Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples front nine marine landing sites and two coastal inland aquaculture farms were screened for the Vihrio spp and assessed their resistance to twenty-two different antibiotics, which arc commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of' the total 119 Vihrio isolates, 16. 8'7(, were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rilanipicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimcthoprinr, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Multiple Antibiotic Resistances of Vibrio Isolates from Coastal and Brackish Water Areas

An experiment was designed to assess the occurrence of multiple antibiotic resistances in Vibrio sp from different (brackish and marine) environments. Water samples from nine marine landing sites and two coastal inland aquaculture farms were screened for the Vibrio spp and assessed their resistance to twenty-two different antibiotics, which are commonly encountered in the aquatic ecosystem. Tissue samples (shrimp, mussel and sepia) were tested from the sampling site with highest antibiotic resistance. Of the total 119 Vibrio isolates, 16. 8% were susceptible to all antibiotics. Of the resistant (83.19%) Vibrio strains, 30.3% were resistant against three antibiotics, 55.5% were resistant against 4-10 antibiotics, 14.14% were resistant against more than 10 antibiotics and 54% have shown multiple antibiotics resistance (MAR). Antibiotic resistance index was higher in Coastal 3, 6, Aqua farm 2 in isolates from water samples and all the tissues tested. Interestingly, incidence of antibiotic resistance in isolates from water samples was comparatively lower in aquaculture farms than that observed in coastal areas. Highest incidence of antibiotic resistance was evident against Amoxycillin, Ampicillin, Carbencillin and Cefuroxime followed by Rifampicin and Streptomycin and lowest against Chloramphenicol, Tetracycline, Chlortetracycline, Furazolidone, Nalidixic acid, Gentamycin Sulphafurazole, Trimethoprirn, Neomycin and Amikacin irrespective of the sampling sites. Results from various tissue samples collected from the sites of highest antibiotic resistance indicated that antibiotic resistance Vibrio spp collected from fish and tissue samples were higher than that of water samples. Overall results indicated that persistent use of antibiotics against diseases in human beings and other life forms may pollute the aquatic system and their impact on developing antibiotic resistant Vibrio sp may be a serious threat in addition to the use of antibiotics in aquaculture farms.

Potential public health significance of faecal contamination and multidrug-resistant Escherichia coli and Salmonella serotypes in a lake in India

To assess the prevalence of faecal coliform bacteria and multiple drug resistance among Escherichia coli and Salmonella serotypes from Vembanadu Lake. Study design: Systematic microbiological testing. Methods: Monthly collection of water samples were made from ten stations on the southern and northern parts of a salt water regulator constructed in Vembanadu Lake in order to prevent incursion of seawater during certain periods of the year. Density of faecal colifrom bacteria was estimated. E. coli and Salmonella were isolated and their different serotypes were identified. Antibiotic resistance analysis of E. coli and Salmonella serotypes was done and the MAR index of individual isolates was calculated. Results: Density of faecal coliform bacteria ranged from mean MPN value 2900 -7100/100ml. Results showed multiple drug resistance pattern among the bacterial isolates. E. coli showed more than 50% resistance to amickacin, oxytetracycline, streptomycin, tetracycline and kanamycin while Salmonella showed high resistance to oxytetracycline, streptomycin, tetracycline and ampicillin. The MAR indexing of the isolates showed that they have originated from high risk source such as humans, poultry and dairy cows. Conclusions: The high density of faecal coliform bacteria and prevalence of multi drug resistant E. coli and Salmonella serotypes in the lake may pose severe public health risk through related water borne and food borne outbreaks

Antibiotic Resistance of Escherichia coli Serotypes from Cochin Estuary

This study aimed at detecting the prevalence of antibiotic-resistant serotypes of Escherichia coli in Cochin estuary, India. E. coli strains were isolated during the period January 2010–December 2011 from five different stations set at Cochin estuary. Water samples from five different stations in Cochin estuary were collected on a monthly basis for a period of two years. Isolates were serotyped, antibiogram-phenotyped for twelve antimicrobial agents, and genotyped by polymerase chain reaction for uid gene that codes for β-D-glucuronidase. These E. coli strains from Cochin estuary were tested against twelve antibiotics to determine the prevalence of multiple antibiotic resistance among them. The results revealed that more than 53.33% of the isolates were multiple antibiotic resistant. Thirteen isolates showed resistance to sulphonamides and two of them contained the sul 1 gene. Class 1 integrons were detected in two E. coli strains which were resistant to more than seven antibiotics. In the present study, O serotyping, antibiotic sensitivity, and polymerase chain reaction were employed with the purpose of establishing the present distribution of multiple antibiotic-resistant serotypes, associated with E. coli isolated from different parts of Cochin estuary.

