteria, Ampicillin, Amoxicillin. TÓM TẮT Đề tài này đánh giá về hiện trạng kháng thuốc kháng sinh của các khuẩn Gram âm trên sông Đồng Nai Sài Gòn. Mẫu nước được thu thập từ 4 khu vực khác nhau trên sông Đồng Nai bao gồm: Rừng Nam Cát Tiên, huyện La Ngà (ti nh Đồng Nai), sông Sài Gòn (Tp. HCM) và huyện Cần Giờ (Tp. Hồ Chí Minh). Các vị trí lấy mẫu gần với các khu vực nuôi trồng thủy sản lớn, kéo dài từ thượng nguồn cho tới hạ nguồn của sông. Kết quả phân lập được 20 dòng vi khuẩn Gram âm, được xác định thuộc 5 loài vi khuẩn: Vibrio sp, Serratia sp, Enterobacteriaceae sp, Pseudomonas sp và Escherichia coli. Sự nhạy cảm đối với kháng sinh được xác định theo bốn loại thuốc kháng sinh phổ biến sử dụng trong nuôi trồng thuỷ sản bao gồm Ampicillin, Amoxicillin, Chloramphenicol (CHL), Tetracycline (TET) và Kanamycin (KAN). Tỷ lệ vi sinh vật kháng kháng sinh AMP là cao nhất (55%), và thấp nhất là TET (25%). Hầu hết các chủng bị cô lập (80%) kháng với ít nhất một kháng sinh, trong đó có 3 loài kháng với tất cả các kháng sinh. Tỷ lệ kháng kháng sinh trong năm loài khuẩn được cô lập đã cho thấy cần quan tâm nhiều hơn nữa trong việc bảo vệ môi trường và sức khoe con người, đặc biệt là thuốc kháng sinh sử dụng trong nông nghiệp và nuôi trồng thủy sản. Từ khoá: Sông Đồng Nai, kháng kháng sinh, vi khuẩn Gram âm, Ampicillin, Amoxicillin INTRODUCTION Vietnam is known as a country providing the large amount of aquatic product with the third ranked in the world (Tran Van Hoa, 2016). While many aquatic products are exported, the demand in domestic markets is also high as a necessary part of the daily diet of Vietnamese. Consequently, many aquaculture farms in Vietnam are highly intensive to increase the yield per area unit. An important factor that affects the yield is disease outbreaks, which in turn are irritated by the high stock density of intensive farming (Subasinghe et al., 2001; Nguyen and Ford, 2010). In attempt to protect farms and

79 improve yields, farmers must use antibiotics to prevent and treat diseases for animals. Hence, aquaculture productions become one of the main sources of pollution of veterinary medicines into the environment (Pruden et al., 2013; Rico and Van den Brink, 2014). As a result, many water resources have been rendered unwholesome and dangerous to man and other living organism systems (Bakare et al., 2003). In additionally, awareness of utilizing antibiotics in aquatic ecosystems is improving increase in many countries around the world. Recently, Vietnam has also published new regulations to prevent ungoverned antibiotic usage in domestic aquaculture (Official Decision No. 2625 / QD-BNN-TY, 21/6/2017). However, the antibiotic usage surveillance in livestock and aquaculture in the local areas is still not effective enough which causes concerns for antibiotic resistance in pathogenic microorganism. Bacteria cultured from the collected water sources were tested for resistance against antibiotics (Tetracycline, Ampicillin, Kanamycin and Chloramphenicol) and effect with short term exposure (Pham Kim Dang et al.,2015). The resistance was displayed against the antibiotics tested among the bacteria cultured from the water. This may indicate that the existence of antibiotic residue could enter in the aquatic systems and cause a potential threat to ecosystem function and influence on human health (Costanzo SD et al., 2004). Potential consequences of antibiotic use in animal feeds are the development of drug resistant bacteria, resistance transfer to pathogenic bacteria, and reduced efficiency of antibiotic treatment for animal diseases caused by resistant pathogens (Frappaolo and Guest, 1986). It is worth considering that, in many ways, the resistance is already well promoted in bacterial populations in human, pest and the environment and also cause the disease with the resistance gene (Salyers and Amabile-Cuevas, 1997). A recent study conducted on antibiotic contamination in southern Vietnam indicates the presence of four major antibiotics