Alicia Gibello

Multiplex PCR Assay for Detection of Bacterial Pathogens Associated with Warm-Water Streptococcosis in Fish

ABSTRACT A multiplex PCR-based method was designed for the simultaneous detection of the main pathogens involved in warm-water streptococcosis in fish (Streptococcus iniae, Streptococcus difficilis, Streptococcus parauberis, and Lactococcus garvieae). Each of the four pairs of oligonucleotide primers exclusively amplified the targeted gene of the specific microorganism. The sensitivity of the multiplex PCR using purified DNA was 25 pg for S. iniae, 12.5 pg for S. difficilis, 50 pg for S. parauberis, and 30 pg for L. garvieae. The multiplex PCR assay was useful for the specific detection of the four species of bacteria not only in pure culture but also in inoculated fish tissue homogenates and naturally infected fish. Therefore, this method could be a useful alternative to the culture-based method for the routine diagnosis of warm-water streptococcal infections in fish.

Winter disease outbreak in sea bream (Sparus aurata) associated with Pseudomonas anguilliseptica infection

Abstract The nature of an outbreak of ‘winter disease’ affecting both juvenile and adult sea-bream ( Sparus aurata ) in several Iberian Peninsula farms from January to April of 1996 is described. The average mortality rate was approximately 10–15%, although in some fish farms mortality reached 30%. Pure cultures of aerobic Gram-negative filamentous rods were isolated from the kidney of diseased fish, as well as from some of the liver and ascitic fluid samples. 16S rRNA gene sequence analysis identified the isolates from diseased fish as Pseudomonas anguilliseptica . The results of biochemical and physiological tests of clinical isolates and the type strain of P. anguilliseptica were entirely consistent with the genotypic identification. The isolation of P. anguilliseptica exclusively from the diseased fish of all affected fish farms, together with the failure to isolate this bacterium from non-affected fish of the same farms, suggest that P. anguilliseptica is likely to be the agent responsible for the ‘winter disease’ outbreak in the sea-bream examined. The relationship of the ‘winter disease’ outbreaks to stressful environmental conditions is discussed.

Catabolism of 3- and 4-hydroxyphenylacetic acid by Klebsiella pneumoniae

Klebsiella pneumoniae catabolizes both 4-hydroxyphenylacetic acid and 3-hydroxyphenylacetic acid via meta-cleavage of 3,4-dihydroxyphenylacetic acid, ultimately yielding pyruvate and succinate. The organism can synthesize two hydroxylases catalysing 3,4-dihydroxyphenylacetic acid formation, which differ in substrate specificity, cofactor requirement, kinetics and regulation. Five enzymes sequentially involved in the catabolism of 3,4-dihydroxyphenylacetic acid are encoded on a 7 kbp fragment of the K. pneumoniae chromosome that has been isolated in a recombinant plasmid.

Molecular cloning and analysis of the genes encoding the 4-hydroxyphenylacetate hydroxylase from Klebsiella pneumoniae

Abstract The Klebsiella pneumoniae genes encoding the hydroxylase involved in the meta-cleavage pathway of 4-hydroxyphenylacetic acid (4-HPA) were cloned, and the DNA fragment from the region essential for hydroxylase activity was sequenced. K. pneumoniae 4-HPA hydroxylase was composed of two proteins (HpaA and HpaH) with different molecular masses. HpaA seems to be a flavin-containing hydroxylase with a molecular mass of 58,781 Da. HpaH, with a molecular mass of 18,680 Da, seems to be a “helper” protein required for productive hydroxylation of the substrate. The hpa genes were expressed and the hydroxylase was active in Escherichia coli. Comparison of the enzyme with other monooxygenases indicates that K. pneumoniae 4-HPA hydroxylase is a member of a new family of hydroxylases.

