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Arch. Pol. Fish. (2013) 21: 29-39DOI 10.2478/aopf-2013-0004 Transferable drug-resistant coliforms in fish exposed to sewage
Sutapa Sanyal, Samir Banerjee
Received – 21 October 2012/Accepted – 19 February 2013. Published online: 31 March 2013; Inland Fisheries Institute in Olsztyn, PolandCitation: Sanyal S., Banerjee S. 2013 – Transferable drug-resistant coliforms in fish exposed to sewage – Arch. Pol. Fish. 21: 29-39.
Abstract. In this study, the thermotolerent fecal coliform (Th
FC) bacterial population (n = 81) in a waste-fed aquaculturesystem was examined for multiple antibiotic resistance and Wastewater-fed aquaculture provides an opportu- the possession of transferable drug resistance factors (Rfactors). Multiple antibiotic resistant (MAR) coliforms were nity to treat wastewater with integrated material-flow found to be common in the sewage-fed pond environment, recycling. Several goals are achieved simultaneously: with 83% of the screened MAR isolates harboring plasmids of on the one hand valuable goods such as foodstuffs, > 10 kilo base pair (kb). The transfer of resistance was animal feeds, raw materials, ornamental plants and confirmed by mating experiments in 92% of plasmid-positive animals, mainly fish, are produced, and on the other MAR coliforms with a nalidixic acid (NA) resistant strain, utilizable gray water, or purified and hygienized Escherichia coli ATCC (American Tissue Type Cell Culture)14948 wastewater, is produced. Nowadays, recycling and (Deoxyribonuclease). Antibiotic resistance profiles of some reusing wastes through aquaculture present several mated progenies (70.83%) indicated that transfer was concerns that are mainly related to health hazards unidirectional. DNase-treated cell-free supernatants did not generated by the use of water recovered from sewer- transform, which excluded transduction. A DNase-resistant age systems. This reclaimed water contains parasites, conjugation-like mechanism probably plays a major role in bacteria, viruses, various types of pharmaceuticals the transfer of resistance factors. Physical evidence oftransmissible resistance factors in fish suggests a potential such as antibiotics, estrogens, and active ingredients health risk to humans and animals, including farmed fish.
of drugs at relatively low concentrations. Althoughindividual antibiotic concentrations are low, there Keywords: fish, health risk, multiple-antibiotic-resistant
are so many different ones that when combined they isolates, transferable drug resistance factors can pose serious health and environmental problems(Koplin 2002). These lower concentrations of antibi-otics are sufficient to affect susceptible bacteria(Al-Ahmad et al. 1999). Therefore, the occurrence ofsuch antibiotic concentrations in sewage has the po-tential to select for antibiotic resistance.
In the field of aquaculture, both environmental Department of Zoology, Bethune College, 181 Bidhan SaraniKolkata - 700006, West Bengal, India and therapeutic problems are addressed since Tel. + 91 9874075312; e-mail: sutapa2007.sanyal@gmail.com, antimicrobial agents are released into surroundingwaters of waste-fed fish ponds through domestic S. BanerjeeDepartment of Zoology, University of Calcutta, 35-Ballygunge sewage (Aoki 1992). Some of the major concerns Circular Road, Kolkata 700019, West Bengal, India presented by antibiotics in the aquatic environment Sutapa Sanyal, Samir Banerjee are that entire trophic levels of bacteria can be elimi- bacterial population (more than one antibiotic) ex- nated in some ecosystems, or that multiple hibits R factors, and which portion of them is capable drug-resistant bacteria flourishes and make their of transferring R factors and at what rates to sensitive way into the food chain. Commercial fishes residing in wastewaters can act as carriers of antibiotic resis-tant bacteria prompting health risks to consumers(Schwartz et al. 2003, Pathak and Gopal 2005, Materials and Methods
Newaj et al. 2008). The occurrence of antibiotic resis-tance might signal the occurrence of plasmid transferin the microbial milieu of sewage systems (Schluter Study area
et al. 2003). The high concentrations of bacteria, nu-trients, and suspended solids in sewage-fed ponds A wetland located in Bandipur, Rahara, North 24 are all factors that enhance horizontal gene transfer Parganas, (22°44' N and 88°24' E), where untreated (Lorenz and Wackernagel 1994). Wastewaters and domestic wastewater is utilized for fish farming, was the fishes inhabiting them are, therefore, a potent the study site. The raw sewage is entirely of domestic source of antibiotic-resistant bacteria, which, in turn, origin, and comes from the adjacent town of Titagarh, can transfer their resistance genes to nonresistant a municipal area in North 24 Parganas, West Bengal, bacteria. Today, transferable drug resistance repre- sents a major threat to the treatment of infectious dis-eases in both humans and animals, including farmed Sampling and dissection
fish (Schwartz et al. 2003).
