1 Qualitative and Quantitative Comparison of Bacterial Flora Associated with Hatchery-reared and Wild-caught Shrimp Postlarvae Celia R. Lavilla-Pitogo, Leobert D. de la Peña and Milagros R. Paner Fish Health Section, Southeast Asian Fisheries Development Center Tigbauan 5021, Iloilo, Philippines Abstract- Because of high mortality recorded in pond-reared shrimps due to luminescent vibriosis infection, a study was conducted to determine if postlarvae (PLs) could be major sources of luminescent bacteria (LB). Batches of hatchery-reared (PL12 to 18) and wild-caught Penaeus monodon PLs were examined to determine their bacterial load. Results show that although all PLs have associated Vibrio spp., not all of them harbored detectable levels of LB. Fifty eight percent of wild-caught postlarval batches did not have associated LB compared with only 23-44% of hatchery-reared postlarvae. A significant difference in quantitative LB load was noted between hatchery reared and wild-caught PLs with the former harboring up to 3.0 x 105 cfu LB/postlarva. Wild- caught PLs had only up to 3.5 x 102 cfu LB/postlarva. Antimicrobial sensitivity tests using disc diffusion method show significant resistance to Chloramphenicol and Oxytetracycline among isolates from hatchery-reared PLs (33 and 44%) compared with bacteria from wild-caught PLs (3 and 6%) and near shore seawater (0 and 12%). The differences between the quantitative and qualitative bacterial flora of hatchery-reared and wild-caught PLs may have contributed to the occurrence of luminescent vibriosis in grow-out ponds, which generally make use of hatchery-reared postlarvae. Keywords – bacterial flora, Penaeus monodon, postlarvae, antibiotic resistance I. INTRODUCTION Protocols for disease prevention and chemotherapy employed in shrimp, Penaeus monodon, hatcheries may influence the selection and establishment of associated bacterial flora. Selection and dominance of opportunistic bacteria in postlarvae (PLs) may have potential harmful effects during grow-out culture in ponds due to the carry over of gut-associated drug-resistant bacteria upon stocking. The spate of bacterial disease outbreaks in shrimp culture systems [1, 2, 3] has caused a slump in the industry despite chemotherapeutic applications of antibacterial [4]. The use of antimicrobial agents in aquaculture has resulted in higher frequency of strains resistant to these agents [5, 6, 7, 8] and antibiotic resistant vibrios have been reported in the hatchery and grow-out phases of shrimp culture [6, 9]. Because of the widespread use of antimicrobials in shrimp hatcheries, it is important to address the effects of these treatment regimes on the bacterial flora of shrimp postlarvae (PLs) about to be stocked in ponds. It should be noted that in both hatchery and grow-out phases of shrimp culture, similar antimicrobials are used [4]. The main objectives of this study was to compare the bacterial flora of hatchery-reared and wild-caught shrimp postlarvae, and to determine differences in their qualitative bacterial populations based on presumptive Vibrio population and reactions to antibiotics. II. METHODOLOGY A. Sources of Postlarvae Hatchery-reared postlarvae (PL 12-18) of Penaeus monodon were obtained from various hatcheries in different provinces in central Philippines. Several batches of wild- caught PLs were obtained from various natural grounds in Panay Island, Philippines during the abundant season from March to July. The number of PLs in each batch ranged from 20-100 pieces. Water from hatcheries and natural fry grounds were also sampled for microbial analysis. Since age determination is different for hatchery-reared and wild- caught PLs, classification of their developmental sub-stage were based upon published morphological characteristics [10]. B. Bacteriological Examination Postlarvae were washed thoroughly with sterile seawater (SSW) prior to processing to minimize surface- attached bacteria. The bacterial flora of whole postlarvae was quantified by thoroughly homogenizing pooled samples of 10-20 PLs. The macerated tissues were suspended in SSW at the rate of 1 PL/ml, serially diluted, and plated on nutrient agar (NA) and the selective medium for vibrios, thiosulfate citrate bile salts sucrose agar (TCBS). Incubation was at room temperature (28-30°C) for 24 h, after which the bacterial colonies were counted. Qualitative classification of bacterial flora of PLs from was done up genus level only based on their growth on the selective medium. Strains that were purified for antibiotic sensitivity tests were picked at random from plates containing between 30-300 colonies. C. Antimicrobial Sensitivity Tests The antibacterial sensitivity