MATERIALS AND METHODS

Isolation of multidrug resistant strains: Urine samples were collected from community acquired UTI patient in the month of August 2009. Isolated strains from the samples were identified based on staining and biochemical tests. From this, multidrug resistant strains were isolated by conducting antimicrobial susceptibility test of Kirby et al. (1957). Bauer disk diffusion method (1957) following the definition of the National Committee of Clinical Laboratory Standards (NCCLS 46) against common antimicrobial agents such as Ampicillin, Cephalexin, Ciprofloxacin, Cotrimoxazole, Gentamycin, Nalidixic acid, Streptomycin and Vancomycin (Himedia Labs, Mumbai, India).

Seaweed extraction and antimicrobial assays: Seaweeds were collected from intertidal rockyshore region in Pudhumadam in the Gulf of Mannar, India during August 2010 and identified using keys provided according to Ganesapandian and Kumaraguru (2008). After air dried, samples were homogenized. Each 25 g of homogenized seaweeds were suspended in 100 mL of methanol for 24 h and the extracts were filtered and concentrated under reduced pressure in a rotary evaporator. The antibacterial activity of the test agent was determined by measuring the diameter of zone of inhibition expressed in millimeter. The experiment was carried out three times and the results were the mean of three replicates (Livermore and Brown, 2001).

RESULTS

Table 1 gives the multi-drug resistance profile of various isolates to the routinely used antibiotics. Four UTI strains were isolated and tested for susceptibility against some antibiotics, many of antibiotics showed resistance to the MDR strains. Among this E. coli and P. aeruginosa were highly resistant to most of the antibiotics, whereas S. aureus exhibited sensitivity to Cephalexin, Ciprofloxacin, Cotrimoxazole and Streptomycin.

Antibacterial activity exhibited in most of the methonol extracts of seaweeds was summarized in Table 2. Extract of Padina tetrastromatica and Grateloupia lithophila showed good inhibiting activity against S. aureus. The extract of Grateloupia lithophila also showed good inhibiting activity against E. coli. Where as, extract of Acanthophora specifera, Dictyota dichotoma, Gracilaria corticata and Chaetomorpha antennina showed weak inhibiting activity against most of the non-MDR strains. Caulerpa peltata and Caulerpa scalpelliformis did not show any inhibiting activity against all non-MDR strains.

Table 1:	Susceptibility pattern of Antibiotic against UTI isolates
R: Resistant, S: Sensitive

Table 2:	Susceptibility pattern of crude methanol seaweeds extracts against Non-MDR
-: No activity, +: Weak activity (1-5mm halo), ++: Moderate activity (6-10mm halo), +++: Good activity (10-15-mm halo)

Table 3:	Susceptibility pattern of crude methanol seaweeds extracts against MDR strain
-: No activity, +: Weak activity (1-5mm halo), ++: Moderate activity (6-10mm halo), +++: Good activity (10-15-mm halo)

Extract of Grateloupia lithophila showed moderate inhibiting activity against MDR strains of S. aureus and Pseudomonas (Table 3). UTIs pathogenic E. coli was resistant to most of the extracts except Grateloupia lithophila. The extract of Chaetomorpha antennina, Dictyota dichotoma, Padina tetrastromatica and Stoechospermum marginatum showed weak inhibiting activity against the MDR isolates. Whereas, methanol extracts of Caulerpa peltata, Caulerpa scalpelliformis, Acanthophora specifera and Gracilaria corticata did not show any inhibiting activity against MDR isolates.