in aquatic environments: trimethoprim, oxytetracycline, sulfamethoxazole and sulfadimidine (Pham Kim Dang et al., 2015). The antibiotic resistance patterns of bacteria are influenced by selective pressure; thus, may be different in other geographical areas and may vary over time (Parveen et al., 1999). Due to the lack of regulation, there is very little information about antibiotics used in aquaculture for almost rivers in Vietnam, especially in Ho Chi Minh city and surrounding regions. That is the reason why Sai Gon - Dong Nai river, which is known as the main river system in the area with many aquatic farms and industrial areas along the river, is considered the antibiotic residue and appearance of the drug resistant bacteria related to the aquaculture activity (Vietnam Environment Administration, 2016). It is known that Dong Nai river system passes through Ho Chi Minh city and other provinces and have more than 250 rivers and streams of over 10km each. The basin system is reach with copious minerals resources and covered by tropical forests with a great biodiversity of tropical ecosystems, which help to protect the area from flood in the rainy season and maintain water lever for Dong Nai river in the dry season. Fast urbanization, high rate of the population growth and the rapid of economic development included the industry and agriculture might make the environment worsened. In the same way, aquaculture activities, especially occurring strongly at the La Nga district, Dong Nai province and Can Gio district, Ho Chi Minh city, have raised the questions about the situation of utilizing antibiotic for the animal and the exuberance of drugs into the environment through wastewater or fecal. Therefore, this project aims to study the situation of Dong Nai river system water and evaluate the level of antibiotic susceptibility of isolated gram negative bacteria from the water sample collected in the Dong Nai river at four different selective locations, extended from upstream to downstream, included Nam Cat Tien forest, La Nga district (Dong Nai province), Sai Gon river and Can Gio district (Ho Chi Minh city).

80 MATERIAL AND METHOD Sampling The sampling sites were chosen based on aquaculture activity along the river, extended from upstream to downstream. The water samples were taken at four locations: Nam Cat Tien forest, Sai Gon river (district 2 and Thu Duc district), La Nga district and Can Gio district (Figure 1). At the Nam Cat Tien rorest, the samplings were collected near waterfront while the others were directly taken at the middle of the stream using a bottle tied with a 2-meters string. Three samples (500ml of water for each sample) at each place were taken into the sterile screw capped bottles to preserve the sample in cold bag until transported to the laboratory for analysis. The samples were transported to laboratory for the isolation procedure within 2-3 hours. a) Sai Gon river b) Shrimp farm (Can Gio district) c) La Nga district d) Nam Cat Tien forest Isolation procedure A 1-ml portion of water samples was suspended in 9ml of 0.85% NaCl, and 7 fold serial dilutions were prepared in 0.85% NaCl. Portion (0.1 ml) of each dilution were inoculated onto Tryptic Soy Agar (TSA) and MacConkey (Mac) agar plates and incubate for 18 24h at 30 0 C (Ehsan, 2015). After incubated, each different colony, which based on the morphologies were recovered from each plate, was streaked into new medium agar plate to isolate purify and single colony. TSA Figure 1. Pictures of sampling sites on this study plates were utilizing to evaluate a bacterial concentration. Each isolated colony was examined by gram staining and morphology under microscope to preliminary choose the gram negative bacteria. Microbial Identification The gram negative bacteria were identified by biochemical tests using IDS 14 GNR Kit (Nam Khoa company, Vietnam). Those isolates exhibiting metabolism were identified by IDS 14 GNR kit (Nam Khoa company, Vietnam) following the instruction.