Multiple antibiotic resistance profiles of various Escherichia coli serotypes isolated from Cochin Estuary

A total of eighty-one Escherichia coli isolates belonging to forty-three different serotypes including several pathogenic strains such as enterotoxigenic E. coli (ETEC), enterohaemorrhagic E. coli (EHEC), enteropathogenic E. coli (EPEC) and uropathogenic E. coli (UPEC) isolated from Cochin estuary between November 2001 and October 2002 were tested against twelve antibiotics to determine the prevalence of multiple antibiotic resistance (MAR) and antimicrobial resistance profiles as a measure of high risk source of contamination. The results revealed that more than 95% of the isolates were multiple antibiotic resistant (resistant to more than three antibiotics). The MAR indexing of the isolates showed that all these strains originated from high risk source of contamination. The incidence of multiple antibiotic resistant E. coli especially the pathogenic strains in natural waters will pose a serious threat to human population

Antibiotic resistance pattern of motile aeromonads from farm raised fresh water fish

Motile aeromonads isolated from the intestines of farm-raised freshwater fish such as Catla catla, Labeo rohita and Ctenopharyngodon idella have been characterized to species level. Morphological and physiological grouping revealed 61% Aeromonas hydrophila, 30% Aeromonas caviae, 7% Aeromonas sobria and 2% which remained unidentified. Hemolytic activity was detected mostly in A. hydrophila, while only half of the A. sobria and A. caviae showed this activity. Antibiotic resistance patterns of the strains revealed that they had acquired a relatively higher resistance to oxytetracycline, amoxycillin, ampicillin, novobiocin and polymixin-B, implicating possible use of these antibiotics in the aquaculture systems.

Prevalence and Antibiotic sensitivity of Escherichia coli in Extensive Brackish water Aquaculture Ponds

Prevalence and antibiotic resistance of Escherichia coli in the water and sediment samples of brackish water aquaculture ponds adjacent to Cochin backwaters was analysed. More than 50% of the water samples and more than 80% of sediment samples from all the sampling stations were tested positive for £. coli. Risk assessment of the E. coli strains was carried out using multiple antibiotic resistance (MAR) indexing. Majority of the strains were found to be multiple antibiotic resistant suggesting their origin from high risk sources of contamination such as human where antibiotics are frequently used. While none of the £. coli strains were resistant against amikacin, chloramphenicol, streptomycin and trimethoprim, considerable levels of resistance was encountered against ampicillin, erythromycin, penicillin G and vancomycin. High prevalence of £. coli in the water and sediment samples of this extensive brackish water ponds indicates high degree of faecal pollution of this environment. The high risk nature of the strains warrants efficient post harvest and processing measures to avoid health risk to consumers

The incidence ,Antibiotic resistence and survival of salmonella and escherichia coli isolated from broiler chicken retail outlets

The incidence of salmonella and escherichia coli in chicken retail outlets in a residential area of coimbatore, Tamilnadu India was studied with the view that accessories may be a source of cross contamination.Accessories like cages,knives ,chopping boards weighing balance trays and the hands of butcher were examined.A toatal of 14 salmonella as well as 31 E.coli were isolated from different sources. The incidence of E.coli was higher than that of Salmonella.The highest incidence of Salmonella was found in chopping boards and the maximum level of E.Coli was detected in cages.

Antibiotic resistance of Aeromonas hydrophila isolated from marketed fish and prawn of South India

A total of 319 strains of Aeromonas hydrophila were isolated from 536 fish and 278 prawns for a 2-year period. All the strains were tested for resistance to 15 antibiotics and 100% of the strains was resistant to methicillin and rifampicin followed by bacitracin and novobiocin (99%). Only 3% of the strains exhibited resistance against chloramphenicol. The multiple antibiotic resistance (MAR) indexing of A. hydrophila strains showed that all of them originated from high-risk sources

Virulence Potential And Antibiotic Susceptibility Pattern Of Motile Aeromonads Associated With Freshwater Ornamental Fish Culture Systems: A Possible Threat To public Health