The potential for antibiotic exposure and resis- The fish samples were caught with a net and were tance development in human and animal gastroin- immediately transferred to the laboratory in contain- ers with pond water. The major Indian carps Labeo abundance in waters contaminated with human and rohita (Hamilton), Cirrhinus mrigala (Hamilton), and animal wastes, make the thermotolerent fecal Oreochromis niloticus (Peters) were subjected to bac- coliform bacteria a logical focal group for studies of teriological assays. Ten live fish of each of the species antibiotic resistance and transfer in waste-fed were selected randomly from the catch at each sam- aquatic environments. Sewage-fed fisheries play pling time (bimonthly from March 2010 to Novem- a great role in improving water quality in tropical ber 2010). The fish were dissected according to Asian countries, and it can be compared to an effi- Buras et al. (1987). Muscles and digestive tract con- cient stabilization pond as far as fish production is tents were isolated and placed in sterile glass vessels.
concerned. Moreover, potential public health haz- The tissues were weighed under sterile conditions, ards have never been considered although good fish ground in a mortar, and suspended in a sodium chlo- production has been achieved (Strauss 1997). In ride (NaCl) physiological solution (9 ml of the solu- view of the public health concern about the safety of tion for each 1 g of muscle or digestive tract content).
fish raised in wastewater ponds, the aim of this study The suspensions were homogenized using a Univer- was to analyze the antibiotic resistance patterns in sal Laboratory Aid Type MPW-309 homogenizer, at thermotolerent fecal bacteria collected from fish and 1000 rpm for 10 min. The homogenates were then the waste-fed aqueous environments they are reared serially diluted (10-1 to 10-8 for muscles and 10-1 to in. The investigation was also undertaken to study 10-10 for digestive tract contents) and inoculated into the transfer of R factors and the frequencies of trans- culture media. The time lag from fish collection to fer under standardized laboratory conditions. Much analyses did not exceed 6 h. Water from the sew- of the work dealing with the transfer of R factors has focused on which portion of an antibiotic-resistant sub-surface, i.e., 15 to 20 cm below the water Transferable drug-resistant coliforms in fish exposed to sewage surface, to avoid surface contamination and analyzed Plasmid profiles of drug-resistant
simultaneously with the fish sampling.
Drug resistant isolates were tested for the availability of plasmids. A rapid alkaline extraction procedureproposed by Sambrook et al. (1989) was followed for The pond water and fish samples were examined for screening plasmid DNA. Electrophoresis was per- thermotolerent fecal coliforms using the three-tube formed using a 0.8% and 1% agarose gel system fermentation technique (APHA 1998). Representa- (Bangalore Genei, India) in tris acetate buffer with tives of typical thermotolerent coliform isolates were molecular weight markers (Hi Media, Mumbai, In- selected randomly by colony morphology from the dia). The gels were stained with ethidium bromide selective culture medium (as described by APHA (0.5 µg ml-1 Sigma). The resolved bands were visual- 1998 and Sanyal et al. 2011). They were streaked ized on a UV-transilluminator at a wavelength of 360 aseptically several times on freshly prepared Nutri- nm, and photographed using a UV gel documenta- ent agar plates to obtain pure isolates for antibiotic tion system (Alpha Imager, Innotech Corporation, resistance tests. Pure fecal isolates were subjected to USA). The concentrations of agarose noted above several biochemical tests which included indole pro- duction, methyl-red, and Voges Proskauer reactions, high-molecular-weight plasmid DNA (up to 25 kb).
