Hazard analysis of antimicrobial resistance associated with asian aquacultural environments

A repetitive DNA element PCR fingerprinting (rep-PCR) protocol was developed for the strain typing of aquaculture-associated bacterial isolates (n= 650). A simple boiled extract of broth cultures acted as a DNA template for the PCR. The (GTG)5 primer was employed in combination with the convenience of a commercial PCR master mix and following amplification, the PCR samples separated under standardised electrophoresis conditions. Numerical analysis of the DNA profiles (GelCompar, Applied Maths, Belgium) allowed individual strain comparisons to be made. Replicates, isolates found to possess identical (GTG)5-PCR profiles originating within the same sample, could be revealed to yield a de-replicated strain set, thus reflecting the true genotypic diversity of the strain collection. Analysis of the DNA profiles was also able to highlight the presence of possible strain types occurring between different samples. The identity of these PCR strain types was later confirmed by macrorestriction analysis, an independent typing technique. The rep-PCR typing method was found to be a suitable as a screening method, was simple to perform and could be universally applied to assess the genotypic variation of a strain collection.

Total of 276 and 30 chloramphenicol resistance and sensitive strains of bacteria collected from shrimp and fish farms in Thailand were isolated and identified to species level. These specimens were kept at the Microbologicla reference laboratory at Gent University, Belgium.

The simple device for sampling pond sediment was developed to fulfil the requirement of the soil sampling technique indicated in the project. The sample consisted of three-piece PVC pipe that could be sterilized with the total length of 230 cm. It was named ASIARESIST sediment sample. This sample was found to be inexpensive (20 Euro), simple to manufacture, user friendly, robust and reliable for sampling even soft sediment.

Molecular Characterization of aquaculture-associated bacteria by (GTG)5 repetitive PCR Epidemiological typing of bacteria or pathogens has become more and more important in public health control. PCR is a powerful technique that has revolutionized the molecular biology. Repetitive-PCR (rep-PCR) is a commonly used method in PCR. In this study, rep-PCR was used to genotype 140 of the aquaculture-associated bacteria isolates, which yield negative results for Cat genes detection. Rep-PCR entails the use of primers complementary to highly conserved, repetitive sequences present in multiple copies on the genome. Because the distance between the repetitive elements varies among strains, PCR amplification of the DNA sequences found between them results in generation of a distinct fingerprint. Prior to amplification, crude genomic DNA of isolates was harvested using boiling method. For the detection and confirmation of PCR products by gel electrophoresis, the amplification product mixture was subjected to electrophoresis. Subsequently, amplified DNA fragments of specific sizes were visualized by UV fluorescence after being stained with ethidium bromide. The (GTG)5 rep-PCR fingerprinting profiles obtained were analyzed with the GelCompare software (version 4.1). The overall achievable patterns were used to construct dendrograms using the UPGMA (unweighted pair group average method) clustering algorithms. To determine the reproducibility of the (GTG)5 rep-PCR analysis procedure, (GTG)5 rep-PCR was repeated at least three times for DNA from each strain, so that the amplification profiles for each attempt could be compared. We found that profiles for a given strain to be highly reproducible, with very little variation from one (GTG)5 rep-PCR analysis to another. (GTG)5 rep-PCR was used as a mean to determine the clonal relatedness of the isolates in this study by their chromosomal polymorphism. Our (GTG)5 rep-PCR analysis revealed homogeneous isolates and of match those of UoS's profile. In summary, results from this study demonstrated that genotyping aquaculture-associated bacteria isolates by using (GTG)5 rep-PCR is feasible for differentiation of various strains. The classical and microbiological methods, based on the identification of phenotypic markers, while perfectly adequate to identify microorganisms on the level of species, are often not reliable enough when it comes to differentiating further into individual strains. Molecular typing procedures like (GTG)5 rep-PCR are applied to show clonal and close relationship between isolates of one species. Thus, it is possible to identify pathogen reservoirs and to follow up the regional and global spread of pathogens in aquaculture. In addition, it gives an insight into the evolutionary dynamics of the bacterial genome. (GTG)5 rep-PCR has shown to be rapid, sensitive, discriminative and cost effective in genotyping the aquaculture-associated bacteria isolates from Malaysia.

