Environmental monitoring of bacterial contamination and antibiotic resistance patterns of the fecal coliforms isolated from Cauvery River, a major drinking water source in Karnataka, India

The present study focuses prudent elucidation of microbial pollution and antibiotic sensitivity profiling of the fecal coliforms isolated from River Cauvery, a major drinking water source in Karnataka, India. Water samples were collected from ten hotspots during the year 2011-2012. The physiochemical characteristics and microbial count of water samples collected from most of the hotspots exhibited greater biological oxygen demand and bacterial count especially coliforms in comparison with control samples (p <= 0.01). The antibiotic sensitivity testing was performed using 48 antibiotics against the bacterial isolates by disk-diffusion assay. The current study showed that out of 848 bacterial isolates, 93.51 % (n=793) of the isolates were found to be multidrug-resistant to most of the current generation antibiotics. Among the major isolates, 96.46 % (n=273) of the isolates were found to be multidrug-resistant to 30 antibiotics and they were identified to be Escherichia coli by 16S rDNA gene sequencing. Similarly, 93.85 % (n=107), 94.49 % (n=103), and 90.22 % (n=157) of the isolates exhibited multiple drug resistance to 32, 40, and 37 antibiotics, and they were identified to be Enterobacter cloacae, Pseudomonas trivialis, and Shigella sonnei, respectively. The molecular studies suggested the prevalence of blaTEM genes in all the four isolates and dhfr gene in Escherichia coli and Sh. sonnei. Analogously, most of the other Gram-negative bacteria were found to be multidrug-resistant and the Gram-positive bacteria, Staphylococcus spp. isolated from the water samples were found to be methicillin and vancomycin-resistant Staphylococcus aureus. This is probably the first study elucidating the bacterial pollution and antibiotic sensitivity profiling of fecal coliforms isolated from River Cauvery, Karnataka, India.

Chronic lung infection by Pseudomonas aeruginosa biofilm is cured by L-Methionine in combination with antibiotic therapy

Bacterial biofilms are associated with 80-90% of infections. Within the biofilm, bacteria are refractile to antibiotics, requiring concentrations >1,000 times the minimum inhibitory concentration. Proteins, carbohydrates and DNA are the major components of biofilm matrix. Pseudomonas aeruginosa (PA) biofilms, which are majorly associated with chronic lung infection, contain extracellular DNA (eDNA) as a major component. Herein, we report for the first time that L-Methionine (L-Met) at 0.5 mu M inhibits Pseudomonas aeruginosa (PA) biofilm formation and disassembles established PA biofilm by inducing DNase expression. Four DNase genes (sbcB, endA, eddB and recJ) were highly up-regulated upon L-Met treatment along with increased DNase activity in the culture supernatant. Since eDNA plays a major role in establishing and maintaining the PA biofilm, DNase activity is effective in disrupting the biofilm. Upon treatment with L-Met, the otherwise recalcitrant PA biofilm now shows susceptibility to ciprofloxacin. This was reflected in vivo, in the murine chronic PA lung infection model. Mice treated with L-Met responded better to antibiotic treatment, leading to enhanced survival as compared to mice treated with ciprofloxacin alone. These results clearly demonstrate that L-Met can be used along with antibiotic as an effective therapeutic against chronic PA biofilm infection.

Successful treatment of biofilm infections using shock waves combined with antibiotic therapy

Many bacteria secrete a highly hydrated framework of extracellular polymer matrix on suitable substrates and embed within the matrix to form a biofilm. Bacterial biofilms are observed on many medical devices, endocarditis, periodontitis and lung infections in cystic fibrosis patients. Bacteria in biofilm are protected from antibiotics and >1,000 times of the minimum inhibitory concentration may be required to treat biofilm infections. Here, we demonstrated that shock waves could be used to remove Salmonella, Pseudomonas and Staphylococcus biofilms in urinary catheters. The studies were extended to a Pseudomonas chronic pneumonia lung infection and Staphylococcus skin suture infection model in mice. The biofilm infections in mice, treated with shock waves became susceptible to antibiotics, unlike untreated biofilms. Mice exposed to shock waves responded to ciprofloxacin treatment, while ciprofloxacin alone was ineffective in treating the infection. These results demonstrate for the first time that, shock waves, combined with antibiotic treatment can be used to treat biofilm infection on medical devices as well as in situ infections.