citrate utilization, and reaction in motility and ureamedium. Eighty-one purified fecal bacterial isolateswere tested for anti-microbial sensitivity using the Mating experiments on the transfer of
disc diffusion method (Bauer et al. 1966). Antibiotic resistance factors in bacteria
impregnated discs of 8 mm diameter were used for A nalidixic acid (NA) resistant strain, E. coli ATCC the test, and the disks contained the following anti- 14948, was used as the recipient in all the mating ex- bacterial agents: ampicillin (AMP 10 µg); amikacin periments. All of the environmental isolates that were not resistant to nalidixic acid were tested as donor cotrimoxazole (CO 25 µg); gentamicin (GEN 30 µg); strains in individual mating assays. Isolates demon- kanamycin (KAN 30 µg); streptomycin (STR 10 µg); strating multiple-drug resistance were processed and tetracycline (TET 25 µg). Resistance was estimated tested separately for the ability to transfer each type of by measuring the inhibition zone as per standards drug resistance factor. Twenty-four plasmid-positive (NCCLS 2002). Reference strains E. coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853, coliforms isolated from the fish and the water of the recommended by NCCLS (1997), were used as con- waste-fed ponds were tested for transferable resis- trol organisms for verification of the disks on Tryptone soya agar plates. All the culture media andantibiotic discs were obtained from Hi Media Labo-ratories Ltd., Mumbai, India. The rationale for choos- Transfer of resistance factors in liquid
ing these compounds as target antibiotics was based on previous studies, (Andersen 1993, Goñi-Urriza etal. 2000, Kim and Diana 2007, Jury et al. 2010) that Prior to mating, individual bacterial cultures were reported the occurrence of AMP, AK, C, CO, GEN, grown overnight at 30°C in Tryptone Soya Broth KAN, STR, and TET in municipal wastewater treat- (TSB) containing 30 ìg ml-1 of the appropriate anti- ment plants. This made it likely that the microbial biotic (NA for E. coli ATCC 14948 and either AMP, biomass in the Bandipur waste-fed ponds also in- C, STR, or TET for the environmental isolates). The cluded bacteria resistant to these antibiotics.
bacterial cells were pelleted by centrifugation for 5 Sutapa Sanyal, Samir Banerjee min at 8,500 g, and resuspended in fresh TSB lack- STR or TET, all at 30 ìg ml-1) and incubated for 24 h ing antibiotic. Mating experiments were conducted at 30°C. When mating progeny was achieved, the an- as described by Bell et al. (1983). Briefly, equal vol- umes (0.1 ml) of donor cells and recipient cells were recombinants were determined with multi tipped mixed in a 1.5 ml tube with 0.8 ml of antibiotic-free disks. Additional mating experiments were also per- TSB. Mated cells in broth were supplemented with formed in the presence of DNase I following the DNase I and MgCl2 at final concentrations of 200 µg method described above with cell-free supernatants ml-1 and 2 mM, respectively (Omar et al. 2007). The of donors that were not filtered. Further experiments mixtures were incubated at 30°C for 24 h. After incu- were performed in the presence of DNase I with bation the mixtures of donor and recipient cells were treated supernatants from co-cultures of donors and diluted 10-fold in 0.9% saline to a 10-7 dilution.
recipient cells.
Samples (0.1 ml) from each dilution were plated ontoTSA containing dual antibiotics (NA and either AMP,C, STR or TET, all at 30 ìg ml-1) and incubated for Measurement of transfer frequency
24 h at 30°C to select for recombinants. When mat-ing progeny was achieved, the antibiotic-resistance Transfer frequency was estimated by dividing the patterns of representative recombinants were deter- number of recombinants per milliliter by the number mined with multi tipped disks. Negative mating con- of donors per milliliter in the mating mixture (Yutaka trol experiments lacking either the donor or recipient et al. 2004). A number of transferable resistant bacteria were included in all mating experiments to coliforms were characterized biochemically to detect check for the mutation of either strain to antibiotic re- possible effects on strain distribution caused by the sistance. No mutation to specific antibiotic resistance discharge of domestic waste effluent in sewage-fed was noted with any donor strains or the universal E. ponds. Biochemical characterizations of the isolates coli ATCC 14948 recipient strain (West et al. 2011).
were performed to identify genus by a standard pro-cedure used for Enterobacteriaceae (Kelly et al.