A Standard Operating Procedure (SOP) was defined for the antibiotic susceptibility testing of project isolates using the disc diffusion (DD) method (SOP-ASIARESIST-ABR). The final version of the SOP was obtained following intensive discussions among partners and was made available to all partners during the lifetime of the project via the restricted access area of the project website. DD testing was performed on Iso-sensitest Agar (ISA) to minimize interactions between medium components and antibiotic gradients as much as possible. In order to increase the relevance of the outcome of the harmonization study with regard to the recommendations of Alderman & Smith on standard methods for susceptibility testing of bacteria associated with fish diseases [Aquaculture 196:211-243 (2001)], a panel of reference strains was designed as follows: Aeromonas hydrophila subsp. hydrophila, Stenotrophomonas maltophilia, Salmonella enteritidis, Escherichia coli, Acinetobacter junii, Aeromonas salmonicida subsp. salmonicida, Yersinia ruckeri, Vibrio alginolyticus, Vibrio anguillarum, and Photobacterium damsela subsp. piscicida. These strains were stored using the cryobeads system in each partner country and, following harmonization testing in each partner lab, a strain verification including purity and recovery check and control of the taxonomic authenticity of the strains by fatty acid analysis was performed. The value of this strain verification control was highlighted by the fact that some subcultures yielded poor recovery or lacked purity, upon which new subcultures were requested from the laboratories concerned. The analysis of the final data set was based on the determination of the arithmetic mean and its standard deviation for each of the 60 possible strain-disk combinations [i.e. 10 control strains tested for 6 antibiotic disks: ampicillin (25 ug), tetracycline (30 ug), oxytetracycline (30 ug), oxolinic acid (2 ug), streptomycin (25 ug), and chloramphenicol (30 ug)]. Ten repeated determinations by the five partners delivered 50 data points per combination, or a total of 300 data sets (i.e. 60 combinations tested in five labs). Overall, the intralaboratory performance of SOP-based DD testing was highly satisfactory as evidenced by a standard deviation (SD) range of 0-2 mm when 79% of all recorded data sets were considered. It was agreed that this range can be defined as the tolerance limit that is acceptable for DD susceptibility testing of the chloramphenicol resistant isolates in the course of the project. In these determinations, E. coli strain LMG 8223 (= ATCC 25922 = NCIMB 12210) was included as a control strain in each analysis batch. In 8% of the data sets, the SD value exceeded 4 mm which in most cases could be attributed to the fact that the data points for a given strain-disk combination were not normally distributed in one of the labs. As could be expected, the SD range at the interlaboratory level was somewhat broader ranging from 0-5 mm for 81% of the SD values based on global averaging of 50 data points per strain-disk combination. This SOP thus provides a validated guide to conduct susceptibility studies at an inter-laboratory level. The results of the harmonization study were integrated in a joint manuscript for the peer-reviewed journal Diseases of Aquatic Organisms 66:197-204 (2005).

6 antibiotic discs from Oxoid was selected as follow: tetracycline, ampicilin, chloramphenicol, nitrofurantoin, norfoxacin and trimethoprim to do the sensitivity test of bacterial samples isolated from shrimp and fish farms in Thailand. Reference strain harmonisation set were - ASIARESIST strain C256-V-OM-A-4-O-1, - Eschericia coli LMG 8223, - Aeromonas hydrophila LMG 2844, - Stenotrophomonas maltophilia LMG 11098, - Acinetobacter junii LMG 10577, - Salmonella enteritidis LMG 10395. After searched on more literatures the second harmonisation set of bacteria for sensitivity test was added as follow: Photobacterium damsella LMG 7892, Vibrio mediterranei LMG 11258, Vibrio alginolyticus LMG 4409, Aeromonas salmonicida LMG 3780, Yersinia ruckeri LMG 3280. Most of the isolates from Thailand show multi-resistance to the antibiotics tested.

Pulsed-field gel electrophoresis of macrorestriction fragments was applied to confirm the clonal relationships of aquaculture-associated genera previously inferred by (GTG)5-PCR typing. Cell lysis, restriction enzymes and electrophoresis running conditions were optimised for each species tested. The majority of isolates yielded suitable profiles for macrorestriction analysis; however, a proportion of the isolates could not be processed due to DNA degradation. Numerical analysis of the resulting pulsotypes, revealed significant agreement between the clonal strain types identified by (GTG)5-PCR typing and macrorestriction analysis. This confirmed the presence of replicates (identical strains occurring within the same sample) and was able to track strains within the aquaculture environment (clonal strain types occurring between different samples) over time, between distinct niches (water, sediment and cultured organism), ponds and even between farm sites. Macrorestriction analysis was found to be a highly discriminatory genotyping method, in detecting the clonal variation present within a strain collection.