Aeromonas spp. are ubiquitous aquatic organisms, associated with multitude of diseases in several species of animals, including fishes and humans. In the present study, water samples from two ornamental fish culture systems were analyzed for the presence of Aeromonas. Nutrient agar was used for Aeromonas isolation, and colonies (60 No) were identified through biochemical characterization. Seven clusters could be generated based on phenotypic characters, analyzed by the programme NTSYSpc, Version 2.02i, and identified as: Aeromonas caviae (33.3%), A. jandaei (38.3%) and A. veronii biovar sobria (28.3%). The strains isolated produced highly active hydrolytic enzymes, haemolytic activity and slime formation in varying proportions. The isolates were also tested for the enterotoxin genes (act, alt and ast), haemolytic toxins (hlyA and aerA), involved in type 3 secretion system (TTSS: ascV, aexT, aopP, aopO, ascF–ascG, and aopH), and glycerophospholipid-cholesterol acyltransferase (gcat). All isolates were found to be associated with at least one virulent gene. Moreover, they were resistant to frequently used antibiotics for human infections. The study demonstrates the pathogenic potential of Aeromonas, associated with ornamental fish culture systems suggesting the emerging threat to public health

Reducing Vibrio load in Artemia nauplii usingantimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

Sequential interactions of Silver-silica nanocomposite (Ag-SiO2NC) with cell wall, metabolism and genetic stability of Psedomonas aeruginosa, a multiple antibiotic resistant bacterium

The study was carried out to understand the effect of silver-silica nanocomposite (Ag-SiO2NC) on the cell wall integrity, metabolism and genetic stability of Pseudomonas aeruginosa, a multiple drugresistant bacterium. Bacterial sensitivity towards antibiotics and Ag-SiO2NC was studied using standard disc diffusion and death rate assay, respectively. The effect of Ag-SiO2NC on cell wall integrity was monitored using SDS assay and fatty acid profile analysis while the effect on metabolism and genetic stability was assayed microscopically, using CTC viability staining and comet assay, respectively. P. aeruginosa was found to be resistant to β-lactamase, glycopeptidase, sulfonamide, quinolones, nitrofurantoin and macrolides classes of antibiotics. Complete mortality of the bacterium was achieved with 80 μgml-1 concentration of Ag-SiO2NC. The cell wall integrity reduced with increasing time and reached a plateau of 70 % in 110 min. Changes were also noticed in the proportion of fatty acids after the treatment. Inside the cytoplasm, a complete inhibition of electron transport system was achieved with 100 μgml-1 Ag-SiO2NC, followed by DNA breakage. The study thus demonstrates that Ag-SiO2NC invades the cytoplasm of the multiple drug-resistant P. aeruginosa by impinging upon the cell wall integrity and kills the cells by interfering with electron transport chain and the genetic stability

Characterization and Pathogenicity of Vibrio cholerae and Vibrio vulnificus from Marine environments