1995). In the present study, the presence of coliforms Transfer of resistance factors using cell
was confirmed using multiplex PCR for the specific amplification of the lacZ gene that encodes â-Dgalactosidase and a portion of the uid A gene for E. Experiments using cell-free culture supernatants as coli detection. For multiplex PCR, a pair of primers a DNA source were done as described below. After the harvested cells of donors and recipients were cen- 5'-CACCATGCCGTGGGTTTCAATATT-3', Bej et al.
trifuged for 5 minutes at 8,500 g, the supernatants 1990) located within the coding region of the lacZ were passed through a filter with a 0.2 µm pore size gene of E. coli and a pair of primers (5'- (Life Sciences Products, Inc.) and then centrifuged for an additional 3 min at 11,500 g. Aliquots of recip- 5'-ACGCGTGGTTACAGTCTTGCG-3', Bej et al.
ient cells (0.1 ml) were mixed in 0.8 ml of antibi- 1991) located within the uid A structural gene of E. otic-free TSB broth tubes together with 0.1 ml of the coli were used. The primers were obtained from treated supernatants of the donor cells. These experi- Messers Bangalore Genei, Bangalore, India. During ments were done in the presence of DNase I, as de- the development of the PCR amplification procedure scribed above. The mixtures were incubated at 30°C for coliform detection, total genomic DNA was ex- for 24 h. After incubation, the mixtures were diluted tracted from the cultures with the procedure by 10-fold in 0.9% saline to a 10-7 dilution. Samples Ausubel et al. (1996). In each PCR amplification, (0.1 ml) from each dilution were plated onto TSA a buffer control, to which no DNA template was containing dual antibiotics (NA and either AMP, C, added, was included as an internal control. DNA Transferable drug-resistant coliforms in fish exposed to sewage Table 1
Antibiotic resistance patterns in thermotolerent fecal coliforms from different sources at a waste-fed farm. Numbers of isolates in
brackets. NA= not analyzed, -ve = negative for plasmid, AMP = ampicillin, AK = amikacin, C = chloramphenicol, CO =
cotrimoxazole, GEN = gentamicin, KAN = kanamycin, STR = streptomycin and TET = tetracycline, MAR= Multiple antibiotic
Single antibiotic Patterns and number of MAR No. of plasmid positive MAR resistant isolates AMP-CO-STR-TET (1) AMP-CO-STR-TET (1) Labeo sp.
AMP-TET (3, 1-ve) templates of coliform bacteria and E. coli extracted associated with multiple resistances. Twelve differ- ent MAR patterns were observed with a wide range of pneumoniae ATCC 27736, E. coli ATCC 25922) multi resistance distribution, to as many as five anti- were included as positive controls.
biotics (Table 1). Most of the isolated strains shared resistance to ampicillin and tetracycline. Of 29 MAR isolates screened, 24 (83%) harbored plasmids (Ta- ble 1). Single high molecular weight plasmid (> 10 kb) possessed by isolates was clearly observed in 0.8 A substantial portion of bacteria isolated from the and 1% agarose gel electrophoresis (Fig. 1a and 1b), fish and pond water were resistant to ampicillin, while 92% of the MAR isolates that harbored chloramphenicol, and tetracycline (except Cirrhinus plasmids were able to transfer all or part of their de- sp.), whereas bacteria resistant to kanamycin (except terminants of antibiotic resistance to E. coli ATCC 14948 (Table 2). However, in none of the successful co-trimoxazole were infrequent (Table 1). No resis- mating experiments was the presence of DNase tance was found for amikacin and gentamicin. Resis- I able to prevent the development of recombinants.
tance to ampicillin alone was very common, whereas The addition of treated, cell-free culture supernatant resistance to each of the other antibiotics was usually of donors in the presence of DNase I did not

Sutapa Sanyal, Samir Banerjee Figure 1. Plasmids of Drug Resistant Coliform isolates. 1a) 0.8% agarose gel (Plasmids of > 10 kilo base pair (kb)). 1b) 1% agarose gel(Plasmids of > 10 kilo base pair (kb)).
Table 2
Resistance pattern observed after gene transfer for non-nalidixic acid isolates.* Marks indicate mated progeny with unidirectional
transfer. AMP = ampicillin, AK = amikacin, C = chloramphenicol, CO = cotrimoxazole, GEN = gentamicin, KAN = kanamycin,
STR = streptomycin and TET = tetracycline
No. of iso-lates trans- Resistance pattern genetically trans- Antibiotic resistance pattern Treated cell-free supernatant of donors Untreated cell-free supernatant of donors Supernatant from co-culture of donors and

Transferable drug-resistant coliforms in fish exposed to sewage transform the recipient cells (Table 2). Additional ex- periments were performed with supernatants thatwere not filtered. The results were the same as thoseobserved with the filtered supernatants. Mating ex- periments were performed with supernatants fromco-cultures of donors and recipients, and the results were the same again (Table 2), with 45% of the iso-lates (10 out of 22) capable of achieving antibiotic re- sistance transfer rates greater than 10-3 per donor organism (Table 2). The following numbers of com-binations of fecal coliforms with transferable R+ de- terminants were found: 13 in the E. coli populations,4 in the Enterobacter populations, 4 in the Klebsiellapopulations, and 1 in the Citrobacter populations(Table 2).