A Gel-Compar-based database (Applied Maths, Belgium) was set up to contain descriptive data of the strains in the ASIARESIST strain collection as well as the experimental identification and typing data. These descriptive data include the full strain code of each isolate, the shortened strain reference code and colonial morphology of each isolate. In the course of the project, experimental data were added to the database from the identification and typing work packages. All ASIARESIST isolates received in Stirling (n= 650) were entered into a strain collection maintained within the Bacteriology Department of the Institute of Aquaculture and processed by (GTG)5-PCR mediating typing. A sub-set of isolates that appeared indistinguishable or highly similar by (GTG)5-PCR typing were also selected for macrorestriction analysis. The Gel-Compar database contains the stored DNA patterns of these two typing techniques as well as plasmid profiles of selected Vietnam isolates. The database allows individual strains to be compared based on the combined DNA fingerprinting attributes and was applied to de-replicate the strain collection and track strains within the aquaculture environment.

Antibiotic susceptibility testing of aquaculture-associated bacteria with the disc diffusion method. The abuse of antimicrobials can result in the development of resistant strains of bacteria in aquaculture systems, which ultimately affect the cultured species, human and environment. Several of these aspects have been well documented by researchers worldwide. Disk diffusion method could be described as very simple, most effective and informative for antimicrobial testing. The present study showed that chloramphenicol (C) is used in the aquaculture farms in Malaysia. The major problem with chloramphenicol is that the bacterial pathogens easily develop plasmid-mediated resistance to it and may also enhance the frequency of new chloramphenicol resistant bacteria in culture system. In aquaculture, management practices such as high stocking densities, chemical treatment, grain based artificial diet and accumulation of organic wastes in the form of excess feed, excreta and dead plankton have residual influence on the antibiotic resistance of aquatic bacteria. Nevertheless, proper water exchange, monitoring and management by controlled use of drugs could lower the risks of antibiotic resistant bacteria. Additionally, the results from MIC (Minimum Inhibitory Concentration) testing carried out in this study indicated that many samples were resistant to a high concentration of chloramphenicol (> 512 ppm). Both methods (disk diffusion and MIC testings) denote the occurrence of multi-resistance bacteria strains. Hence, disk diffusion is a reliable method and easy tool for monitoring the prevalence of resistant or multi-resistant aquaculture-associated bacteria strains.

A multiplex PCR assay was evaluated for the PCR detection of cat1-3 genes (Yoo et al., 2003) among the chloramphenicol resistant strain collection. Primers specific to the three distinct cat genes are combined with a single conserved primer. The individual cat genes are distinguished by virtue of size differences in the PCR products. The presence of cat1 and cat2 PCR products was confirmed by sequencing. No cat3 genes were detected among the strain collection. In the rare instances where more than one cat gene was present within an isolate, the application of a single specific primer was found to be beneficial for clarification purposes. The method is recommended as a screening method for the detection of cat1-3 genes.

Resistance gene detection and horizontal transfer. To increase the rapidity and accuracy of resistance testing, the use of PCR (a genotypic approach and DNA-based assay) for the detection of bacterial resistance was developed. This novel approach is a true revolution because it relies on a completely different concept; testing for resistance instead of testing for susceptibility. In our study, detection of the 136 chloramphenicol resistant isolates (32 for Cat I, 101 for Cat II and 3 for both Cat I and Cat II) using PCR targeting the Cat genes (genes responsible for chloramphenicol resistance) showed that PCR is a rapid, robust, cost-effective and sensitive method. Prior to amplification, crude genomic DNA of isolates was harvested using boiling method, which is a simple, safe and rapid way of DNA extraction. Biochemical Cat assay were carried out to analyze whether the mechanism of chloramphenicol resistant bacteria occurred by enzymatic or non-enzymatic resistance. The isolates, which gave negative results for Cat genes detection, were analyzed for biochemical Cat assay. Resistance to antibiotics is usually a transferable factor and is important to the growth and spread of pathogenic bacteria. Out of 136 positive Cat genes isolates, only 56 were considered as potential donors because of their sensitivity to streptomycin, giving an inhibition zone of more than 6 mm. Antibiotic susceptibility testing showed that the donor transferred sulphonamide (S3 100) and chloramphenicol (C30) resistant genes to recipient and appears as transconjugant. This is evidence that chloramphenicol resistant bacteria detectable in aquaculture do participate in the molecular communication between bacteria of the aquaculture system. The emergence of drug resistance in microorganisms is a serious problem, and several strategies have been proposed to try to overcome it. Prevention should be the ultimate solution, and vaccines have been suggested as a strategy that can be used to slow down the emergence of drug resistance by decreasing the infection rate and hence antibiotic usage. Broad surveillance programs and education of clinicians, pharmacists, veterinarians, drug company representatives, and the public about the spread of antimicrobial resistance and the consequences of antibiotic misuse should also have a significant impact. Restrictive use of newer and broad-spectrum antibiotics has also been applied and advocated. Strict application of therapeutic guidelines might also be useful. Another strategy is the simultaneous rapid genotypic identification of bacteria and their antibiotic resistance genes will have a major impact on the treatment of infectious diseases while contributing to a better control of antimicrobial resistance.