of newly isolated bacteria obtained from various media was tested using procedures based on the standardized disc-agar diffusion method of the National Committee for Clinical Laboratory Standards (11) for antimicrobial susceptibility tests. Antimicrobials were chosen and tested based on what was currently and previously in use in the hatcheries using commercially- 2 available discs (Beckton and Dickinson Co.). Test plates were incubated at 28-30°C and the zones of inhibition were measured after 18-24 h incubation. Control tests using organisms for quality control such as Escherichia coli, Staphylococcus aureus and Pseudomonas aeruginosa were run simultaneously with those isolated from shrimp PLs and their environments. III. RESULTS A total of 272 batches of hatchery-reared shrimp postlarvae (PLs) ranging in age from PL12 to 18 that were obtained from more than 40 hatcheries in central Philippines were processed. Thirty-one batches of wild-caught Penaeus monodon PLs from natural collection grounds in Panay Island, Philippines were examined to determine their total bacterial load and the Vibrio spp. associated with them. Table 1 shows the ranges of bacterial colonies counted from the samples with increasing numbers of bacteria recovered in direct proportion with PL stage. Results further show that although all PLs have associated Vibrio spp. (Table 1), not all of them harbor detectable levels of luminescent bacteria (LB) (Table 2). Fifty eight percent of wild-caught postlarval batches were negative for LB, while only 22.6 to 44% of hatchery-reared postlarval batches did not have associated LB. Sixty one percent of PL12 were negative for LB (Table 2). This table also shows the significant difference in quantitative bacterial load between hatchery reared and wild-caught PLs. While hatchery-reared PLs harbored up to 9.9 x 104 LB colony-forming-units (cfu)/postlarva (PL 16 in Table 2), wild-caught PLs had only up to 3.5 x 102 LB cfu/postlarva. The relative reactions to various antibiotics of randomly selected bacterial strains from postlarvae and near shore seawater are in Table 3. More than 50% of strains associated with postlarvae are resistant to Penicillin and Streptomycin, and sensitive to Chloramphenicol and Norfloxacin. The sensitivity tests also show relatively higher resistance to Chloramphenicol and Oxytetracycline among isolates from hatchery-reared PL (33 and 44%) compared with bacteria from wild-caught PL (3 and 6%) and near shore seawater (0 and 12%). Among the 10 antibiotics used, more than 50% of strains associated with hatchery-reared PLs exhibited resistance to 5 antibiotics, namely Trimethoprim/ Sulfamethoxazole, Streptomycin, Erythromycin, Ampicillin, and Penicillin. Wild-caught PLs were resistant to Streptomycin, Rifampin, and Penicillin, while 50% of strains from near shore seawater showed relatively resistance only to Ampicillin and Penicillin. Fifty percent of strains obtained from near shore seawater remain sensitive to Trimethoprim/Sulfamethoxazole, Nitrofurantoin, Norfloxacin, and Chloramphenicol. More than 50% of strains from wild-caught PLs exhibited relative sensitivity to Trimethoprim/Sulfamethoxazole, Norfloxacin, and Chloramphenicol, and bacteria associated with hatchery-reared PLs to Norfloxacin and Chloramphenicol only. TABLE 1. Range of bacterial population associated with hatchery- reared and wild-caught postlarvae (PLs) Stage Total Bacterial Count Presumptive Vibrio Count Hatchery-reared PLs PL 12 1.4 x 101 – 8.8 x 104 1.0 x 100 – 1.7 x 104 PL 13 3.4 x 103 – 9.0 x 104 3.5 x 101 – 8.9 x 104 PL 14 5.0 x 102 – 7.5 x 104 4.7 x 102 – 2.5 x 104 PL 15 2.7 x 102 – 8.7 x 104 1.0 x 102 – 1.7 x 104 PL 16 3.5 x 101 – 9.9 x 104 5.0 x 100 – 9.9 x 104 PL 17 1.1 x 103 – 7.4 x 104 2.9 x 102 – 4.6 x 104 PL 18 6.7 x 102 – 1.3 x 105 2.0 x 102 – 2.7 x 104 Wild PLs 3.8 x 103 – 3.0 x 105 1.6 x 102 – 1.4 x 105 TABLE 2. Comparison of luminescent bacterial load of hatchery-reared and wild-caught postlarvae. Stage Number of Batches Examined Negative for Luminescent Bacteria (%) Range of Associated Luminescent Bacteria Hatchery-reared postlarvae = 272 batches PL 12 97 59 (60.8) 5.0 x 100 - 1.3 x 104 PL 13 36 12 (33.3) 2.5 x 100 - 8.9 x 104 PL 14 25 11 (44) 5.0 x 100 - 2.5 x 104 PL 15 37 11 (29.7) 7.0 x 101 - 1.7 x 104 PL 16 18 6 (33.3) 5.0 x 100 - 9.9 x 104 PL 17 28 9 (32) 5.0 x 100 - 3.0 x 104 PL 18 31 7 (22.6) 2.0 x 102 - 4.0 x 104 Wild- caught PLs 31 18 (58) 5.0 x 100 - 3.5 x 102 IV. DISCUSSION Studies to determine the bacterial population associated with arthropods, particularly in the intestinal tract, have been done to determine the role of bacteria in the digestion of food [12]. Because of bacterial infection in