81 Antibiotic resistance determination Agar Disk Diffusion Test method was used with the Mueller Hinton agar (Bauer et al., 1966). The isolates were tested for sensitivity to four antibiotics applied with the different dosage for each (µg/ml): Ampicillin (AMP) (10), Kanamycin (KAN) (30), Tetracyline (30), and Chloramphenicol (30). The plates were incubated 24h at 30 o C. The result considered resistant relied on the sign of the good growth on antibiotic plate and the zone inhibition table 1. Table 1. Standard for Antibiotic susceptibility of bacteria (by diameter of zone of inhibition in mm) from Clinical and Laboratory Standards Institute 2015. Resistance Intermediate Sensitivity AMP 13 14-16 17 CHL 12 13-17 18 TET 11 12-14 15 KAN 13 14-17 18 Result Sampling The water samples were taken from four stations as mentioned in the table 2. Each place has different characteristic to test distinctive feature might affect the number of bacteria even the specificity strain. The samples at first, second and third stations have nearly the same ph value with around 6.7 to 7.0. Similarly, the salinity is zero of the first three place. However, at the fourth station, the ph value is slightly higher than the others with the average value is about 7.5. In that period, the water and the soil in Can Gio district was still influenced by salt intrusion. Therefore, the salinity value is very great, over 10. Those disparities in ph and salinity value could affect the number of bacteria. Table 2. Features of the water sampling sites Sampling Stations Characteristic 16 th September 2016 16 th September 2016 30 th September 2016 30 th September 2016 Nam Cat Tien forest (1) La Nga district (2) Sai Gon river (3) Can Gio district (4) Headwater Fish farm Downstream Shrimp farm Station Water ph Salinity ( ) 1.1 6.76 0 1.2 6.81 0 1.3 6.79 0 2.1 6.84 0 2.2 6.9 0 2.3 6.91 0 3.1 7.01 0 3.2 6.94 0 3.3 6.98 0 4.1 7.52 14 4.2 7.64 17 Isolation and Identification After isolation procedure, all the gram negative bacteria strains were taken for microbial identification steps using the biochemical reactions by IDS 14 GNR Kit provided by Nam Khoa company. The Kit uses 14 different biochemical reactions caused by bacteria to identify the rod-shape Gramnegative bacterium after 24 hours of incubation. The table 3 shows the ratio of strains between the gram-positive bacteria and gramnegative bacteria are isolated at four locations. Although there were total 23 isolated strains, three were one-gram positive bacterium at first

82 station and two at second station (13.04%). Most of the bacteria were the gram negative microorganism (86.96%). Gram positive bacteria were eliminated leads to the first station have five strains of gram negative bacteria while the second exist eight strains. Besides, the third places have four isolated gram-negative microorganism, the other have three strains. In total, there were 20 purified isolated strains of gram negative animalcule at four locations (Table 4). Stations Isolation number Table 3. Ratio of isolated bacteria Bacteria Gram Positive Gram Negative No. % No. % 1 6 1 16.67 5 83.33 2 10 2 20 8 80 3 4 0 0 4 100 4 3 0 0 3 100 TOTAL 23 3 13.04 20 86.96 The IDS 14 GNR Kit was used to determine the microorganism by following strictly the instruction. The result was listed in Table 4 below. Table 4. Microbial Identification Station Series Microbial Identification 1 1 Serratia marcescen 1 2 Vibrio metschinikovii 1 3 Vibrio metschinikovii 1 4 Serratia plymuthica 1 5 Vibrio mimicus 2 6 Vibrio hollisae 2 7 Serratia plymuthica 2 8 Serratia plymuthica 2 9 Vibrio mimicus 2 10 Serratia fonticola 2 11 Serratia marcescen 2 12 Pragia fontium 2 13 Vibrio metschinikovii 3 14 Vibrio vulnificus 3 15 Vibrio metschinikovii 3 16 Escherichia coli 3 17 Vibrio metschinikovii 4 18 Pseudomonas chlororaphis 4 19 Vibrio fluvialis 4 20 Vibrio metschinikovii Antibiotic sensitivity testing A total of 20 bacteria colonies were isolated to purified strains of gram negative bacteria. The Kit was used to identify the microorganism. The final steps were examined antibiotic susceptibility with four types of drug: Ampicillin, Chloramphenicol, Kanamycin and Tetracycline. Table 5 shows the stations particularly with the amount of antibiotic resistance strains isolated from each place. The resultshown15 isolated strains from all sampling locations resist to at least one type of antibiotic. Particularly, La Nga Village which was the main farmed fishing, had the highest number isolated microorganism (8 strains) accompany five were resistant to antibiotic. The other places had less the resistant strains than La Nga District. Table 5. The number of resistant bacteria strains isolated in the stations Stations Isolates Antibiotic resistance strains Nam Cat Tien forest (1) 5 4/5 La Nga district (2) 8 5/8 Sai Gon river (3) 4 4/4 Can Gio district (4) 3 2/3 Total 20 15/20

