Monitoring and surveillance of antimicrobial resistance in microorganisms associated with aquatic animals.

The World Organisation for Animal Health (OIE) Aquatic Animal Health Code recommends that programmes forthe monitoring and surveillance of antimicrobial resistance in microorganisms associated with aquatic animals be initiated by the appropriate authorities. This paper discusses the classes of bacteria to be studied in such programmes and the methods of sample collection to be employed. It also discusses the susceptibility test protocols appropriate for use in such programmes, the interpretive criteria that should be applied to the data they generate and the form in which the output of such programmes should be reported. The authors argue that it is essential that all monitoring and surveillance programmes should employ standardised and internationally harmonised susceptibility test methods to the greatest extent possible. With respect to bacteria capable of infecting aquatic animals, it is recommended that the set of consensus-based standards and guidelines published by the Clinical and Laboratory Standards Institute be adopted as the basis for international harmonisation of test protocols, as they are significantly more developed than any alternatives. It is further recommended that, for the purpose of evaluating antimicrobial resistance trends, such as emerging resistance, the data generated by these protocols should be interpreted by the application of epidemiological cut-off values. However, as yet, internationally agreed cut-off values have been produced for only one species. Thus, for many species, authorities will be obliged to set their own local and laboratory-specific cut-off values. It is recommended that laboratories use a statistical and standardised method of establishing such local cut-off values. Internationally harmonised standard test protocols and interpretive criteria have, to a large extent, been developed to monitor antimicrobial resistance in bacterial species capable of infecting humans. These methods can also be applied to microorganisms capable of infecting humans that are isolated from aquatic animals reared for human consumption or for sale as companion animals.

Molecular typing of Vibrio parahaemolyticus strains isolated from the Philippines by PCR-based methods.

AIM: The main aim of the present study was to use three PCR-based techniques for the analysis of genetic variability among Vibrio parahaemolyticus strains isolated from the Philippines. METHODS AND RESULTS: Seventeen strains of V. parahaemolyticus isolated from shrimps (Penaeus monodon) and from the environments where these shrimps are being cultivated were analysed by random amplified polymorphic DNA PCR (RAPD-PCR), enterobacterial repetitive intergenic consensus sequence PCR (ERIC-PCR) and repetitive extragenic palindromic PCR (REP-PCR). The results of this work have demonstrated genetic variability within the V. parahaemolyticus strains that were isolated from the Philippines. In addition, RAPD, ERIC and REP-PCR are suitable rapid typing methods for V. parahaemolyticus. All three methods have good discriminative ability and can be used as a rapid means of comparing V. parahaemolyticus strains for epidemiological investigation. Based on the results of this study, we could say that REP-PCR is inferior to RAPD and ERIC-PCR owing to the fact that it is less reproducible. Moreover, the REP-PCR analysis yielded a relatively small number of products. This may suggests that the REP sequences may not be widely distributed in the V. parahaemolyticus genome. CONCLUSIONS: Genetic variability within V. parahaemolyticus strains isolated in the Philippines has been demonstrated. The presence of ERIC and REP sequences in the genome of this bacterial species was confirmed. SIGNIFICANCE AND IMPACT OF THE STUDY: The RAPD, ERIC and REP-PCR techniques are useful methods for molecular typing of V. parahaemolyticus strains. To our knowledge this is the first study of this kind carried out on V. parahaemolyticus strains isolated from the Philippines.

Occurrence, characterisation and detection of potential virulence determinants of emerging aquatic bacterial pathogens from the Philippines and Thailand.

Strains of Aeromonas spp., 'non-cholera vibrios' (NCVs) and Plesiomonas shigelloides isolated from aquatic environments, fish and human diarrhoeal cases in the Philippines and Thailand were characterised for potential virulence markers. Thus, the production of cytotoxin, cell-associated and cell-free haemolysin and their capacity to adhere to human intestinal (Henle 407) cells in vitro was investigated. In addition, the occurrence of tlh and tdh haemolysin genes and urease activity among V. parahaemolyticus strains was investigated. The results showed that strains recovered from clinical sources (human and fish) produced these virulence factors, whereas these are absent in environmental strains.

Occurrence, histopathology and experimental transmission of hepatopancreatic parvovirus infection in Penaeus monodon postlarvae.

Hepatopancreatic parvovirus (HPV) was detected in samples of Penaeus monodon post-larvae (PL-13, PL-18, PL-19, PL-26) from 2 hatcheries in 2 provinces (Samar and Iloilo) in the Philippines. The percentage of infection was 20 to 100% in postlarvae obtained from the hatchery in Samar in August 2001. Postlarvae from the hatchery in Iloilo, sampled in October and November 2001, had 70 to 99% HPV infection. Wet mounts of squashed hepatopancreatic tissue stained with malachite green (wet-mount technique) and histopathology revealed the presence of large, usually single, basophilic intranuclear inclusion bodies in the distal tubules, which led to displacement of the nucleoli. Light microscopy showed ovoid to spherical inclusion bodies, 5 to 11 mmicrom in diameter. Transmission electron microscopy revealed that the inclusion bodies were composed of electron-dense granular material and virions. The virions appeared roughly spherical and averaged 18 to 22 nm in diameter. An experiment was undertaken to induce HPV infection by feeding P. monodon postlarvae with virus-infected postlarvae. P. monodon postlarvae (PL-16), initially determined as free from HPV, were found HPV-positive 24 h after being fed with infected material. The percentage of infection ranged from 30% at Day 1 post-infection (p.i.) to 100% at Day 7 p.i. determined by the wet-mount technique and by histopathology. This is the first report of a successful horizontal transmission of HPV in P. monodon postlarvae.