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.

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.

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

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.

Persistence of a wild type Escherichia coli and its multiple antibiotic-resistant (MAR) derivatives in the abattoir and on chilled pig carcasses

The aim of this study was to evaluate the ability of an Escherichia coli with the multiple antibiotic resistance (MAR) phenotype to withstand the stresses of slaughter compared to an isogenic progenitor strain. A wild type E. coli isolate (345-2RifC) of porcine origin was used to derive 3 isogenic MAR mutants. Escherichia coli 345-2RifC and its MAR derivatives were inoculated into separate groups of pigs. Once colonisation was established, the pigs were slaughtered and persistence of the E. coli strains in the abattoir environment and on the pig carcasses was monitored and compared. No significant difference (P>0.05) was detected between the shedding of the different E. coli strains from the live pigs. Both the parent strain and its MAR derivatives persisted in the abattoir environment, however the parent strain was recovered from 6 of the 13 locations sampled while the MAR derivatives were recovered from 11 of 13 and the number of MAR E. coil recovered was 10-fold higher than the parent strain at half of the locations. The parent strain was not recovered from any of the 6 chilled carcasses whereas the MAR derivatives were recovered from 3 out of 5 (P<0.001). This study demonstrates that the expression of MAR in 345-2RifC increased its ability to survive the stresses of the slaughter and chilling processes. Therefore in E. coli, MAR can give a selective advantage, compared to non-MAR strains, for persistence on chilled carcasses thereby facilitating transit of these strains through the food chain. (C) 2010 Elsevier B.V. All rights reserved.

Periplasmic adaptor protein AcrA has a distinct role in the antibiotic resistance and virulence of Salmonella enterica serovar Typhimurium

Objectives: AcrA can function as the periplasmic adaptor protein (PAP) in several RND tripartite efflux pumps, of which AcrAB-TolC is considered the most important. This system confers innate multiple antibiotic resistance. Disruption of acrB or tolC impairs the ability of Salmonella Typhimurium to colonize and persist in the host. The aim of this study was to investigate the role of AcrA alone in multidrug resistance and pathogenicity. Methods: The acrA gene was inactivated in Salmonella Typhimurium SL1344 by insertion of the aph gene and this mutant complemented with pWKS30acrA. The antimicrobial susceptibility of the mutant to six antibiotics as well as various dyes and detergents was determined. In addition, efflux activity was quantified. The ability of the mutant to adhere to, and invade, tissue culture cells in vitro was measured. Results: Following disruption of acrA, RT-PCR and western blotting confirmed that acrB/AcrB was still expressed when acrA was disrupted. The acrA mutant was hypersusceptible to antibiotics, dyes and detergents. In some cases, lower MICs were seen than for the acrB or tolC mutants. Efflux of the fluorescent dye Hoechst H33342 was less than in wild-type following disruption of acrA. acrA was also required for adherence to, and invasion of, tissue culture cells. Conclusions: Inactivation of acrA conferred a phenotype distinct to that of acrB::aph and tolC::aph. These data indicate a role for AcrA distinct to that of other protein partners in both efflux of substrates and virulence.

Fitness and dissemination of disinfectant-selected multiple-antibiotic-resistant (MAR) strains of Salmonella enterica serovar Typhimurium in chickens

Objectives: The aims of this study were to determine whether strains of Salmonella enterica serovar Typhimurium which had acquired low-level multiple antibiotic resistance (MAR) through repeated exposure to farm disinfectants were able to colonize and transmit between chicks as easily as the parent strain and, if such strains were less susceptible to fluoroquinolones, would high-level resistance be selected after fluoroquinolone treatment. Methods: Two mutants were compared with the isogenic parent. In the first experiment, day-old chicks were co-infected with both the parent and a mutant to determine their relative fitness. In the second experiment, parent and mutant strains (in separate groups of chicks) were assessed for their ability to transmit from infected (contact) to non-infected (naive) birds and with respect to their susceptibility to fluoroquinolone treatment. Birds were regularly monitored for the presence of Salmonella in caecal contents. Replica plating was used to monitor for the selection of antibiotic-resistant strains. Results: The parent strain was shown to be significantly fitter than the two mutants and was more rapidly disseminated to naive birds. Antibiotic treatment did not preferentially select for the two mutants or for resistant strains. Conclusions: The disinfectant-exposed strains, although MAR, were less fit, less able to disseminate than the parent strain and were not preferentially selected by therapeutic antibiotic treatment. As such, these strains are unlikely to present a greater problem than other salmonellae in chickens.

Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR. Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains. Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP. Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.

Presence of new mecA and mph(C) variants conferring antibiotic resistance in Staphylococcus spp. isolated from the skin of horses before and after clinic admission

Because of the frequency of multiple antibiotic resistance, Staphylococcus species often represent a challenge in incisional infections of horses undergoing colic surgery. To investigate the evolution of antibiotic resistance patterns before and after preventative peri- and postoperative penicillin treatment, staphylococci were isolated from skin and wound samples at different times during hospitalization. Most staphylococci were normal skin commensals and belonged to the common coagulase-negative group. In some cases they turned out to be opportunistic pathogens present in wound infections. MICs were determined for 12 antibiotics, and antibiotic resistance genes were detected by microarray. At hospital admission, horses harbored staphylococci that were susceptible to antibiotics or resistant to one group of drugs, mainly due to the presence of new variants of the methicillin and macrolide resistance genes mecA and mph(C), respectively. After 3 days, the percentage of Staphylococcus isolates displaying antibiotic resistance, as well as the number of resistance genes per isolate, increased moderately in hospitalized horses without surgery or penicillin treatment but dramatically in hospitalized horses after colic surgery as well as penicillin treatment. Staphylococcus species displaying multiple resistance were found to harbor mainly genes conferring resistance to beta-lactams (mecA and blaZ), aminoglycosides [str and aac(6')-Ie-aph(2')-Ia], and trimethoprim [dfr(A) and dfr(D)]. Additional genes conferring resistance to macrolides [mph(C), erm(C), and erm(B)], tetracycline [tet(K) and tet(M)], chloramphenicol [cat(pC221) and cat(pC223)], and streptothricin (sat4) appeared in several strains. Hospitalization and preventive penicillin use were shown to act as selection agents for multidrug-resistant commensal staphylococcal flora.

The need for continued monitoring of antibiotic resistance patterns in clinical isolates of Staphylococcus aureus from London and Malta

Antibiotic resistance is an increasing problem in isolates of Staphylococcus aureus (S. aureus) worldwide. In 2001 The National Health Service in the UK introduced a mandatory bacteraemia surveillance scheme for the reporting of S. aureus and methicillin-resistant S. aureus (MRSA). This surveillance initiative reports on the percentage of isolates that are methicillin resistant. However, resistance to other antibiotics is not currently reported and therefore the scale of emerging resistance is currently unclear in the UK. In this study, multiple antibiotic resistance (MAR) profiles against fourteen antimicrobial drugs were investigated for 705 isolates of S. aureus collected from two European study sites in the UK (London) and Malta.

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.

Concurrence of cat and tet genes in multiple antibiotic-resistant bacteria isolated from a sea cucumber and sea urchin mariculture farm in China

A basic understanding of abundance and diversity of antibiotic-resistant microbes and their genetic determinants is necessary for finding a way to prevent and control the spread of antibiotic resistance. For this purpose, chloramphenicol and multiple antibiotic-resistant bacteria were screened from a mariculture farm in northern China. Both sea cucumber and sea urchin rearing ponds were populated with abundant antibiotic-resistant bacteria, especially marine vibrios. Sixty-five percent chloramphenicol-resistant isolates from sea cucumber harbored a cat gene, either cat IV or cat II, whereas 35% sea urchin isolates harbored a cat gene, actually cat II. The predominant resistance determinant cat IV gene mainly occurred in isolates related to Vibrio tasmaniensis or Pseudoalteromonas atlantica, and the cat II gene mainly occurred in Vibrio splendidus-like isolates. All the cat-positive isolates also harbored one or two of the tet genes, tet(D), tet(B), or tet(A). As no chloramphenicol-related antibiotic was ever used, coselection of the cat genes by other antibiotics, especially oxytetracycline, might be the cause of the high incidence of cat genes in the mariculture farm studied.