83 Moreover, Figure 3 exposes the comparison among those antibiotics. Four strains of bacteria were not shown the resistance while three were resist to all four types antibiotic. There are seven strains resist to two types of antibiotics. Figure 2. Antibiotic resistance of bacterial strains isolated from Saigon Dong Nai river. Identification by Kit has been indicated the genera of species combined with the results of the antibiotic susceptibility test were illuminated in Table 6. Among the sampling places, the highest antibiotic resistance could be seen in the second and third station. The fourth station has the lowest resistance, especially only two types of antibiotic indicates the resistance. In further comparison, Vibrio sp. appear in all stations with the high ratio of resistance. There was difference that Vibrio sp. was resistant with mainly AMP and KAN in the first station, CHL and TET in the fourth station, but in the second and the third stations, the resistance appears with all four types of antibiotic. Serratia sp has also shown the resistant in the first and the second stations with the highest aspect scale at the second place. The Enterobacriaceae sp. existed in the water of second location did not show the drug resistant. This was the same situation with the Pseudomonas sp. in the fourth station, did not have the resistant. However, in the third station, Escherichia coli has resistant to all of drug. In general, AMP is the antibiotic which has the highest resistant bacteria ratio (55%) resistance whilst the TET has lowest ratio of resistant bacteria (25%). Table 6. Antibiotic resistances levels of isolates in the stations The percentage of the number of antimicrobial bacteria over the total number of bacteria Bacteria groups Station Isolates Resistance to antibiotic (%) AMP CHL KAN TET Vibrio sp 1 3 67 0 100 0 Serratia sp 1 2 50 50 0 0 Total 1 5 60 20 60 0 Vibrio sp 2 3 50 34 34 67 Serratia sp 2 4 50 75 50 25 Enterobacteriaceae sp 2 1 0 0 0 0 Total 2 8 63 50 38 38

84 Bacteria groups Station Isolates Resistance to antibiotic (%) AMP CHL KAN TET Vibrio sp 3 3 67 34 67 34 Escherichia coli 3 1 100 100 100 100 Total 3 4 75 50 75 25 Vibrio sp 4 2 0 100 0 50 Pseudomonas sp 4 1 0 0 0 0 Total 4 3 0 67 0 34 Total 20 55 45 45 25 Discussion The samples were taken at four locations with distinct feature leads the bacteria were isolated differently. Although the ph value might not affect to the number increasing or decreasing of the bacteria (Toroglu et al., 2005), the salinity could be agent that cause the unlike microorganism between Can Gio district and the other places. For instance, at the first and second station, the genus of the microorganism is the same of Vibrio sp and Serratia sp, but at the fourth station (Can Gio), there are the Vibrio sp in small number and the existence of the Pseudomonas sp. This may the outcome of the high value in the salinity. During the high or low salinity period, species of bacteria could not disperse to survive either with the difficulty in reproduction (Mills and Geddes 1980) or dormant burgeon further (Williams 1985; Brock et al., 2003). Furthermore, the geographic coordinate might also affect to the survival of biota (Parveen et al., 1999). Escheria coli prevails only at the Sai Gon river and resist to all four types of drug is one of the things that needs to be concerned. It is said that the E.coli causes many diseases for the human like the diarrhea or anemia, kidney failure can lead to the death (Darfeuille Michaud et al., 1998). Besides, the occurrence of multiple drug resistant in E.coli in animal, which have been examined many years ago, shown that 83% of the samples containing E.coli were resistant to one or more antibiotics (Jackson, 1981; Mat-yaret al., 2004). People got an E.coli infection when drinking or eating the contaminated water or food. The antibiotic was usually prescribed by the doctors to cure the disease. In recent years, Ho Chi Minh city is facing the serious pollution, including the water source. The wastewater that is discharged from daily activities of citizens is very big quantity (1.750.000 m 3 /day by the report of Vietnam Meteorological and Hydrology Administration, 2017). Nevertheless, a little part of water raw is treated before going into canals, rivers or seas. By the report of Vietnam Environment Administration in 2016, the concentration of antibiotic in Saigon Dong Nai river water is not high, however those antibiotics have moved and accumulate mainly in downstream sediments of Sai Gon and Dong Nai river basins. The accumulation of those antibiotics in the river sediment for a long time can cause the local bacteria gradually resisting to those