shrimps, the associated gut bacterial flora of pond-grown shrimp, specifically Vibrio spp., was studied to know if disease occurrence was related to the dominance of one species [13]. The finding that shrimp postlarvae are always closely associated with the presence of vibrios is similar to that found in the guts of two salt marsh detritivore prawns [14] wherein Vibrio and Pseudomonas comprised the dominant genera. Given this, it is important to know if potentially pathogenic vibrios, like luminescent V. harveyi [15], do not become dominant and cause disease. The present findings wherein relatively higher luminescent bacteria are associated with hatchery-reared PLs is a cause of concern because some practices, like application of antibiotics, obviously affects the quantitative and qualitative bacterial flora of hatchery-reared PLs. This has certainly contributed to the occurrence of luminescent vibriosis in grow-out ponds, which generally make use of hatchery-reared postlarvae. Because it may be argued that considerable variations in the methods used to measure sensitivity of strains can occur [5], the values presented herein should be considered 3 in relation to the reactions of all strains used in the tests. The existence of more antibiotic resistant strains closely associated with hatchery-reared PLs should give us urgent reasons to find alternatives methods of disease control to substitute antibiotics. TABLE 3. Summary of resistant, intermediate and sensitive reactions to various antibiotics* of bacteria from wild-caught and hatchery-reared postlarvae, and from near shore seawater. Source of Bacteria/ Antibiotics Resistant Intermediate Sensitive Wild-caught postlarvae = 32 isolates TE-30 2 (6.25) 17 (53.13) 13 (40.63) SXT 2 (6.25) 4 (12.5) 26 (81.25) F/M 300 9 (28.13) 13 (40.63) 10 (31.25) S-10 17 (53.13) 9 (28.13) 6 (18.75) NOR-10 0 (0) 3 (9.38) 29 (90.63) E-15 15 (46.88) 17 (53.13) 0 (0) AM-10 14 (43.75) 6 (18.75) 12 (37.50) RA-5 18 (56.25) 7 (21.88) 7 (21.88) P-10 19 (59.38) 0 (0) 13 (40.63) C-30 1 (3.13) 3 (9.38) 28 (87.50) Hatchery-reared postlarvae = 48 isolates TE-30 21 (43.75) 14 (29.17) 13 (27.08) SXT 24 (50) 4 (8.33) 20 (41.67) F/M 300 6 (12.5) 25 (52.08) 17 (35.42) S-10 36 (75) 8 (16.67) 4 (8.33) NOR-10 0 (0) 9 (18.75) 39 (81.25) E-15 25 (52.08) 22 (45.83) 1 (2.08) AM-10 37 (77.08) 0 (0) 11 (22.92) RA-5 18 (37.5) 19 (39.58) 11 (22.92) P-10 38 (79.17) 0 (0) 10 (20.83) C-30 16 (33.33) 6 (12.5) 26 (54.17) Near shore seawater = 17 isolates TE-30 2 (11.76) 8 (47.06) 7 (41.12) SXT 2 (11.76) 4 (23.53) 11 (64.71) F/M 300 1 (5.88) 6 (35.29) 10 (58.82) S-10 8 (47.06) 7 (41.12) 2 (11.76) NOR-10 0 (0) 3 (17.65) 14 (82.45) E-15 8 (47.06) 9 (18.75) 0 (0) AM-10 13 (76.47) 2 (11.76) 2 (11.76) RA-5 6 (35.29) 5 (29.41) 6 (35.29) P-10 13 (76.47) 0 (0) 4 (23.53) C-30 0 (0) 0 (0) 17 (100) *TE = Tetracycline, SXT = Trimethoprim/Sulfamethoxazole; F/M = Nitrofurantoin; S = Streptomycin; NOR = Norfloxacin; E = Erythromycin; AM = Ampicillin; RA = Rifampin; P = Penicillin; C = Chloramphenicol V. CONCLUSIONS The following conclusions can be derived from the study: • The total bacterial populations that are associated with hatchery-reared and wild-caught Penaeus monodon postlarvae increase in proportion with their age. • Although all postlarvae have associated vibrios, not all of them harbor luminescent bacteria and lower populations of the latter are associated with wild- caught PLs. • Based on the reaction of bacterial strains from various sources, relatively higher percentages of antibiotic resistance are exhibited by bacteria associated with hatchery-reared than wild-caught PLs in more kinds of antibiotics. • There is an urgent need to find and implement alternatives methods of disease control to substitute antibiotic usage. ACKNOWLEDGMENTS Most of the hatchery-reared postlarvae were submitted for quality analysis by fish farmers to the Diagnostic Service Laboratory of the Fish Health Section, SEAFDEC Aquaculture Department and were processed with the assistance of Remia Traviña. C.R.L.P thanks the Government of Japan Regional Fish Disease Project for funding her travel to Chiang Mai, Thailand to present this paper at the AsiaResist Meeting. REFERENCES [1] C.R. Lavilla-Pitogo, M.C.L. Baticados, E.R. Cruz-Lacierda, L.D. de la Peña. 1990. Occurrence of luminous bacterial disease of Penaeus monodon larvae in the Philippines. Aquaculture 91:1-13. [2] I. Karunasagar, R. Pai, G.R. Malathi, and I. Karunasagar, 1994. Mass mortality of Penaeus monodon larvae due to antibiotic-resistant Vibrio harveyi infection. Aquaculture 128:203-209. [3] C.R. Lavilla-Pitogo, E.M. Leaño, and M.G. Paner. 1998. 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