Using wildlife activity and antibiotic resistance analysis to model bacterial water quality in coastal ponds

Models that help predict fecal coliform bacteria (FCB) levels in environmental waters can be important tools for resource managers. In this study, we used animal activity along with antibiotic resistance analysis (ARA), land cover, and other variables to build models that predict bacteria levels in coastal ponds that discharge into an estuary. Photographic wildlife monitoring was used to estimate terrestrial and aquatic wildlife activity prior to sampling. Increased duck activity was an important predictor of increased FCB in coastal ponds. Terrestrial animals like deer and raccoon, although abundant, were not significant in our model. Various land cover types, rainfall, tide, solar irradiation, air temperature, and season parameters, in combination with duck activity, were significant predictors of increased FCB. It appears that tidal ponds allow for settling of bacteria under most conditions. We propose that these models can be used to test different development styles and wildlife management techniques to reduce bacterial loading into downstream shellfish harvesting and contact recreation areas.

Survival of antibiotic resistant Pseudomonas strains in different types of water

A study was conducted to investigate the survival of five Pseudomonas strains resistant to antibiotics in different types of water. The selected Pseudomonas strains were designated as strain P1 (CT-29), strain P2 (CT-25), strain P3 (CT-36), strain P4 (CT-20) and strain P5 (CT-27) which were only recovered from farmed fishes. Six types of water viz., distilled water, saline water, tap water, deionized water, pond water and river water were used. Among these experimental waters, river water was found to be the most suitable for long-term survival of these strains. Deionized water did not support survival of all these Pseudomonas strains. Pond water, tap water and distilled water were moderately suitable for strain P1 and strain P4. Saline water was also found to be highly suitable for long-term survival in case of the strain P3 and moderately suitable for normal survival of strain P2 and strain P5.

Survival of antibiotic resistant Aeromonas strains in different water conditions

Studies were conducted to find out the survival of three antibiotic resistant Aeromonas strains in different types of water. The selected Aeromonas strains were A. hydrophila (local), A. sobria (local) and A. hydrophila (Thai), which were only recovered from farmed fishes. Seven types of water were used. Among these experimental water, lake water, distilled water and fish farm pond water had supported the long time survival of A. hydrophila. In contrast, private fish farm pond water was the most favorable for A. sobria. Deionized water was found not to support the survival of any species but Masjid pond water and FRI pond water were found to be moderately suitable for all the species. However, the survival pattern of Thai strain of A. hydrophila was found to have similarity with the survival of the local strain of A. hydrophila.

Pathogenicity of Aeromonas sobria to Thai silver barb (Barbodes gonionotus Bleeker) and its sensitivity to some antibiotic agents

Five isolates of Aeromonas sobria, collected from the diseased fish were selected for detection the pathogenicity following water-born infection method on silver barbs (Barbodes gonionotus) at the selected exposure dose 2.5x10⁸ CFU/ml which was standardized by preliminary test. In the experimental condition lesion and mortality were found in fishes. Among the isolate, Ass17 Ass19, Ass31 and Ass36 were successfully infected 20-60% fishes. Another isolate Ass20 was found non-pathogenic. Drug sensitivity test was performed by six antibiotics viz. Oxytetracycline, Oxolinic acid, Chloramphenicol, Stilphamethozazole, Streptomycin, Erythromycin. All the isolates showed variable reaction patterns to antibiotics. Most of the isolates were found sensitive to Oxytetracycline (OT), Oxolinic acid (OA) and Chloramphenicol (C) but resistance to Erythromycin and Sulphamethoxazole (SXT). Isolate Ass31 found resistant to Oxolinic acid.

Antibiotic sensitivity of Staphylococcus aureus strains isolated from fishery products

During the course of an investigation on the incidence of Staphylococcus aureus contamination in frozen crab meat, frozen prawns and dried fishes from Cochin, 116 strains of S. aureus were isolated. The sensitivity of the isolated strains towards nine antibiotics showed that all the S. aureus strains were sensitive to kanamycin and streptomycin (100%). Sensitivity to other antibiotics like chloramphenicol, polymyxin-B, erythromycin, tetracycline, neomycin, penicillin and ampicillin were shown by 98.28, 93.10, 87.93, 68.10, 67.24, 56.90 and 55.17 percent of the isolates respectively.

