Garvicins AG1 and AG2: Two Novel Class IId Bacteriocins of Lactococcus garvieae Lg-Granada.

Lactococcus garvieae causes infectious diseases in animals and is considered an emerging zoonotic pathogen involved in human clinical conditions. In silico analysis of plasmid pLG50 of L. garvieae Lg-Granada, an isolate from a patient with endocarditis, revealed the presence of two gene clusters (orf46-47 and orf48-49), each one encoding a novel putative bacteriocin, i.e., garvicin AG1 (GarAG1; orf46) and garvicin AG2 (GarAG2; orf48), and their corresponding immunity proteins (orf47 and orf49). The chemically synthesised bacteriocins GarAG1 and GarAG2 presented inhibitory activity against pathogenic L. garvieae strains, with AG2 also being active against Listeria monocytogenes, Listeria ivanovii and Enterococcus faecalis. Genetic organisation, amino acid sequences and antimicrobial activities of GarAG1 and GarAG2 indicate that they belong to linear non-pediocin-like one-peptide class IId bacteriocins. Gram-positive bacteria that were sensitive to GarAG2 were also able to ferment mannose, suggesting that this bacteriocin could use the mannose phosphotransferase transport system (Man-PTS) involved in mannose uptake as a receptor in sensitive strains. Intriguingly, GarAG1 and GarAG2 were highly active against their own host, L. garvieae Lg-Granada, which could be envisaged as a new strategy to combat pathogens via their own weapons.

Spleen and head kidney differential gene expression patterns in trout infected with Lactococcus garvieae correlate with spleen granulomas.

Lactococcus garvieae is a significant pathogen in aquaculture with a potential zoonotic risk. To begin to characterize the late immune response of trout to lactococcosis, we selected infected individuals showing clinical signs of lactococcosis. At the time lactococcosis clinical signs appeared, infection by L. garvieae induced a robust inflammatory response in the spleen of rainbow trout, which correlated with abundant granulomatous lesions. The response in kidney goes in parallel with that of spleen, and most of the gene regulations are similar in both organs. A correlation existed between the early inflammatory granulomas in spleen (containing macrophages with internalized L. garvieae) and up-regulated gene sets, which defined the presence of macrophages and neutrophils. This is the first analysis of the immune transcriptome of rainbow trout following L. garvieae infection during the initiation of adaptive immune mechanisms and shows a transcriptome induction of antibody response by both IgM (+) and IgT (+) spleen B cells to respond to systemic infection. These results increase our understanding of lactococcosis and pave the way for future research to improve control measures of lactococcosis on fish farms.

Garvicin A, a novel class IId bacteriocin from Lactococcus garvieae that inhibits septum formation in L. garvieae strains.

Lactococcus garvieae 21881, isolated in a human clinical case, produces a novel class IId bacteriocin, garvicin A (GarA), which is specifically active against other L. garvieae strains, including fish- and bovine-pathogenic isolates. Purification from active supernatants, sequence analyses, and plasmid-curing experiments identified pGL5, one of the five plasmids found in L. garvieae [M. Aguado-Urda et al., PLoS One 7(6):e40119, 2012], as the coding plasmid for the structural gene of GarA (lgnA), its putative immunity protein (lgnI), and the ABC transporter and its accessory protein (lgnC and lgnD). Interestingly, pGL5-cured strains were still resistant to GarA. Other putative bacteriocins encoded by the remaining plasmids were not detected during purification, pointing to GarA as the main inhibitor secreted by L. garvieae 21881. Mode-of-action studies revealed a potent bactericidal activity of GarA. Moreover, transmission microscopy showed that GarA seems to act by inhibiting septum formation in L. garvieae cells. This potent and species-specific inhibition by GarA holds promise for applications in the prevention or treatment of infections caused by pathogenic strains of L. garvieae in both veterinary and clinical settings.

Clonal and plasmid-mediated flow of ESBL/AmpC genes in Escherichia coli in a commercial laying hen farm.