Reference strains for MIC work were - ASIARESIST Strain C256-V-OM-A-4-O-1, - Eschericia coli LMG 8223, - Aeromonas hydrophila LMG 28444, - Stenotrophomonas maltophilia LGM 11092, - Acinetobactor junii LGM 10577, and - Salmonella enteritidis LGM 10395. The results on MIC show that many resistance strains of Thai bacterial samples that collected from shrimp and fish farms were resisted to a very high concentration of chloramphenicol (> 512 ppm). Therefore the concentrations of chloramphenicol was increased to find the MIC end point of that particular resistance isolates. The results showed that the highest concentration of MIC of chloramphenicol was 2,048 ppm.

A Standard Operating Procedure (SOP) was developed for determination of Minimum Inhibitory Concentrations (MICs) for a number of antibiotics with reference to the antibacterial agents tested in Southeast-Asian aquaculture practices, i.e. SOP-ASIARESIST-MIC. The choice of these agents depends on the outcome of disc diffusion testing and can include one or more of the following agents: tetracycline, ampicillin, chloramphenicol, nitrofurantoin, norfloxacin, and trimethoprim/ sulfamethoxazole 1:19 (Co-trimoxazole). The SOP document is based on the use of (micro)broth dilution methodology. The final version of the SOP was obtained following intensive discussions among partners and was made available to all partners during the lifetime of the project via the restricted access area of the project website.

Five fish species representing the diversity of important species cultured in the Mekong river delta, Viet Nam including Pangasius catfish (Pangasius hypophthalmus), Tilapia (Oreochromis niloticus), Common carp (Cyprinus carpio), Snakeskin gouramy (Strichogaster pectoralis) and giant gouramy (Osphronemuss goramy) were sampled for chloramphenicol (CHL) resistance bacteria during the period between June to December, 2002. A total of 240 CHL resistant isolates and 50 CHL susceptible bacterial isolates were collected and preserved in cryopreservation system. Our collection includes 10 highly CHL resistant strains which growth was observed at 256 ppm CHL. These isolates are stores in cryopreservation at our lab and at the Microbiological reference laboratory, Gent University, Belgium. The isolates have been subjected for a series of studies including taxonomy, susceptibility testing to antimicrobials used in aquaculture, epidemiological relationship amongst the collection of bacteria and the potential of genetic exchange between resistant and susceptible isolates. The generating data will be used to define strategic approaches to monitoring antibiotic resistance on fish farms in the Delta.

The information on MIC from SEA partners showed that the heterotrophic bacterial population in aquaculture sources comprised was both susceptible and resistant to CHL. Overall; the three country sub collections of CHL-resistant bacteria were similar. The majority of CHL-resistant isolates (82.6%) showed a MIC =>512 ppm. They also exhibited multi-resistance to CHL and ampicillin (AM), tetracycline (TE), nitrofurantoin (F/M) and trimethoprim/sulfamethoxazole (SXT) at various frequencies, but were relatively susceptible to norfloxacin (only 28% being resistant to this antibiotic). The most common profiles were of resistance to 3 and 4 antibiotics (14% and 15% of tested isolates, respectively), the commonest resistance profiles being CHL-AM-TE-SXT and CHL-AM-TE-SXT-F/M. Resistance to CHL alone was rare (3%). (Annex VI) Moreover, there was a tendency for CHL-resistant isolates with higher MIC values to be resistant to greater numbers of antibiotics than those with lower MICs. Similarly, bacterial isolates from integrated farms showed resistance to more antibiotics than those obtained from monoculture farms (99% confidence interval).