drugs. Those things can make the consideration to human health because the water from Dong Nai river, especially Sai Gon river (upstream river), is providing supplies to over 20 million citizens of Ho Chi Minh city and surrounding regions. Therefore, the consumption of antibiotic in either animals and patients need to be restricted in a threshold allowed to prevent the resistance to antibiotic of microorganism and save the people. In this study, 20 strains of gram negative bacteria were isolated, after eliminated three strains of gram positive organism at four locations throughout the Sai Gon - Dong Nai river. This is a respectable result for the continuous steps in this project. In theory, among the four sampling places, only La Nga district and Can Gio district have the strong aquaculture activities nearly connect with antibiotics, the ratio of antibiotic resistant bacteria should be

85 higher. However, in headwater location, this also appear drug- resistant microorganisms with four per five ratios. This can be explained that the biotas live in water at Nam Cat Tien forest also have the resistance genes available. If the water of the Nam Cat Tien forest somehow contacts with the antibiotic contaminants, it can be a serious threat to the human health in the future. Further studies on this possibility should be performed to have a final conclusion on this hypothesis. In Vietnam, drug is available for the people can practice self administration and further increase the inclination of drug resistant bacteria strains (Adewoye and Lateef, 2004). The farmers do not know explicitly how to use antibiotic effectively or abusing the effect of antibiotics on the aquaculture farming (Tran, 2017). In La Nga district, fish farms with the fish cages are constructed in the middle of the river. Most of the raw or the wastewater are directly put into the river and cause the pollution to the flow with many danger factors. Those may be the reasons why there were the number of isolates in La Nga is more than the others locations. In the same way, the tendency of antimicrobial resistance is also higher. Furthermore, economic development in this area is faster than the construction of the infrastructures. This puts more and more pressure on the environment. It is considered that both of industry and agriculture can cause bad affect to environment. The government needs to have the programs to contribute the awareness of the farmers with the use of antibiotic used for the aquatic animals and the solutions for the long-term bad effects caused by the accumulating of antibiotic in the rivers. CONCLUSION The relatively high level of resistance to antimicrobial agent is a reverberation of encroach or imposition of these to the environment. It is the reason why the present of high number of antibiotic resistant microorganism at four stations of Sai Gon - Dong Nai river. The appearance of those antibiotic resistant bacteria may cause by the activities of aquaculture farming along the rivers. The increasing of the bacteria with high resistant to antibiotics may cause a lot of problems for the population in the future. There will be further study to research specific strains by modern technology to detect exactly the gene of bacteria and also resistance genes for the scientists to find out the solutions for every specific problem. Finally, the utilization of antibiotics for animals and people should be restricted to prevent the multiple drug resistance. Acknowledgement This research is funded by International University, VNU-HCM under grant number SV2016-BT-12. REFERENCES Bakare N., Rickerts V., Bargon J., & Just Nübling G. (2003). Prevalence of Aspergillus fumigatus and other fungal species in the sputum of adult patients with cystic fibrosis. Mycoses, 46(1-2), 19-23. Brown, M. R., & Williams, P. (1985). The influence of environment on envelope properties affecting survival of bacteria in infections. Annual Reviews in Microbiology, 39(1), 527-556. Clinical and Laboratory Standards Institute (2015). Performance Standards for Antimicrobial Susceptibility Testing; Twenty-Fifth Informational Supplement. Online Manual. Costanzo S. D., O Donohue M. J., & Dennison W. C. (2004). Assessing the influence and distribution of shrimp pond effluent in a tidal mangrove creek in north-east Australia. Marine Pollution Bulletin, 48(5), 514-525. Darfeuille-Michaud, A., Neut, C., Barnich, N., Lederman, E., Di Martino, P., Desreumaux, P.,... & Colombel, J. F. (1998). Presence of adherent Escherichia coli strains in ileal mucosa of patients with Crohn s disease. Gastroenterology, 115(6), 1405-1413. Ehsan H., Aqsa B., Atif U.R., Sajjad A., Nodia S. (2015). Isolation and Identification of Coliform Bacteria from Drinking Water

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