Resistance to third- and fourth-generation cephalosporins in Escherichia coli is mainly due to extended-spectrum beta-lactamases (ESBL) and AmpC cephalosporinases, which have been increasingly reported, mainly in isolates from humans and poultry. The aim of this study was to address the flow of antimicrobial resistance determinants in the full laying hen production cycle (four batches followed from day-old chicks to 83/84-week-old layers), using cephalosporin-resistant E. coli as a model and their characterization using whole genome sequencing (WGS). Fifteen out of 22 samples analysed yielded growth on MacConkey agar with cefotaxime (1 mg/L). Of these, 141 isolates were identified as E. coli and 47 were characterized by WGS. Genes detected were three ESBL (blaCTX-M-1 (n = 19); blaCTX-M-14 (n = 1); and blaSHV-12 (n = 9)) and one AmpC (blaCMY-2 (n = 13)). Some isolates only harboured blaTEM-1B (n = 2) or blaTEM-1D (n = 1). IncI1 plasmids were the main platform for ESBL/AmpC genes. In addition, five clones were identified harbouring blaCTX-M-1 (two), blaSHV-12 (one) and blaCMY-2 (two), drawing a clone-plasmid mixed flow model. Gene blaCTX-M-1 was found in the chromosomal DNA of clone 1 over 14 months, and in IncI1/ST3 plasmids over six months; over six months blaSHV-12 was found harboured by clone 3 (IncI1/ST26 plasmids), and 15 months later in a non-replicon detected plasmid. Finally, blaCMY-2 spread for at least 16 months, mainly by IncK2 (including clone 4) and IncI1/ST12 (clone 5) plasmids. Proper use of antimicrobials should be combined with other farm management strategies for the effective control of cephalosporin-resistant E. coli isolates in commercial layer farms.

Comparative genomics and evolutionary analysis of Lactococcus garvieae isolated from human endocarditis.

Lactococcus garvieae is a well-known pathogen of fish, but is rarely involved in infections in humans and other mammals. In humans, the main clinical manifestation of L. garvieae infections is endocarditis usually related to the ingestion of contaminated food, such as undercooked fish and shellfish. This study presents the first complete genomic sequence of a clinical L. garvieae strain isolated from a patient with endocarditis and its comparative analysis with other genomes. This human isolate contains a circular chromosome of 2 099 060 bp and one plasmid of 50 557 bp. In comparison with other fully sequenced L. garvieae strains, the chromosomal DNA of L. garvieae Lg-Granada carries a low proportion of insertion sequence elements and a higher number of putative prophages. Our results show that, in general, L. garvieae is a highly recombinogenic species with an open pangenome in which almost 30 % of its genome has undergone horizontal transfers. Within the genus Lactococcus, L. lactis is the main donor of genetic components to L. garvieae but, taking Lg-Granada as a representative, this bacterium tends to import more genes from Bacilli taxa than from other Lactococcus species.

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%.

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.

Pseudomonas composti sp. nov., isolated from compost samples.

Two unusual, Gram-negative, catalase- and oxidase-positive rods, designated C2(T) and C5, were isolated from compost samples. Comparative 16S rRNA gene sequencing studies demonstrated that both isolates were members of the genus Pseudomonas and belonged to the Pseudomonas aeruginosa group. Strain C2(T) was most closely related to Pseudomonas cuatrocienegasensis 1N(T) and Pseudomonas borbori R-20821(T) (97.9 and 97.8% 16S rRNA gene sequence similarity, respectively). However, phylogenetic analysis based on rpoD gene sequences revealed that both isolates could be discriminated from members of the P. aeruginosa group that exhibited >97% 16S rRNA gene sequence similarity. The DNA G+C content of strain C2(T) was 61.5 mol%. The major fatty acids of strain C2(T) were a summed feature (C(16:1)ω7c and/or iso-C(15:0) 2-OH), C(18:1)ω7c/12t/9t, C(16:0) and C(12:0), which supported the isolates' affiliation with the genus Pseudomonas. Moreover, strain C2(T) could be distinguished from its closest phylogenetic neighbours of the genus Pseudomonas by DNA-DNA hybridization studies and biochemical tests. On the basis of both phenotypic and phylogenetic findings, it is proposed that the isolates be classified as a novel species, with the name Pseudomonas composti sp. nov. The type strain is C2(T) (=CECT 7516(T)=CCUG 59231(T)).