A mini-prep plasmid extraction protocol was optimised for varied genera encountered within the aquaculture environment based on the alkaline lysis method. The modifications allowed for the extraction of high molecular weight plasmid DNA (> 100 kb) and avoided the use of commercial purification columns and hazardous solvents. As such the method is ideal as a cost-effective, simple method to screen for plasmid DNA. Although the protocol was originally designed for plasmid profiling chloramphenicol resistant bacteria from SE Asian aquaculture sites, the method can be adapted for other bacterial groups using different bacterial growth conditions. Troubleshooting notes are incorporated into the method as well as recommended agarose gel electrophoresis conditions for the separation of the plasmid DNA samples.

A plate-based conjugation assay was optimised using a known donor isolate, E. coli RC85 (chloramphenicol resistant) and a known recipient E. coli isolate HB101 (streptomycin resistant). Chloramphenicol resistant project isolates shown to be susceptible (> 6 mm zone size) to streptomycin by disc diffusion could be included as donor (test) isolates with the recipient isolate E. coli isolate HB101. Transconjugants were confirmed by a series of phenotypic (appearance on MacConkey agar, antibiotic resistance profiling) and genotypic tests (plasmid content and rep-PCR typing) to exclude the possibility of mutant donors or recipients. Transconjugants typically originated from donors belonging to the family Enterobacteriaceae and involved the transfer of resistance of sulphonamide, tetracycline as well as chloramphenicol. Plasmid profiling of the transconjugants revealed that large plasmids (> 100 kb) were commonly associated with the transfer of resistance to the recipient. The conjugation assay was shown to be useful in the screening of resistance gene transfer occurring within Enterobacteriaceae isolates.

A biochemical CAT assay was optimised for the detection of CAT activity from aquaculture-associated genera. The method was adapted to a microtitre plate format to minimise cost. A change in colour from colourless to bright yellow indicated CAT activity. A strategy to confirm suspected CAT negative reactions was proposed. The assay was used to screen for the presence of CAT activity in those project isolates without known cat1 or cat2 genes.

A Standard Operating Procedure (SOP) document was defined for the preservation of isolates that were obtained in the course of the project. The use of a commercial cryobeads system was proposed to store strains for a prolonged period as defined in SOP-ASIARESIST-PRES. The final version of the SOP was obtained following intensive discussions among partners and was made available to all partners during the lifetime of the project via the restricted access area of the project website. To validate this SOP, two harmonization strain sets were send to all partners that participated in the sampling and sample processing activities. The partners were asked to - subculture the strain set, - prepare cryovials for long-term preservation, and - send subcultures recovered from cryopreservation to Partner 2. The strains in the first set consisted of organisms that represented different bacterial groups known to be (pre)dominant in aquaculture environments i.e. Aeromonas hydrophila, Vibrio harveyi, Stenotrophomonas maltophilia, Salmonella enteritidis, Escherichia coli and Acinetobacter junii. A second harmonization strain set was send to represent organisms that are considered to be involved in aquaculture infections including Aeromonas salmonicida, Photobacterium damsela, Yersinia ruckeri, Vibrio mediterranei and Vibrio alginolyticus. These control strains were stored using the cryobeads system in each partner country and subsequently checked for recovery and send back to Ghent University for confirmation of the taxonomic identity with fatty acid analysis. No major inconsistencies were noted in this regard, thus indicating that the document is a valuable guide towards the global preservation of bacteriological material in geographically separated labs.

