The genus Shigella is composed of four species, S. dysenteriae, S. boydii, S. flexneri, and S. sonnei, and is usually transmitted to humans by ingestion of contaminated water and foods. The infective dose of Shigella spp. is very low, varying from [10.sup.1] to [10.sup.4] organisms (Min, Park & Kim, 2002). Shigellosis is endemic throughout the world; it is among the most common causes of bacterial diarrhea diseases; and it is responsible for approximately 165 million cases of diarrhea annually. Of those cases, 163 million are in developing countries and 1.5 million are in industrialized countries. It is estimated that 1.1 million people die annually from Shigella infection and nearly 580,000 cases of shigellosis are reported among travelers from industrialized countries. The frequency of S. flexneri, S. sonnei, S. boydii and S. dysenteriae was 60%, 15%, 6% and 6% (30% of S. dysenteriae cases were type 1) in developing countries, respectively; and 16%, 77%, 2%, and 1% in developed ones, respectively (Kotloff et al., 1999; Peirano, Souza, & Rodrigues, 2006).
Many of the bacterial virulence determinants that mediate the complex interactions are encoded by large plasmids (Askhenazi, Levy, & Kazaronovski, 2003). Determination of plasmid profile has been shown to be a powerful too in epidemiological studies when used as a fingerprint for a strain (Askhenazi, Levy, & Kazaronovski, 2003). Plasmid profile may aid in differentiation of strains, identifying a source of infection, or evaluating the efficiency control measures (Litwin & Ryan, 1991).
Indiscriminate use of drugs and horizontal gene transfer have led to the resistance of Shigella species patterns are influenced by geographic location, year of isolation, classes of antimicrobial agents, and pressure exerted by antimicrobial use. It was noticed that over past decades, Shigella strains have progressively become resistant to most of the widely used antimicrobials, such as ampicillin, chloramphenicol, tetracycline, and trimethoprim-sulfamethoxazole (Askhenazi, Levy, & Kazaronovski, 2003). Also, quite striking geographical differences exist in the corresponding resistance rates. This may be due to the occurrence and spread of antimicrobial-resistant clones.
People from India and other developing countries use river water directly not only for drinking but also for various recreational purposes. Narmada is the fifth largest river of India. It originates from Amarkantak (M.P.) and merges into the Arabian Sea at Dahej (Gujarat). Its catchment areas sustain various agricultural and industrial communities. This water system also serves as a main source of water to the large rural population that resides in this region (Sharma & Khokale, 1999). The aim of this study was to identify Shigella spp. isolated from fresh water and to compare antibiotic resistance, plasmid profile, and phenotypic virulent strains by Congo red binding and production of hemolysin.
Materials and Methods
The river Narmada (latitude 21[degrees] 23' to 24[degrees] 46' N, longitude 72[degrees] 32' to 81[degrees] 46' E) is the largest west-flowing river in the Indian peninsula. Total length of the river from the head to its outfall into the sea is 1,312 km. It originates from Amarkantak at 1,151 m altitude (latitude 22[degrees] 40' N and longitude 81[degrees] 46' E) in the Shahdol district from the Maikal ranges in Madhya Pradesh, flowing through different cities (Dindori, Mandla, Jabalpur, Narsinghpur, Hoshangabad, Omkareshwar, Koral, Neelkantheshwar, Ankleshwar, and Dahej), which are surrounded with dense human population, agricultural farms and large and small industries.
Water samples were collected from 11 different stations of the river from its origin (Amarkantak) to end (Dahej) on a seasonal basis from July 2005 to June 2006.Samples of 1000 ml were collected in sterilized bottles from the surface and subsurface (approximately 1.5 feet below the surface) from all the sampling stations, brought to the laboratory under ice-cold conditions, and analyzed immediately for the presence of Shigella.
Isoaltion and Identification of Shigella
Water samples of 1000 ml were filtered through a Sartorius membrane filter (0.22) [micro] pore size, 47 mm diameter). The membrane filter was transferred into a flask containing 100 ml of sterile nutrient broth and incubated at 37 [+ or -]2[degrees]C for six hours under shaking conditions (Faruque et al., 2002). After enrichment it was streaked onto the XLD agar plate and incubated at 37[+ or -]2[degrees]C for 18 to 24 hours. Red-headed pink colonies were isolated, transferred to nutrient agar, and incubated at 37 [+ or -]2[degrees]C overnight.
All the isolates were examined for the following biochemical characteristics: oxidase production, urease production, Methyl Red and Vogues Proskauer test, indole production, citrate production, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, gluconate, malonate, acid production from arabinose, dulcitol, glucose, lactose, maltose, mannitol, raffinose, rhamnose, sorbitol, sucrose, trehalose, and xylose (MacFaddin, 1980). They were tentatively identified with the help of Bergey's Manual of Systematic Bacteriology (Krieg,Sneath, Staley, & Holt, 1984) and Probabilistic Identification of Bacteria (PIB) computer kit (Bryant, 2003), and confirmed on the basis of molecular studies (Random Amplified Polymorphic DNA [RAPD] and Amplified Ribosomal DNA Restriction Analysis [ARDRA]) (Silvia et al., 1998; Villalobo & Torres, 1998).
Forty isolates confirmed as Shigella spp. were subjected to serotyping by slide agglutination test with poly A, poly B, Cl, C2, C3, and D antigens (Edwards &r Ewing, 1972).
Disk diffusion assay was performed to assess the antibiotic resistance/sensitivity pattern (Clinical Laboratory Standard Institute [CLSI], 2002). Susceptibility to antimicrobial agents was tested on Mueller-Hinton agar using commercially available disks (Himedia, India): Amikacin, .30 mcg; Amoxicillin...