The PCR amplification of oligonucleotide prim- ers yielded a detectable DNA fragment of an ex-pected molecular weight (876 bp for coliformbacteria; 876 bp and 147 bp for E. coli) only in the presence of their respective DNA templates. How- ever, the amplified products of Klebsiella spp. with Figure 2. Agarose gel (2%) electrophoresis of PCR amplified prod- lacZ primer were larger than those of E. coli, ucts from various pure bacterial DNAs, using optimized multi- Citrobacter spp., and Enterobacter spp., indicating plex PCR. 2a) Lane M: molecular size marker (100 base pairs a difference between the target lacZ among these or- {bp} DNA ladder); lane 1 Klebsiella spp., lanes 4, 7 and 8 E. coli,lane 9, Klebsiella spp., lanes 10 and 11 Citrobacter spp. respec- ganisms (Fig. 2).
tively. 2b) Lane M: molecular size marker (100 bp DNA ladder);lane 2 positive control, lane 3 Enterobacter spp., lane 7 E. coli,Lane 10 Citrobacter spp., lanes 11 and 12 E. coli. chloramphenicol because of its genotoxicity, em- A high resistance to ampicillin reflects the influence bryo- and fetotoxicity, carcinogenic potential, and the of humans on the environment (Andersen 1993). In- lack of a dose response relationship for aplastic ane- terestingly, in the present study the fish exhibited mia in humans. In addition, the Aquaculture Author- high antibiotic resistance to chloramphenicol and ity of India banned 19 antibiotics, including chloramphenicol, from aquaculture (Aquaculture aquaculture. There are no reports available on the News 2003). On the other hand, even when there is use of these drugs applied either as feed supple- high cotrimoxazole resistance in the water, low resis- ments or as drugs used in aquaculture practice in the tance was reported in fish (Table 1). This indicates Bandipur waste-fed farm. It can be presumed that that although the drug is effective, it exerts low selec- tion pressure (Ogbondeminu and Olayemi 1993).
antimicrobial-resistant bacteria enter the fish ponds The sensitivity exhibited for gentamicin is a signal of with the sewage effluents, thus, these anthropogenic the effectiveness of broad-spectrum antibiotics of the factors might have influenced the fecal coliforms in present generation (Pathak and Gopal 2005).
their acquisition of resistance. Fish and pond water The high recovery rates of thermotolerent fecal are not expected to have high resistance to coliforms from Oreochromis sp. and Labeo sp. noted Sutapa Sanyal, Samir Banerjee in the present study, with the simultaneous resis- completely destroyed extracellular DNA and pre- tance to three to four antibiotics, suggest changes in nutritionally beneficial microflora with unexpected transduction, at least for the isolates studied (Table 2).
Zemelman 2001). The high recovery rate of antibi- To confirm that the DNase-resistant recombination otic-resistant bacteria from all the three fish species was not due to protein-coated DNA or DNA present in has immense ecological and public health implica- membrane blebs, experiments were performed with tions; specially, if the resistance is plasmid mediated, cell-free supernatants that were not filtered. The re- then there could be a problem associated with the sults were the same as those observed with filtered transfer of resistance determinants to human patho- supernatants. To exclude the possibility that the re- genic bacteria that could enter the human population sults were because of a phage induced by the through fish consumption (Miranda and Zemelman co-culturing of strains, experiments were also per- 2001). The high proportion of multiple antibi- formed with supernatants from co-cultures of donors otic-resistance in Oreochromis sp. in comparison and recipients. The results were the same again (Table with the detritivorous Cirrhinus sp. suggests that the 2); the transfer was not affected by the presence of fecal bacterial populations in Oreochromis sp. were deoxyribonuclease I, which suggested the conjugative subjected to conditions that fostered the acquisition nature of it. The unidirectional process of DNA trans- of multiple-resistance determinants.