Isolation of chloramphenicol resistant bacteria from aquaculture farms in Malaysia. Antimicrobial agents have been widely used in aquaculture worldwide to treat infections caused by a variety of bacterial pathogens. This study undertaken is the evidence of the occurrence of antibiotic-resistant bacteria specifically chloramphenicol resistant bacteria in the local aquaculture ecosystem. Sampling for the chloramphenicol resistant bacteria was conducted at three farms, two of which were fish farms (Tilapia integrated with Chicken Farm and Big Head Carp Farm) in the State of Perak and one shrimp farm (Banting Shrimp Farm) in the State of Selangor. The farms were selected for sampling after investigations revealed higher than 35 ppm chloramphenicol resistant isolates. At each site, sampling was performed four times at 2 weeks interval. During each sampling the following data were recorded, depth at which water and sediment samples were taken, physico-chemical parameters of the water (including temperature, pH, salinity, total ammonia nitrogen (N-NH4) and total suspension solid). The sediment samples were described according to the soil type (clay, sandy, loam, mixed). As a result, a total of 166 chloramphenicol-resistant strains were successfully isolated according to the SOP guidelines. This resistance can disseminate through the environment and can be transmitted to a variety of bacterial species, including bacteria that can infect humans. Bacteria, present in aquaculture settings may be transmitted to humans who come in contact with this ecosystem. Hence, alternative steps could be introduced, such as probiotics, vaccines and good aquaculture practices to control bacterial infections as this option could prove to be useful in our aquaculture since the prolonged use of antibiotics may result in selection of antibiotic-resistant bacteria.

A central backup collection of all isolates collected in this project was established that is accessible to all partners of the project. The principle of storage is based on cryopreservation using the SOP-ASIARESIST-PRES document. The final strain collection consists of 724 chloramphenicol-resistant (CHLR) isolates (C isolates) and 102 chloramphenicol-susceptible isolates (N isolates). Upon receipt, all isolates were checked for purity and single colony morphology prior to cryopreservation. A number of cultures could not be recovered or purified upon arrival and were resent by the involved partners. According to colony morphology, a number of isolates displayed the typical morphology of yeasts. This was further checked by microscopic analysis of cell morphology and determination of resistance to the eukaryotic inhibitor cycloheximide (0.02%). Likewise the isolates that failed to recover after repeated subculturing, isolates that were classified as yeasts, were discarded from the strain collection. At regular intervals, subsets of checked isolates were sent to University of Stirling (UK) for strain typing. After the end of the project, a representative set of C isolates will be deposited in the public BCCM/LMG Bacteria Collection (http://bccm.belspo.be/db/bacteria_search.htm).

The project database has been designed to host all project data and it is accessible by all partners from the page 'Data Base' of the restricted access project web site. It is protected by a different password from the one necessary to enter the web site, resulting in two passwords required to access the DB. The general structure of the project DB proposed is a relational one. It is based on the main entity strain, which can be connected with many other entities containing various features of the strain itself. Data insertion and queries on this database have been standardised with a consequent creation of a standard data interchange format based on a common protocol for data sharing and a standardised user interface. The implementation has been performed in Microsoft SQL-Sever 2000 and is recorded at the Medinfo server at Department of Communication Computer and System Science - University of Genova. All the information described in the enclosed entity-relation diagram (in the document section), is stored in the relational database, together with the strain code. In order to avoid excessive manipulation of data, it was decided by all partners that each partner has the right of inserting data only into the database related to his/her country but once inserted, they can only be deleted by the database administrators. To implement this and to avoid external access to data, the first page of the web site is a login page asking for a USER ID and a PASSWORD. The user is allowed to enter only if the inserted data matches with the ones in the USER table of the database. This method allows the server to recognise a user and to give him/her certain rights over data, for example the administrator can access data deletion pages, while the other users can only access data insertion pages with country restrictions. At the first release the User database contained only the responsible of each country, so that they were the only authorised persons to access the database. Afterwards they authorised other persons of their own staff, but only for manipulating data related to their country. Each bacterial strain stored in the DB is given a code, on the basis of data insertion and according to a Standard Operating Procedure for sampling agreed among all partners. The strain code contains information about: - Resistance / Non resistance to a certain concentration of chloramphenicol antibiotic - Country of origin - Sampling Site - Fish farm pond - Sampling period - Sample type - Colony number