DISCUSSION

Drug resistance is one of nature’s never ending process by which the organisms develop tolerance to new environmental conditions. It may be due to a pre-existing factor in the organisms or due to acquired factor(s). Rella and Haas (1982) first reported nalidixic acid resistant P. aeruginosa of UTI which showed resistance to β-lactam antibiotics. A significant increase in resistance of UTI pathogenic strains to Ampicillin has been found worldwide (Sahm et al., 2001). Therefore these agents should not be recommended for first line empirical treatment of UTI (Warren et al., 1999). Nalidixic acid has been recommended as drug of choice for the treatment of uncomplicated UTIs (Onifade et al., 2005; Akortha and Ibadin, 2008) because of its activity against several different types of Gram negative bacteria such as E. coli, Klebsiella sp. etc. The findings of this study coincide with the findings of Shittu and Mandere (1999) that S. aureus strains were highly resistant to naladixic acid. All the isolates in this study showed resistance to at least 5-7 different antibiotics, indicating the presence of strong selective pressures from the antibiotics in the community. Brown et al. (2003) have reported that horizontal gene transfer is a factor in the occurrence of antibiotic resistance in clinical isolates and suggested that the high prevalence of resistance to a particular antibiotic does not always reflect antibiotic consumption as previously suggested by Nwanze et al. (2007).

In this study, a multidrug resistancHoppee strain was found which are resistant to most of the antimicrobial agent tested. This reflected the fact that ampicillin, tetracyclin and streptomycin were the most commonly prescribed antibiotics in the hospitals even before the results of urine analyses and also the most easily available in the market without prescription because they are also cheap in terms of cost. The widespread use and more often the misuse of antimicrobial drugs has led to a general rise in the emergence of resistant bacteria. Higher resistant strains were reported in the USA for ampicillin and cotrimoxazole (Sahm et al., 2001), where as few ciprofloxacin resistant strains were found in other countries (Diekema et al., 2004; Mohammadi et al., 2010). This study also noticed ciprofloxacin-resistant E. coli from UTIs. Ciprofloxacin as an option for therapy to UTIs has been considered, since its multiple mechanisms of action seem to have enabled it to retain potent activity against E. coli. Ciprofloxacin has high level of activity against UTI isolates of E. coli compared with other commonly used agents, such as Ampicillin and SXT (Gupta et al., 1999).

Gonzalez del val et al. (2001) reported that the methanolic extract of Padina showed antibacterial activity only against B. subtilis. However, in the present study the methanol extract of P. tetrastromatica inhibited the growth of K. pneumoniae and S. aureus and it was unable to inhibit the growth of E. coli. Similar to this study Tuney et al. (2006) reported acetone, methanol and diethyl ether extracts of Padina had no antibacterial activity but ethanol extract of Padina had weak activity against E. faecalis, P. aeruginosa and E. coli. In the present study, it was observed that E. coli was resistant to methanol extract of all algae except Grateloupia lithophila and Stoechospermum marginatum. Salvador et al. (2007) also reported that E. coli was resistant to the extracts of Dictyota dichotoma, Malopteris filicine, Cladostephus spongiosus, F. verticillatus and Ulva rigida. It may be due to the methanol used as an organic solvent for extracting compounds providing a higher efficiency of antimicrobial activity.

The current study revealed that the inhibitory activity of extract of Grateloupia lithophila showed higher in the MDR isolates of Pseudomonas and S. aureus and Non-MDR isolates of E. coli, Klebsiella and S. aureus than the inhibitory activity exhibited by vancomycin and gentamycin disc. The MDR isolate E. coli, was also inhibited by the same extract but had smaller inhibition zone with 1mm which is less than the inhibition activity of vancomycin disc. The methanol extract of Grateloupia lithophila exhibited intense activity against both MDR and non-MDR strains. Kandhasamy and Arunachalam (2008) reported that methanolic extract of Gracilaria and Caulerpa inhibited the growth of all pathogenic bacteria except E. coli. Rao and Parekh (1981) reported extracts of Enteromorpha intestinalis and G. corticata have antibacterial activity. In contrast to these investigations, our study showed that Caulerpa have less activity where as G. corticata did not show antibacterial activity as reported by Hornsey and Hide (1985).

CONCLUSION

These results indicate the possibility that different quantities of active secondary products were synthesized under different conditions. Differences between the results of the present investigation and results of other studies may be due to the production of bioactive compounds related to e seasonal variations, extraction methods, organic solvents used for extraction of bioactive compounds and different bioassay methods. Finally it can be concluded that extracts of algal species used in the present investigation showed better antibacterial activity against MDR pathogens. Seaweeds are the potential sources of bioactive compounds and should be investigated for natural antibiotics. This study recommends seaweed extracts as antibacterial substance for treating multidrug resistant microbes causing acquired infections.