Occurrence of the oribatid mite Trhypochthoniellus longisetus longisetus (Acari: Trhypochthoniidae) on tilapia Oreochromis niloticus.

Mites as parasites infesting fish have been described in a few case reports involving Histiostoma anguillarum, H. papillata, and Schwiebea estradai. We describe the unexpected occurrence of oribatid mites of the genus Trhypochthoniellus on farmed tilapia Oreochromis niloticus. The fish had mites on the skin, fins, and gills, as well as in the mouth. The morphological characteristics of the mites, observed by optical and scanning electron microscopy, were consistent with those described for T. longisetus longisetus. All stages of development were observed, suggesting that the mites were able to actively reproduce on fish.

Analysis of the genome content of Lactococcus garvieae by genomic interspecies microarray hybridization.

BACKGROUND: Lactococcus garvieae is a bacterial pathogen that affects different animal species in addition to humans. Despite the widespread distribution and emerging clinical significance of L. garvieae in both veterinary and human medicine, there is almost a complete lack of knowledge about the genetic content of this microorganism. In the present study, the genomic content of L. garvieae CECT 4531 was analysed using bioinformatics tools and microarray-based comparative genomic hybridization (CGH) experiments. Lactococcus lactis subsp. lactis IL1403 and Streptococcus pneumoniae TIGR4 were used as reference microorganisms. RESULTS: The combination and integration of in silico analyses and in vitro CGH experiments, performed in comparison with the reference microorganisms, allowed establishment of an inter-species hybridization framework with a detection threshold based on a sequence similarity of >or= 70%. With this threshold value, 267 genes were identified as having an analogue in L. garvieae, most of which (n = 258) have been documented for the first time in this pathogen. Most of the genes are related to ribosomal, sugar metabolism or energy conversion systems. Some of the identified genes, such as als and mycA, could be involved in the pathogenesis of L. garvieae infections. CONCLUSIONS: In this study, we identified 267 genes that were potentially present in L. garvieae CECT 4531. Some of the identified genes could be involved in the pathogenesis of L. garvieae infections. These results provide the first insight into the genome content of L. garvieae.

Utilization of lactose and presence of the phospho-ß-galactosidase (lacG) gene in Lactococcus garvieae isolates from different sources.

This study evaluates the utilization of lactose (Lac) and the presence of the phospho-ß-galactosidase (lacG) gene as markers for distinguishing between fish (Lac-/lacG-) and dairy isolates (Lac+/lacG+) of Lactococcus garvieae, using a panel of L. garvieae isolates from different sources. None of the fish isolates produced acid from lactose (Lac-), however Lac-/lacG- isolates were observed in pigs, cows, birds and humans. Most of the dairy isolates (77.8%) were Lac+/lacG+, but some dairy isolates did not produce acid from this sugar. Data in the present study show that the ability to metabolize lactose and the presence of the lacG gene are heterogeneously scattered among L. garvieae isolates of different sources. Therefore, the use of these criteria as markers to differentiate between L. garvieae isolates of dairy and fish origin should be considered with caution.

Potentially human-virulent Vibrio vulnificus isolates from diseased great pompano (Trachinotus goodei).

Vibrio vulnificus is an opportunistic human pathogen responsible for the majority of seafood-associated deaths worldwide and is also a relevant fish pathogen for the aquaculture industry. In addition to infections in aquatic livestock, V. vulnificus also represents a risk to aquarium animals. For the first time, this work describes an important mortality outbreak in Trachinotus goodei in a zoo aquarium, with the isolation of Vibrio vulnificus (Vv) from the internal organs of the diseased fish. The isolates were identified by MALDI-TOF MS, serotyped and characterized by pulsed-field gel electrophoresis (PFGE). Although the isolates from great pompanos did not belong to pathovar piscis (formerly biotype 2) or to any of the fish-related serovars, they all had identical phenotypes, antimicrobial susceptibility profiles and PFGE patterns, which together with their isolation in pure culture from internal organs is strongly indicative of their clinical significance. Moreover, Vv isolates harboured important genetic markers of human virulence potential: they had the clinical variant of the vcg gene, gave the 338 bp DNA amplification product of the pilF gene and resisted the bactericidal activity of human serum. All these results strongly suggest that these Vv isolates should be considered potentially virulent for humans. These results extend the range of fish species affected by V. vulnificus, confirm the threat that this pathogen represents to aquatic animals and highlight the risk that this bacterial pathogen poses to human health.