fer was confirmed in 70.83% plasmid positive resis- The simultaneous resistance of thermotolerent tant progeny in the presence of DNase I by resistance to secondary antibiotic markers, which is consistent aminoglycoside could be the result of the dissemina- with natural conjugation (Table 2). Mating experi- tion of antibiotic-resistant plasmids in the aquatic en- ments were not performed to confirm that the devel- vironment (Miranda and Zemelman 2001). Of the opment of resistant progeny in the presence of DNase multidrug-resistant isolates, most exhibited resistance required cell-to-cell contact. A similar process of to combinations of antimicrobial drugs that included DNase-resistant transformation not involving conjugal ple-resistance genes may coexist on one plasmid gonorrhoeae (Catlin 1981).
(Sayah et al. 2005), a single conjugative transposon The results for the frequencies of transfer for the (Pembroke et al. 2002), or an integron (Mazel 2006).
different mating experiments were in general agree- This condition is particularly disconcerting given that ment with those of Bell et al. (1983). Most of the iso- exposure to one antibiotic agent may result in resis- lates were capable of achieving resistance transfer tance to others without previous exposure (Sayah et al.
rates within a range of 10-1 to 10-4 (Table 2), which is 2005) or cost to bacterial fitness (Aminov et al. 2002).
consistent with the results of Mach and Grimes Plasmids, conjugative transposons, and integrons (1982). Transfer rates varied between biotypes of the make it possible for new antibiotic resistance genes to same genus used as donors and between different spread through bacterial populations by the process of genera (Walter and Vennes 1985). Intragenetic lateral gene transfer (Scott 1993). High rates of suc- transfer might be the reason for the high transfer po- cess of resistance gene transfer, as was observed in the tentiality of E. coli noted in the present investigation present assay (92% overall), suggests that regardless (Table 2); however, the limited number of strains of the physical location of resistance genes, i.e. chro- used in the mating experiments does not permit mosome, plasmid or integrons within transposons, drawing broad conclusions on the frequency of such these environmental isolates have the ability to move transfers occurring in nature. In the development of copies of themselves from one bacterial cell to another a resistant clone under natural conditions, the (Jury et al. 2010). The concentration of DNase I (200 amount of R plasmid donor cells would be substan- µg ml-1) was so high in the present experiments that it tially smaller than that in the present mating Transferable drug-resistant coliforms in fish exposed to sewage mixtures. However, the number of donor cells would Banerjee for his guidance and invaluable constructive increase over time under continued antimicrobial criticism during manuscript preparation.
pressure. While in present investigation transferswere achieved with only a few strains, more strains Author contributions. S.S. conceived of and designed
might be positive under different conditions or with the experiments, performed the experiments, and ana-lyzed the data, S.S. and S.B. interpreted the data and other recipients.
wrote the paper.
The mating experiments were performed at 30°C, a temperature that is common in tropicalaquatic systems, thus, their transfer in voided ex-creta, sewage, and polluted rivers is a definite possi- bility. However, the intent was to use a method inwhich laboratory manipulation has the least impact Al-Ahmad A., Daschner F.D., Kümmerer K. 1999 – on both resistance genes and bacteria, and which Biodegradability of cefotiam, ciprofloxacin, meropenem,penicillin G, and sulfamethoxazole and inhibition of provides a better indication of what can happen in waste water bacteria – Arch. Environ. Contam. Toxicol.
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Acknowledgments. This investigation received finan-
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University Grants Commission, New Delhi, Govern- Bej A.K., McCarty S.C., Atlas R.M. 1991 – Detection of ment of India). The authors are thankful to the Head of coliform bacteria and Escherichia coli by multiplex poly- the Department of Zoology of University of Calcutta merase chain reaction: comparison with defined sub- who permitted the research work to be conducted in the strate and plating methods for water quality monitoring – department. The first author is grateful to Prof. Samir Appl. Environ. Microbiol. 57: 2429-2432.
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Fast food and takeaway outlets toolkit

Takeaways Toolkit Tools, interventions and case studies to help local authorities develop a response to the health impacts of fast food takeaways A London Food Board and Chartered Institute of Environmental Health publication Based on a consultancy report by Food Matters Updated June 2014 In 2010 the issue of fast food takeaways hit the headlines when a number of London