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

Variation in clonality and antibiotic-resistance genes among multiresistant Salmonella enterica serotype typhimurium phage-type U302 (MR U302) from humans, animals, and foods

Since 1990 multiresistant (MR) Salmonella enterica serotype Typhimurium definitive phage-type (DT) 104 (MR DT104) and closely related phage types have emerged as a worldwide health problem in humans and food animals. In this study the presence of the bla(CARB-2) (ampicillin), cmlA (chloramphenicol), aadA2 (streptomycin/spectinomycin), sul1 (sulphonamide), and tetG (tetracycline) resistance genes in isolates of one such phage type, U302, have been determined. In addition bla(TEM) I primers have been used for the detection of TEM-type beta-lactamases. Isolates have also been characterized by plasmid profile and pulsed field gel electrophoresis (PFGE). Thirty-three of 39 isolates were positive for blaCARB-2, cmlA, aadA2, sul1 and tetG, four for bla(TEM), aadA2 and sul1, one for aadA2 and sul1, and one for blaTEM only. bla(TEM)-mediated ampicillin resistance was transferred to Escherichia coli K12 from three isolates along with other resistance markers, including resistance to chloramphenicol, streptomycin, spectinomycin, sulphonamides, and tetracyclines. Strains carried up to 6 plasmids and 34 plasmid profiles were identified. Although the majority of strains (33/39) produced a PFGE profile identical to that predominant in MR DT104, six different patterns were generated demonstrating the presence of various clones within MR U302. The results show that the majority of the MR U302 strains studied possessed the same antibiotic resistance genes as MR DT104. However, isolates with distinctive PFGE patterns can have different mechanisms of resistance to ampicillin, chloramphenicol, streptomycin, sulphonamides, and tetracyclines. Such resistance genes may be borne on transmissible plasmids.

Increased genome instability in Escherichia coli lon mutants: relation to emergence of multiple-antibiotic-resistant (Mar) mutants caused by insertion sequence elements and large tandem genomic amplifications

Thirteen spontaneous multiple-antibiotic-resistant (Mar) mutants of Escherichia coli AG100 were isolated on Luria-Bertani (LB) agar in the presence of tetracycline (4 microg/ml). The phenotype was linked to insertion sequence (IS) insertions in marR or acrR or unstable large tandem genomic amplifications which included acrAB and which were bordered by IS3 or IS5 sequences. Five different lon mutations, not related to the Mar phenotype, were also found in 12 of the 13 mutants. Under specific selective conditions, most drug-resistant mutants appearing late on the selective plates evolved from a subpopulation of AG100 with lon mutations. That the lon locus was involved in the evolution to low levels of multidrug resistance was supported by the following findings: (i) AG100 grown in LB broth had an important spontaneous subpopulation (about 3.7x10(-4)) of lon::IS186 mutants, (ii) new lon mutants appeared during the selection on antibiotic-containing agar plates, (iii) lon mutants could slowly grow in the presence of low amounts (about 2x MIC of the wild type) of chloramphenicol or tetracycline, and (iv) a lon mutation conferred a mutator phenotype which increased IS transposition and genome rearrangements. The association between lon mutations and mutations causing the Mar phenotype was dependent on the medium (LB versus MacConkey medium) and the antibiotic used for the selection. A previously reported unstable amplifiable high-level resistance observed after the prolonged growth of Mar mutants in a low concentration of tetracycline or chloramphenicol can be explained by genomic amplification.