STUDIES ON THE ISOLATION, CHARACTERIZATION, ANTIBIOTIC SUSCEPTIBILITY, AND TOXIGENIC ACTIVITY OF AEROMONAS SPP. ISOLATED FROM DIFFERENT SOURCES OF DRINI(ING WATER

Abstract

The present work was a cross sectional study conducted to establish baseline data regarding the presence of Aeromonas spp. in drinking water sources in Pakistan. Aeromonas spp. is a water borne gram-negative bacterial group that is increa singly been associated with human diseases worldwide but unfortunately local data regarding the presence of Aeromonas spp. in drinking water was lacking. A total of 1482 water samples originating from 16 different sources were sorted in six groups; five of drinking water and one of recreational water source were included. Aeromonas spp. was isolated alone as well as along with coliforms and thermotolerant (fecal) coliforms . The water in which coliforms were present was considered as failed in quality; out of 1482 water samples 817 (55.13%, P<0.05) samples failed in quality while 665 (44.87%) samples passed in quality test. In underground water, Aeromonas spp. was found in 221 (35.36%, P<0.05) including 203 (45.51%) failed and 18 (10%) passed samples; in surfaces water 23 (53.5%, P>0.05) including 21 (61 .8%) failed and ' 2 (22 .2%) passed samples; in distributions network (municipal) 96 (16.99%, P<0.05) including 84 (30 .76%) failed and 12 (4%) passed samples; in reservoi rs 26 (33 .3%, P<0.05) including 18 (4 1.86%) fa iled and 8 (22 .86%) passed samples; in bottled 7 (5.88%, P<0.05) includ ing 6 (31 .57%) fai led and 1 (1 %) passed samples, and in recreational water 1 (1.92%, P<0.05) from quality passed samples. In total 374/1 482 (25.23%, P<0.05) strains of Aeromonas spp. were isolated from 332/817 (40 .64%, P<0.05) quality failed samples in different combinations with coliforms and thermotolerant (fecal) coliforms while, 42/665 (6.32%, P<0.05) strains were isolated from quality passed samples. The 374 strains of Aeromonas comprised of A. hydrophila (249, 66.57%), A. caviae (94, 25%) and A. sobria (31 , 8.28%). Aeromonas spp. was found in 6.32% quality pass water samples, this was a significant finding (P<0 .05) as it showed the possible risk to human health Samples were from 48 cities and towns of Pakistan (four regions including NorthEastern region: Islamabad, Punjab, NWFP and AJK). The quality of water was: samples passed in quality in Islamabad region 51%, in Punjab 31 .72%, in NWFP 16%, and in AJK 32.14%. In Islamabad, Aeromonas spp. was found in 233 (22 .23%, P<0.05) samples, including 203 (39.5%) in quality failed and 30 (5.6%) in quality passed samples; in Punjab 120 (32 .25%, P<0.05) samples, 109 (42 .91%) in quality failed and 11 (9.4%) in quality passed samples; in NWFP 6 (24%, P<0.05) samples, in 6 (28 .57%) in quality failed samples and none in quality passed sample, and in AJK 15 (40 .54%, P>0.05) including 14 (50%) in quality failed and 1 (11.11 %) in quality passed sample. During the first eleven months (Feb 2006 to Dec 2006) , Aeromonas spp. was found in 194 (26.25%) out of 739 total samples, in 172 (38.3%) quality failed samples and 22 (7 .58%) quality passed samples. During this period their presence was significant (P<0 .05) in May, June, July, August, September, October and December; and nonsignificant (P>0.05) in February, March, April and November, 2006. In the next seven months (Jan 2007 to Jul 2007) Aeromonas spp. was found in 180 (24 .22%) out of 743 total samples, 160 (43.47%) in quality failed samples and 20 (5.33%) in quality passed samples respectively. During this period presence of Aeromonas spp. was significant (P<0 .05) in February, March, April , May, June and July. Selected twelve isolates were evaluated for their in vitro and in vivo toxigenic abilities. Strains found to be ~-hemolytic , cytotoxic and enterotoxic were: A. hydrophila 12.5% (1 of 8), A. caviae 66.66% (2 of 3) and A. sobria 100% (1 of 1). Hemolysin titer in vitro on 1 % rabbit erythrocytes was observed in 33.33% samples of ~-hemolytic species, in the range of 2048 HU/mL in case of A. hydrophila, 1024-2048 HU/mL in A. caviae, and 1024 HU/mL in A. sobria. Cytotoxic activity in vitro on MRC-5 cells , Vero cells, MDBK cells, and HEp-2 cel ls was observed in the same ~ - hemolytic stra ins of Aeromonas spp. The production of enterotoxin was studied in suckling mice and it was found that the ~hemolytic and cytotoxic strains were also enterotoxic. The antibiotic susceptibilities of these strains were studied by using disc diffusion method. The isolates were mostly susceptible to commonly used antibiotics; 58.33% strains were susceptible to tetracycline, 83.33% to ciprofloxacin, 75% to co-trimaxozole, 83.33% to pipracillin, 83.33% to tobramycin, 91.66% to amikacin , 41.66% to methicillin, and 100% susceptible to ofloxacin, imipenem, aztreonam, nalidixic acid , while 8.3% was intermediate-sensitive to co-trimaxozole and amikacin , 16.6% to tobramycin and ampicillin. The antibiotic susceptibility test of Aeromonas isolates showed that A. sobria was susceptible to all antibiotics studied except ampicillin. It was also found that A. caviae was more susceptible than A. hydrophila. This cross sectional study concluded the occurrence of toxigenic Aeromonas spp. throughout the year in the drinking water sources of selected regions of Pakistan. It requires a further in depth investigation of possible sources of contaminations, failure of chlorination and inadequate microbiological analysis of drinking water quality.