Application of fluorescence in situ hybridization technique to detect simazine-degrading bacteria in soil samples.

We propose a new approach to evaluate the natural attenuation capacity of soil by using fluorescence in situ hybridization (FISH). A specific oligonucleotide probe AtzB1 was designed based on the sequence data of the atzB gene involved in the hydrolytic deamination of s-triazines; this gene, located in a multiple copy plasmid was detected by the optimized FISH protocol. Two agricultural soils (Lodi and Henares) with a history of simazine treatments, and two natural soils (Soto and Monza), without previous exposure to simazine, were studied. AtzB1 probe-target cells were found only in the agricultural soils and, in a greater percentage, in the Lodi soil, compared to the Henares one. Moreover, the greatest percentage of AtzB1 probe-target cells in Lodi was accompanied by a greater mineralization rate, compared to the Henares soil. The FISH method used in this study was suitable for the detection of simazine-degrading bacteria and could be a useful indicator of the potential of soil bioremediation.

The zoonotic potential of Lactococcus garvieae: An overview on microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Lactococcus garvieae is a relevant worldwide fish pathogen affecting various farmed and wild marine and freshwater species. It has also been isolated from other animals, such as ruminants with subclinical mastitis and pigs with pneumonia. From the early 90s, L. garvieae has been associated with different human infections, mainly endocarditis. During the last five years, human infections by this bacterium appear to be increasing, likely due to the improvement in microbiological methods for bacterial identification and the alertness of this bacterium by physicians. Human L. garvieae infections have been associated with the consumption or the handling of contaminated raw fish or seafood, and recently, a genetic study showed that meat, raw milk and dairy products may also be food sources of human L. garvieae infections. However, the status of L. garvieae as a potential zoonotic bacterium is still controversial to date. In this work, we describe four new human infections by L. garvieae in elderly and inmunocompromised patients, and we show an overview on L. garvieae microbiology, epidemiology, virulence factors and relationship with its presence in foods.

Genetic analysis of human clinical isolates of Lactococcus garvieae: Relatedness with isolates from foods.

Lactococcus garvieae is a Gram-positive bacterium well-known as an important pathogen in aquaculture, and it is also a human pathogen of increasing clinical significance. Forty-three human L. garvieae isolates from clinical specimens were characterized by Multilocus Sequence Typing (MLST). Twenty-six different sequence types (STs) were identified among the human isolates, of which 20 were novel STs. Most human isolates clustered into four clonal complexes, with a predominance of CC3. Within CC3, ST10 was the most common genotype, indicating the existence of a circulating genetic lineage among the human isolates analyzed. The four CCs also grouped L. garvieae strains isolated from meat, dairy and fish, indicating a genetic overlap between isolates from human and these foods. Genetic relatedness among human and food L. garvieae isolates was confirmed by phylogenetic analysis based on the concatenated sequences of the seven MLST genes. These results represent the first evidence of genetic relatedness between isolates of L. garvieae of human and those isolated meat, milk and dairy products and suggest that, in addition to fish and seafood, these foods might represent important sources of human L. garvieae infections.

Lactococcus garvieae: a small bacteria and a big data world.