The genus Vibrioof the family Vibrionaceae are Gram negative, oxidasepositive, rod- or curved- rodshaped facultative anaerobes, widespread in marine and estuarine environments. Vibrio species are opportunistic human pathogens responsible for diarrhoeal disease, gastroenteritis, septicaemia and wound infections and are also pathogens of aquatic organisms, causing infections to crustaceans, bivalves and fishes. In the present study, marine environmental samples like seafood and water and sediment samples from aquafarms and mangroves were screened for the presence of Vibrio species. Of the134 isolates obtained from the various samples, 45 were segregated to the genus Vibrio on the basis of phenotypic characterization.like Gram staining, oxidase test, MoF test and salinity tolerance. Partial 16S rDNA sequence analysis was utilized for species level identification of the isolates and the strains were identified as V. cholerae(N=21), V. vulnificus(N=18), V. parahaemolyticus(N=3), V. alginolyticus (N=2) and V. azureus (N=1). The genetic relatedness and variations among the 45 Vibrio isolates were elucidated based on 16S rDNA sequences. Phenotypic characterization of the isolates was based on their response to 12 biochemical tests namely Voges-Proskauers’s (VP test), arginine dihydrolase , tolerance to 3% NaCl test, ONPG test that detects β-galactosidase activity, and tests for utilization of citrate, ornithine, mannitol, arabinose, sucrose, glucose, salicin and cellobiose. The isolates exhibited diverse biochemical patterns, some specific for the species and others indicative of their environmental source.Antibiogram for the isolates was determined subsequent to testing their susceptibility to 12 antibiotics by the disc diffusion method. Varying degrees of resistance to gentamycin (2.22%), ampicillin(62.22%), nalidixic acid (4.44%), vancomycin (86.66), cefixime (17.77%), rifampicin (20%), tetracycline (42.22%) and chloramphenicol (2.22%) was exhibited. All the isolates were susceptible to streptomycin, co-trimoxazole, trimethoprim and azithromycin. Isolates from all the three marine environments exhibited multiple antibiotic resistance, with high MAR index value. The molecular typing methods such as ERIC PCR and BOX PCR revealed intraspecies relatedness and genetic heterogeneity within the environmental isolatesof V. cholerae and V. vulnificus. The 21 strains of V. choleraewere serogroupedas non O1/ non O139 by screening for the presence O1rfb and O139 rfb marker genes by PCR. The virulence/virulence associated genes namely ctxA, ctxB, ace, VPI, hlyA, ompU, rtxA, toxR, zot, nagst, tcpA, nin and nanwere screened in V. cholerae and V. vulnificusstrains.The V. vulnificusstrains were also screened for three species specific genes viz., cps, vvhand viu. In V. cholerae strains, the virulence associated genes like VPI, hlyA, rtxA, ompU and toxR were confirmed by PCR. All the isolates, except for strain BTOS6, harbored at least one or a combination of the tested genes and V. choleraestrain BTPR5 isolated from prawn hosted the highest number of virulence associated genes. Among the V. vulnificusstrains, only 3 virulence genes, VPI, toxR and cps, were confirmed out of the 16 tested and only 7 of the isolates had these genes in one or more combinations. Strain BTPS6 from aquafarm and strain BTVE4 from mangrove samples yielded positive amplification for the three genes. The toxRgene from 9 strains of V. choleraeand 3 strains of V. vulnificus were cloned and sequenced for phylogenetic analysis based on nucleotide and the amino acid sequences. Multiple sequence alignment of the nucleotide sequences and amino acid sequences of the environmental strains of V. choleraerevealed that the toxRgene in the environmental strains are 100% homologous to themselves and to the V. choleraetoxR gene sequence available in the Genbank database. The 3 strains of V. vulnificus displayed high nucleotide and amino acid sequence similarity among themselves and to the sequences of V. cholerae and V. harveyi obtained from the GenBank database, but exhibited only 72% homology to the sequences of its close relative V. vulnificus. Structure prediction of the ToxR protein of Vibrio cholerae strain BTMA5 was by PHYRE2 software. The deduced amino acid sequence showed maximum resemblance with the structure of DNA-binding domain of response regulator2 from Escherichia coli k-12 Template based homology modelling in PHYRE2 successfully modelled the predicted protein and its secondary structure based on protein data bank (PDB) template c3zq7A. The pathogenicity studies were performed using the nematode Caenorhabditiselegansas a model system. The assessment of pathogenicity of environmental strain of V. choleraewas conducted with E. coli strain OP50 as the food source in control plates, environmental V. cholerae strain BTOS6, negative for all tested virulence genes, to check for the suitability of Vibrio sp. as a food source for the nematode;V. cholerae Co 366 ElTor, a clinical pathogenic strain and V. cholerae strain BTPR5 from seafood (Prawn) and positive for the tested virulence genes like VPI, hlyA, ompU,rtxA and toxR. It was found that V. cholerae strain BTOS6 could serve as a food source in place of E. coli strain OP50 but behavioral aberrations like sluggish movement and lawn avoidance and morphological abnormalities like pharyngeal and intestinal distensions and bagging were exhibited by the worms fed on V. cholerae Co 366 ElTor strain and environmental BTPR5 indicating their pathogenicity to the nematode. Assessment of pathogenicity of the environmental strains of V. vulnificus was performed with V. vulnificus strain BTPS6 which tested positive for 3 virulence genes, namely, cps, toxRand VPI, and V. vulnificus strain BTMM7 that did not possess any of the tested virulence genes. A reduction was observed in the life span of worms fed on environmental strain of V. vulnificusBTMM7 rather than on the ordinary laboratory food source, E. coli OP50. Behavioral abnormalities like sluggish movement, lawn avoidance and bagging were also observed in the worms fed with strain BTPS6, but the pharynx and the intestine were intact. The presence of multi drug resistant environmental Vibrio strainsthat constitute a major reservoir of diverse virulence genes are to be dealt with caution as they play a decisive role in pathogenicity and horizontal gene transfer in the marine environments.