Antibiotic sensitivity of Vibrio parahaemolyticus strains

The strains of Vibrio parahaemolyticus isolated from water, sediment, plankton, fish and prawn of Cochin backwater were tested for sensitivity to ten antibiotics namely, ampicillin, chloramphenicol, gentamycin, kanamycin, neomycin, oxytetracycline, penicillin, polymyxin-B, streptomycin and sulphadiazine. Of the 120 isolates tested, 96.7 and 93.3% were sensitive to gentamycin and chloramphenicol respectively. No strain was sensitive to penicillin and only 5% were sensitive to kanamycin. Isolates from fish and prawn showed higher resistance to ampicillin and none of them was sensitive to kanamycin. Multiple resistant V. parahaemolyticus strains were more in prawn than in other samples.

Antibiotic resistance of Staphylococcus aureus strains isolated from fish processing factory workers

One hundred and twenty two strains of Staphylococcus aureus isolated from throats and palms of 39 workers from 6 fish processing factories situated in and around Cochin were tested for their sensitivity to nine commonly used antibiotics-ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin, penicillin, polymyxin-B, streptomycin and tetracycline. Highest percentage of resistance was observed towards ampicillin followed by penicillin i.e. 64.75% and 59.84%. Resistance towards other antibiotics like tetracycline, polymyxin-B, erythromycin, kanamycin, neomycin, chloramphenicol and streptomycin were shown by 22.95, 16.39, 7.38, 5.74, 3.28 and 1.64% of the isolates respectively.

Effects of dietary supplementation of commercial human probiotic and antibiotic on the growth rate and content of intestinal microflora in ornamental fishes

The effects of dietary supplementation of commercial human probiotic, Lactobacil and antibiotic, oxytetracycline on the growth, survival, disease resistance and content of intestinal microflora in two ornamental fishes, viz., goldfish, Carassius auratus and swordtail, Xiphophorus helleri were studied. The total wet weight gain, food conversion ratio and specific growth rate of C. auratus did not vary significantly (p>0.05) among treatments. While in X. helleri, significant differences existed in the total wet weight gain, survival, food conversion ratio and specific growth rate among treatment groups (p<0.05). The counts of antibiotic resistant bacteria in fish gut increased with days of culture in all the treatments and the increase was more in antibiotic fed fishes. A reduction in the development of antibiotic resistance among the bacterial flora of fish gut was noticed in probiotic fed groups of C auratus and X. helleri. The results of the present study revealed that the effects of human probiotic on the growth, survival and disease resistance of ornamental fishes are variable and difficult to reproduce the similar effect on different species.

Cecum lymph node dendritic cells harbor slow-growing bacteria phenotypically tolerant to antibiotic treatment.

In vivo, antibiotics are often much less efficient than ex vivo and relapses can occur. The reasons for poor in vivo activity are still not completely understood. We have studied the fluoroquinolone antibiotic ciprofloxacin in an animal model for complicated Salmonellosis. High-dose ciprofloxacin treatment efficiently reduced pathogen loads in feces and most organs. However, the cecum draining lymph node (cLN), the gut tissue, and the spleen retained surviving bacteria. In cLN, approximately 10%-20% of the bacteria remained viable. These phenotypically tolerant bacteria lodged mostly within CD103⁺CX₃CR1⁻CD11c⁺ dendritic cells, remained genetically susceptible to ciprofloxacin, were sufficient to reinitiate infection after the end of the therapy, and displayed an extremely slow growth rate, as shown by mathematical analysis of infections with mixed inocula and segregative plasmid experiments. The slow growth was sufficient to explain recalcitrance to antibiotics treatment. Therefore, slow-growing antibiotic-tolerant bacteria lodged within dendritic cells can explain poor in vivo antibiotic activity and relapse. Administration of LPS or CpG, known elicitors of innate immune defense, reduced the loads of tolerant bacteria. Thus, manipulating innate immunity may augment the in vivo activity of antibiotics.

New antibiotic with typical plant anthraquinone structure obtained studying terrestrial and marine Streptomycetes

In our screening for new antibiotics from bacteria, the streptomycete isolate M097 from Jiaozhou Bay in China was found to produce aloesaponarin II (1a) and 1,6-dihydroxy-8-hydroxymethyl-anthraquinone (2). Similarly, a terrestrial streptomycete GW24/1694 produced 1a and its methyl ether, the new compound 1-hydroxy-6-methoxy-8-methyl-anthraquinone (1b). All structures were derived by spectrochemical analysis and by comparison with reference data. The results showed that the marine streptomycete isolate M097 and the terrestrial streptomycete GW24/1694 could be a promising material for studying the biosynthetic pathway of polyketides.