The project web site has been structured into two parts: one part open to general public and one part whose access have been restricted to partners only during the project. After the publication of the results of the project some parts of the restricted area site will be open to scientific community to share our project experience. A further site has been added in the last year of the project to announce to all interested people the Thailand Workshop. The project WEB site (www.dist.unige.it/asiaresist) was developed in the first months of the project and after partners' comments and revision, the first release was put on the DIST server in April 2002 but it has been constantly updated during the project life. The present version of the public web site contains the following sections: - Home page: information about the EU contract, program and project period; - Background: general description of the background of the project; - Objectives: description of project objectives; - Outputs: description of project outputs; - Activities: description of project activities; - Work plan: description project work plan with direct links to two more pages, WP Time table and DB download; - Partners: description of the project consortium, logo, name and address of each partner, with links to each partner specific www address. Two pages are connected to this page: - Partner Photo Gallery: photo gallery of pictures taken at project meetings; - Farm Photo Gallery: photo gallery of pictures taken at some of the farm sites from where samples have been collected; - Symposia: collection of info relating to conferences in the field - Publications: page dealing with the publications within the project - Workshop: page dealing with the final workshop announcement, linked to the specific workshop web site (described below). From this page it is possible to download the workshop proceedings. A restricted access project WEB site was designed and put on the DIST server (address www.dist.unige.it/asiaresist1). It was aimed for restricted internal discussions between partners, and it contains 9 pages structured in the same way as the public part. The main pages are: - Home page - Meetings: a list of the project meetings - DOCs: document download page - Forum: bulletin board for electronic discussion among partners - Data Base Login: access page to the database described in another section of TIP. The main section of this part is the discussion forum. From the Forum main page the user can have two possibilities: to join a conversation already started or to start a new conversation by clicking on the 'new thread' button. There is also the possibility to add a file from its own PC that will be added to the message and sent to the Forum hosting server by clicking on the 'post' button. Many threads and messages indicate the wide use of Forum in this project. A web site open to public and linked to the project public web site through its page 'workshop' was constructed specifically for the final project workshop dissemination (www.dist.unige.it/asiaresist/workshop). It consists of 13 pages, specifically: - Home page: information about the ASIARESIST project; - Topics: list of topics considered in the workshop; - Committee: list of members of the workshop scientific committee; - Registration: workshop registration fee and modalities for the registration on-line; - Contact: main contact for additional information about the workshop; - Accommodation: information and link to the hotel of the workshop; - Thailand: info about the country - Chiang Mai: info about the workshop venue - Map: info about the workshop venue - Deadlines: info about the registration and paper submission deadlines - Paper submission: author instruction and modalities for on line paper submission; - Workshop programme: info about the workshop programme - Workshop output: download page for Workshop Presentations

A total of 196 chloramphenicol (CHL) resistant bacterial isolates originating from aquaculture farms in the Mekong river Delta of Viet nam was tested against six antimicrobials used in aquaculture, agriculture and human medicine. Results of antibiotic susceptibility testing by the disk diffusion method showed that resistance to CHL alone was relatively rare (2%). The majority of isolates tested (59%) exhibited multi-resistance to three or four antibiotics in addition to CHL. The most common profiles (34%) included resistance to CHL, ampicillin, tetracyline, trimethoprim/sulfamethoxazole with/or without nitrofurantoin. In contrast, the CHL-resistant isolates (29%) were found to be relatively more susceptible to norfloxacin.

A total of 558 chloramphenicol (CHL) resistant bacterial isolates originating from aquaculture farms in Thailand, Malaysia and Vietnam were tested against 6 antimicrobials used in aquaculture, agriculture and human medicine. Results of antibiotic susceptibility testing by the disk diffusion method showed that resistance to CHL alone was relatively rare (4%). The majority of isolates tested (59%) exhibited multi-resistance to three or four antibiotics in addition to CHL and this finding was common to all the countries and sample types including water, sediment and cultured organisms. A common component of the most dominant profiles (42%) was resistance to CHL, ampicillin, tetracycline with/or without resistance to trimethoprim/sulfamethoxazole. In contrast, the CHL-resistant isolates (28%) were found to be relatively more susceptible to norfloxacin. There is evidence that Gram-negative bacteria displayed a significant difference on CHL resistance to that of Gram positive and the same pattern was found between families of Gram-negative. The minimum inhibitory concentrations (MICs) for CHL were also determined for the resistant isolates using a broth macrodilution method. Eighty three percent of the resistant isolates showed high CHL MIC ³ 512 ppm. There was a tendency for MIC to increase in the more multi-resistant isolates (resistant to CHL + 3 - 5 antibiotics). This finding is also true to CHL resistant isolates belong to Gram-negative bacteria. Distribution of MICs values was significant difference between Gram-negative and Gram-positive isolates as well as between families of Gram-negative. However, there are no statistical differences of MICs values between Gram-positive families.