The role of a groundwater bacterial community in the degradation of the herbicide terbuthylazine

A bacterial community in an aquifer contaminated by s-triazines was studied. Groundwater microcosms were treated with terbuthylazine at a concentration of 100 microg L(-1) and degradation of the herbicide was assessed. The bacterial community structure (abundance and phylogenetic composition) and function (carbon production and cell viability) were analysed. The bacterial community was able to degrade the terbuthylazine; in particular, Betaproteobacteria were involved in the herbicide biotransformation. Identification of some bacterial isolates by PCR amplification of the 16S rRNA gene revealed the presence of two Betaproteobacteria species able to degrade the herbicide: Advenella incenata and Janthinobacterium lividum. PCR detection of the genes encoding s-triazine-degrading enzymes indicated the presence of the atzA and atzB genes in A. incenata and the atzB and atzC genes in J. lividum. The nucleotide sequences of the PCR fragments of the atz genes from these strains were 100% identical to the homologous genes of the Pseudomonas sp. strain ADP. In conclusion, the results show the potential for the use of a natural attenuation strategy in the treatment of aquifers polluted with the terbuthylazine. The two bacteria isolated could facilitate the implementation of effective bioremediation protocols, especially in the case of the significant amounts of herbicide that can be found in groundwater as a result of accidental spills.

Propachlor Removal by Pseudomonas Strain GCH1 in an Immobilized-Cell System

ABSTRACT A bacterial strain capable of growing on propachlor (2-chloro-N-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genusPseudomonas. Washed-cell suspensions of strain GCH1 accumulated N-isopropylacetanilide, acetanilide, acetamide, and catechol. Pseudomonas strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 ± 0.15 μmol h−1. Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 ± 0.02 mg of protein μmol−1. GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.

Genome Sequence of Lactococcus garvieae 21881, Isolated in a Case of Human Septicemia

Lactococcus garvieae is a Gram-positive bacterium considered an important opportunistic emerging human pathogen and also a well-recognized fish pathogen. Here, we present the draft genome sequence of Lactococcus garvieae strain 21881 (2,164,557 bp, with a G+C content of 37.9%), which represents the first report of a genome sequence on Lactococcus garvieae.

Genome Sequence of Lactococcus garvieae 8831, Isolated from Rainbow Trout Lactococcosis Outbreaks in Spain

Lactococcus garvieae is the etiological agent of lactococcosis, one of the most important disease threats to the sustainability of the rainbow trout farming industry. Here, we present the draft genome sequence of Lactococcus garvieae strain 8831, isolated from diseased rainbow trout, which is composed of 2,087,276 bp with a G+C content of 38%.

A new fluorescent oligonucleotide probe for in situ detection of s-triazine-degrading Rhodococcus wratislaviensis in contaminated groundwater and soil samples.

A bacterial strain (FPA1) capable of using terbuthylazine, simazine, atrazine, 2-hydroxysimazine, deethylatrazine, isopropylamine or ethylamine as its sole carbon source was isolated from a shallow aquifer chronically contaminated with s-triazine herbicides. Based on its 16S rDNA sequence analysis, the strain FPA1 was identified as Rhodococcus wratislaviensis. The disappearance time of 50% of the initial terbuthylazine concentration in the presence of this strain (DT(50)) was 62days. This strain was also able to mineralise the [U-ring (14)C] triazine-ring, albeit at a slow rate. A 16S rRNA target oligonucleotide probe (RhLu) was designed, and the FISH protocol was optimised, in order to detect R. wratislaviensis in s-triazine-contaminated sites. The RhLu probe gave a positive signal (expressed as % of total DAPI-positive cells) in both the groundwater (2.19+/-0.41%) and soil (2.10+/-0.96%) samples analysed. Using the RhLu probe, R. wratislaviensis can be readily detected, and its population dynamics can be easily monitored, in soil and in water ecosystems contaminated with s-triazine. To the best of our knowledge, this is the first report showing the isolation, from groundwater, of a bacterial strain able to degrade s-triazines.

First identification of Streptococcus phocae isolated from atlantic salmon (Salmo salar)

Different genera and species of gram-positive, catalase-negative cocci are pathogenic to fish. The numbers of infections caused by these microorganisms have increased during the last decade, and such infections are responsible for significant economic losses in the fish farm industry ([9][1]).