OBJECTIVE: To describe the importance of bioinformatics tools to analyze the big data yielded from new "omics" generation-methods, with the aim of unraveling the biology of the pathogen bacteria Lactococcus garvieae. METHODS: The paper provides the vision of the large volume of data generated from genome sequences, gene expression profiles by microarrays and other experimental methods that require biomedical informatics methods for management and analysis. RESULTS: The use of biomedical informatics methods improves the analysis of big data in order to obtain a comprehensive characterization and understanding of the biology of pathogenic organisms, such as L. garvieae. CONCLUSIONS: The "Big Data" concepts of high volume, veracity and variety are nowadays part of the research in microbiology associated with the use of multiple methods in the "omic" era. The use of biomedical informatics methods is a requisite necessary to improve the analysis of these data.

Skin and subcutaneous mycoses in tilapia (Oreochromis niloticus) caused by Fusarium oxysporum in coinfection with Aeromonas hydrophila.

Subcutaneous mycoses in freshwater fish are rare infections usually caused by oomycetes of the genus Saprolegnia and some filamentous fungi. To date, Fusarium infections in farmed fish have only been described in marine fish. Here, we report the presence of Fusarium oxysporum in subcutaneous lesions of Nile tilapia (Oreochromis niloticus). Histopathologic evaluation revealed granuloma formation with fungal structures, and the identity of the etiological agent was demonstrated by morphological and molecular analyses. Some of the animals died as a result of systemic coinfection with Aeromonas hydrophila.

Development of a PCR assay for Streptococcus iniae based on the lactate oxidase (lctO) gene with potential diagnostic value.

Streptococcus iniae is a well-known pathogen of both fish and humans that is difficult to identify by conventional biochemical tests. A PCR assay based on the lactate oxidase (lctO) gene of S. iniae was developed for the rapid and specific detection and identification of this pathogen from different sources. The PCR assay had a detection limit of 62-31 cells, and 25 pg of DNA per PCR reaction mixture. The PCR was also effective in detecting the bacterium from inoculated tissue homogenates, suggesting its potential use for a rapid and accurate diagnosis of S. iniae infections.

PCR detection and PFGE DNA macrorestriction analyses of clinical isolates of Pseudomonas anguilliseptica from winter disease outbreaks in sea bream Sparus aurata.

A PCR-based detection system for Pseudomonas anguilliseptica was evaluated. The primer combination PAF-PAR (forward primer PAF = 5'-GACCTCGCCATTA-3', reverse primer PAR = 5'-CTCAGCAGTTTTGAAAG-3') gave a unique and specific amplification product of 439 bp at an annealing temperature of 46 degrees C with all the P. anguilliseptica isolates and strains (n = 56) but no amplification products were observed with any other Pseudomonas species or phylogenetically related bacteria tested. The PCR assay had a detection limit of 170 to 200 cells per PCR tube, which was improved 8-fold when the PCR amplification product was used as a nonradioacfive probe in blotting hybridization experiments. The PCR assay allowed the specific and reliable detection of P. anguilliseptica within 8 h, compared with up to 10 d required for its isolation and further characterization by conventional microbiological approaches. Clinical isolates of P. anguilliseptica recovered from several winter disease (WD) outbreaks diagnosed in sea bream Sparus aurata in Spain and Portugal between 1996 and 2001 were characterized by pulse field-gel electrophoresis (PFGE) macrorestriction analysis. The 54 clinical isolates analyzed were included in 4 different pulsotypes. Pulsotypes B and C represented 54 and 25% of the isolates, respectively, and were responsible for most of the WD outbreaks diagnosed in Spain between 1996 and 2001. The implication of asymptomatic infected carriers in the dissemination and spread of WD is discussed.

Post-stained Western blotting, a useful approach in immunoproteomic studies.

The precise localisation of immunogenic proteins on stained two-dimensional electrophoresis (2DE) gels is occasionally difficult, contributing to the erroneous identification of unrelated non-immunogenic proteins, which is expensive and time consuming. This inconvenience can be solved by performing immunoblotting using previously stained polyacrylamide gels. This approach was proposed nearly 20 years ago but is now almost forgotten. We have evaluated the suitability of this approach to identify immunogenic proteins from Lactococcus garvieae. Some of the immunogenic proteins identified in L. garvieae, such as Gls24, have been considered important as immunotarget in different bacterial species. Post-staining western blotting facilitated the correct selection of immunogenic proteins of interest in 2D gels before their identification.