A Standard Operating Procedure (SOP) was defined for sampling and sample processing of aquaculture environments (SOP-ASIARESIST-SAMPLE). This SOP was developed based on the decision to examine chloramphenicol (CHL) resistance. The final version of the SOP was made available to all partners via the project website. A critical step in this matter concerned the decision on the breakpoint concentration, i.e. the concentration CHL to be added to the isolation medium for selection of acquired resistances rather than low-level intrinsic resistances. Following experimental work by SEA partners and taking into account literature data, it was decided to set the breakpoint concentration at 35 ppm. A consensus version of SOP-ASIARESIST-SAMPLE was formulated in which clear indications are given on the number of sampling points that need to be identified in Thailand, Malaysia, and Vietnam, the choice of the sampling equipment for water, sediment, and organism samples, the number of isolates that should be generated per site and per country, and the strain designation code to be used as a key to enter data in the project database.

The minimal inhibitory concentration (MIC) for CHL was also measured for 196 chloramphenicol (CHL) resistant bacterial isolates originating from aquaculture farms in the Mekong river Delta of Vietnam using a broth macrodilution method. Ninety-one percent of the resistant isolates were shown to possess a MIC to CHL between 512 to 1.024 and > 1.024 ppm. There is a statistical evidence (p < 0.05) on the fact that strains with few resistance (1-2) has lowest MIC values than strains with many resistances (3-6).

At the end of the project, the data contained into the DB are summarised in the following tables. There are 826 strains in all of which 623 are confirmed. 166 come from Malaysia, 360 from Thailand, and 300 from Vietnam. Of the confirmed strains, 558 are Chloramphenicol resistant, and 65 are Chloramphenicol sensible. A first data analysis through the Project Central Data Base was performed during the writing of the paper entitled "Intra- and inter-laboratory performance of antibiotic disk diffusion susceptibility testing of bacterial control strains of relevance for monitoring aquaculture environments". In order to single out sets of raw data in which some error occur, the data analysis was performed, and the reply was yes: - we have calculated for each group of raw data on a single strain and antibiotic average and standard deviation; - we have applied the Gaussian model with these parameters; - we have calculated the Euclidean distance of the real data from the estimations obtained with this model; - when this distance was higher than an optimized binarizing threshold (Otsu method) the raw data can be considered as non normally distributed (that is with sum type of errors inside); - for normally distributed sets we calculate the confidence interval for the average of the population with the t-student test; - for non normally distributed sets we calculated the confidence interval for the average of the population with Chebyshev theorem; After that, we calculated for each couple strain-antibiotic the group of statistically similar set: - when all involved sets are normal we can use the one factor -fixed effects ANOVA test combined with the multiple range test of Duncan; - when some sets (even only one) are not normal we can use Kruskal-Wallis test for independent samples; Different types of data analysis were performed by each partner using the search engine and our statistical elaboration, these results are reported in their respective final reports.

A reliable identification programme was carried out for the taxonomic characterization of C isolates collected during the project. Replicate or clonally related isolates were first identified by DNA fingerprinting using repetitive chromosomal element-PCR analysis (rep-PCR). On the basis of the relatedness of the profiles obtained, 92 - possible duplicate - isolates were identified and not considered further. Following dereplication, a first step in the identification strategy included gas-liquid chromatographic analysis of cellular fatty acid methyl esters (FAMEs). The extraction and analysis of FAMEs was performed according to the standardized protocol of the Sherlock Identification System (Microbial ID Inc., Newark, DE, USA). For identification, FAME profiles of unknown isolates were compared with the general TSBA database (MIDI) for heterotrophs. Depending on the taxonomic diversity of the strain collection, second choice techniques such as API, AFLP fingerprinting and 16S rDNA sequencing were included for species identifications of specific bacterial groups. FAME analysis allowed to classify all 558 investigated isolates at least at the bacterial family level. Distribution analysis of the identification results revealed that the majority of the C isolates belong to six bacterial families i.e. the Enterobacteriaceae (n=350; 63%) followed by members of the Pseudomonadaceae (n=67; 12%), Moraxellaceae (n=62; 11%), Bacillaceae (n=33; 6%), Micrococcaceae (n=19; 3%) and Aeromonadaceae (n=11; 2%). A total of 15 C isolates belonged to other families i.e. Alcaligenaceae, Comamonadaceae, Enterococcaceae, Flavobacteriaceae and Sphingobacteriaceae. One isolate belonging to the g-Proteobacteria could not be unequivocally placed in a specific bacterial family. For the first time, a comprehensive view was generated on the taxonomic diversity of CHL-resistant heterotrophs in Southeast Asian aquaculture environments.

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