Microorganisms in ballast water: Disinfection, community dynamics, and implications for management.

Increasing concerns have accelerated the development of international regulations and methods for ballast water management to limit the introduction of non-indigenous species. The transport of microorganisms with ballast water has received scientific attention in recent years. However, few studies have focused on the importance of organisms smaller than 10 µm in diameter. In this work, we review the effects of ballast water transport, disinfection, and the release of microorganisms on ecosystem processes with a special focus on heterotrophic bacteria. It is important to evaluate both direct and indirect effects of ballast water treatment systems, such as the generation of easily degradable substrates and the subsequent regrowth of heterotrophic microorganisms in ballast tanks. Disinfection of water can alter the composition of bacterial communities through selective recolonization in the ballast water or the recipient water, and thereby affects bacterial driven functions that are important for the marine food web. Dissolved organic matter quality and quantity and the ecosystem status of the treated water can also be affected by the disinfection method used. These side effects of disinfection should be further investigated in a broader context and in different scales (laboratory studies, large-scale facilities, and on the ships).

A multiplex polymerase chain reaction assay for genus-, group- and species-specific detection of mycobacteria.

We developed and evaluated a single-step, multiplex polymerase chain reaction (PCR) assay for distinguishing (1) between the Mycobacterium tuberculosis complex (MTBC) and mycobacteria other than tuberculosis (MOTT) and (2) between M. tuberculosis and M. bovis species. The assay targeted the 16S and the 23S rDNA to distinguish between MTBC and MOTT species, and the oxyR gene to distinguish between M. tuberculosis and M. bovis strains. Clinical samples and reference strains (N = 156) comprised 93 strains of M. tuberculosis, 44 of M. bovis, 1 M. africanum strain, and 18 strains representing 9 different species of MOTTs. MOTTs generated only a single PCR product of about 2.5 kilobase; however, all of the MTBC strains produced a 118 base pair (bp) fragment and an additional 270 bp fragment was obtained for M. tuberculosis and M. africanum when the primer pair oxyRTB-2.1/oxyRMT-1 was used. When oxyRTB-2.1/oxyRMB-1 primers were used, the 270 bp fragment was obtained for only M. bovis. The assay needed as little as 1 pg of purified genomic DNA to make a positive identification.

Amplified ribosomal DNA restriction analysis in the differentiation of related species of mycobacteria.

This study explores the potential of the amplified ribosomal DNA restriction analysis (ARDRA) for intra- and interspecies identification of the genus Mycobacteria. A set of primers was used to amplify part of the 16S and 23S rDNA as well as the 16S-23S rDNA spacer from 121 isolates belonging to 13 different mycobacterial species. Restriction analysis was carried out with five different restriction enzymes, namely CfoI, HaeIII, RsaI, MspI and TaqI. Restriction digestion of the PCR product using CfoI enabled differentiation between 9 of the 13 mycobacterial species, whereas the remaining four enzymes differentiated between 7 of these 13 species. None of the five enzymes distinguished between different isolates of Mycobacterium tuberculosis or between species within the M. tuberculosis complex i.e., M. tuberculosis, M. bovis, M. bovis BCG and M. africanum. Although ARDRA analysis of the 16S-23S rDNA does not seem to have a potential for intraspecies differentiation, it has proven to be a rapid and technically relatively simple method to recognise strains belonging to the M. tuberculosis complex as well as to identify mycobacterial species outside this complex.

Sequence analysis in the 23S rDNA region of Mycobacterium tuberculosis and related species.

We sequenced a 516 base pair segment in the 23S rRNA gene of 54 Mycobacterium tuberculosis isolates, 52 of which were clinical isolates from Ethiopia. Sequence polymorphism was observed with 19 of the 54 strains; the polymorphic sites occurred in less than 2% (9/516) of the total sequence positions. The sequence variations represented base pair substitutions (14/23), insertions (9/23) or both (1/23). Insertions occurred at one site only, whereas substitutions were observed in various regions of the gene. There was no relation between mutational sites and drug susceptibility. However, using information from the GenBank database, comparison between the 23S rDNA sequences of M. tuberculosis and the corresponding sequences of other mycobacteria and of related non-mycobacterial species revealed considerable variation, suggesting that this region may provide a target for rapid detection and identification of mycobacteria both at the genus or species level.

Detection of infectious salmon anemia virus in sea water by nested RT-PCR.

A method to detect low levels of infectious salmon anemia virus (ISAV) in environmental samples has been developed. The method is based on concentrating the viruses by tangential flow filtration and ultracentrifugation prior to amplification of the extracted viral RNA by nested reverse transcription polymerase chain reaction (RT-PCR). In the current investigation, seawater samples from salmon holdings were used for ISAV identification. ISAV was detected in seawater samples from a salmon holding site and from a vessel transporting salmon in 2 consecutive trials of the methodology. When known concentrations of ISAV were added to 21 of sea water, 5.5 viruses ml(-1) (corresponding to a tissue culture titer of TCID50 1.6 x 10(-2) ml(-1)) were detected using this method.

Use of PCR-RFLP for genotyping 16S rRNA and characterizing bacteria cultured from halibut fry.

Small subunit ribosomal genes were explored using PCR-RFLP to facilitate the characterization of bacteria cultured from reared fry of the Atlantic halibut (Hippoglossus hippoglossus). Concern has been expressed about pathogen invasion in larvae lacking a counteracting normal flora that may aid the immune system in producing robust noninfected individuals. In this study, pure cultured representatives of normal flora that were previously found to be antagonistic towards a pathogenic Vibrio sp. were subjected to a whole cell PCR protocol amplifying approximately 1500 bp of 16S rDNA. Amplified DNA was digested by AluI, BstUI, CfoI, and RsaI, to generate restriction profiles. Before the isolates were characterized, a survey was performed to test the discriminative efficiency of the RFLP. Efficient detection of polymorphism and the resolution of species and subspecies were achieved. Using the RFLP on 103 isolates generated as many as 22 genotypes. Based on the restriction profiles, a taxonomic tree incorporating 19 reference strains was constructed. Partial sequencing found this tree to be dominated by gamma-Proteobacteria in clusters of Vibrio-, Pseudomonas-, and Alteromonas-affiliated species. Only nine isolates fell outside these genera, including the three isolates Shewanella alga, Deleya marina, and Marinomonas protea. These species have not previously been reported as halibut flora. The most frequently isolated genotype resembled Vibrio salmonicida.