Covadonga R. Arias

Yeast species associated with orange juice: evaluation of different identification methods.

ABSTRACT Five different methods were used to identify yeast isolates from a variety of citrus juice sources. A total of 99 strains, including reference strains, were identified using a partial sequence of the 26S rRNA gene, restriction pattern analysis of the internal transcribed spacer region (5.8S-ITS), classical methodology, the RapID Yeast Plus system, and API 20C AUX. Twenty-three different species were identified representing 11 different genera. Distribution of the species was considerably different depending on the type of sample. Fourteen different species were identified from pasteurized single-strength orange juice that had been contaminated after pasteurization (PSOJ), while only six species were isolated from fresh-squeezed, unpasteurized orange juice (FSOJ). Among PSOJ isolates, Candida intermedia and Candida parapsilosis were the predominant species. Hanseniaspora occidentalis and Hanseniaspora uvarum represented up to 73% of total FSOJ isolates. Partial sequence of the 26S rRNA gene yielded the best results in terms of correct identification, followed by classical techniques and 5.8S-ITS analysis. The commercial identification kits RapID Yeast Plus system and API 20C AUX were able to correctly identify only 35 and 13% of the isolates, respectively. Six new 5.8S-ITS profiles were described, corresponding to Clavispora lusitaniae, Geotrichum citri-aurantii, H. occidentalis, H. vineae, Pichia fermentans, and Saccharomycopsis crataegensis. With the addition of these new profiles to the existing database, the use of 5.8S-ITS sequence became the best tool for rapid and accurate identification of yeast isolates from orange juice.

Diversity of the skin microbiota of fishes: evidence for host species specificity

Skin microbiota of Gulf of Mexico fishes were investigated by ribosomal internal spacer analysis (RISA) and 16S rRNA gene sequencing. A total of 102 fish specimens representing six species (Mugil cephalus, Lutjanus campechanus, Cynoscion nebulosus, Cynoscion arenarius, Micropogonias undulatus, and Lagodon rhomboides) were sampled at regular intervals throughout a year. The skin microbiota from each individual fish was analyzed by RISA and produced complex profiles with 23 bands on average. Similarities between RISA profiles ranged from 97.5% to 4.0%. At 70% similarity, 11 clusters were defined, each grouping individuals from the same fish species. Multidimensional scaling and analysis of similarity correlated the RISA-defined clusters with geographic locality, date, and fish species. Global R values indicated that fish species was the most indicative variable for group separation. Analysis of 16S rRNA gene sequences (from pooled samples of 10 individual fish for each fish species) showed that the Proteobacteria was the predominant phylum in skin microbiota, followed by the Firmicutes and the Actinobacteria. The distribution and abundance of bacterial sequences were different among all species analyzed. Aeribacillus was found in all fish species representing 19% of all clones sequenced, while some genera were fish species-specific (Neorickettsia in M. cephalus and Microbacterium in L. campechanus). Our data provide evidence for the existence of specific skin microbiota associated with particular fish species.

Catfish hybrid Ictalurus punctatus × I. furcatus exhibits higher resistance to columnaris disease than the parental species

We present experimental data on susceptibility to columnaris disease, caused by the bacterium Flavobacterium columnare, in hybrid catfish (female channel catfish Ictalurus punctatus × male blue catfish I. furcatus) (C×B). Under our experimental conditions, C×B hybrids were significantly more resistant to columnaris disease caused by the highly virulent strain of F. columnare BGFS-27 (genomovar II) than channel catfish and blue catfish. Channel and blue catfish cumulative mortalities after immersion challenge were 74 and 87%, respectively, whereas mortality in the C×B hybrid was 31%. Susceptibility to the strain ARS-1 (genomovar I) was lower among all catfishes, although channel catfish was the least resistant species at 32% cumulative mortality. By contrast, C×B hybrid and blue catfishes were strongly resistant to the ARS-1 strain, with <10% mortality. Our data suggest enhanced disease resistance of the C×B hybrid to columnaris disease.

Adhesion dynamics of Flavobacterium columnare to channel catfish Ictalurus punctatus and zebrafish Danio rerio after immersion challenge

The adhesion dynamics of Flavobacterium columnare to fish tissues were evaluated in vivo by immersion challenge followed by bacterial plate count and confirmatory observations of gill-adhered bacterial cells using scanning electron microscopy. Adhesion of F. columnare genomovar I (ARS-1) and II (BGFS-27) strains to skin and gill of channel catfish Ictalurus punctactus and gill of zebrafish Danio rerio was compared. At 0.5 h post-challenge, both strains adhered to gill of channel catfish at comparable levels (10(6) colony forming units [CFU] g(-1)), but significant differences in adhesion were found later in the time course. Channel catfish was able to effectively reduce ARS-1 cells on gill, whereas BGFS-27 persisted in gill beyond the first 24 h post-challenge. No significant difference was found between both strains when adhered to skin, but adhered cell numbers were lower (10(3) CFU g(-1)) than those found in gill and were not detectable at 6 h post-challenge. Adhesion of BGFS-27 cells to gill of zebrafish also occurred at high numbers (> 10(6) CFU g(-1)), while only < 10(2) CFU g(-1) of ARS-1 cells were detected in this fish. The results of the present study show that particular strains of F. columnare exhibit different levels of specificity to their fish hosts and that adhesion to fish tissues is not sufficient to cause columnaris disease.

Biofilm formation by the fish pathogen Flavobacterium columnare: development and parameters affecting surface attachment

ABSTRACT Flavobacterium columnare is a bacterial fish pathogen that affects many freshwater species worldwide. The natural reservoir of this pathogen is unknown, but its resilience in closed aquaculture systems posits biofilm as the source of contagion for farmed fish. The objectives of this study were (i) to characterize the dynamics of biofilm formation and morphology under static and flow conditions and (ii) to evaluate the effects of temperature, pH, salinity, hardness, and carbohydrates on biofilm formation. Nineteen F. columnare strains, including representatives of all of the defined genetic groups (genomovars), were compared in this study. The structure of biofilm was characterized by light microscopy, confocal laser scanning microscopy, and scanning electron microscopy. F. columnare was able to attach to and colonize inert surfaces by producing biofilm. Surface colonization started within 6 h postinoculation, and microcolonies were observed within 24 h. Extracellular polysaccharide substances and water channels were observed in mature biofilms (24 to 48 h). A similar time course was observed when F. columnare formed biofilm in microfluidic chambers under flow conditions. The virulence potential of biofilm was confirmed by cutaneous inoculation of channel catfish fingerlings with mature biofilm. Several physicochemical parameters modulate attachment to surfaces, with the largest influence being exerted by hardness, salinity, and the presence of mannose. Maintenance of hardness and salinity values within certain ranges could prevent biofilm formation by F. columnare in aquaculture systems.

Bacterial loads and microbial composition in high pressure treated oysters during storage.

Analysis of bacterial communities present in high-pressure (HP)-treated, quick-frozen (QF), and raw oysters was carried out during three different seasons. Bacterial numbers and species diversity in each sample were determined at 0, 7, 14, and 21 days of storage. Results showed that numbers of total aerobic bacterial counts (TABC) in treated oysters were significantly lower than in untreated oysters at day 0 by 10 to 10(5) colony forming units per gram of oyster meat (CFU/g) in all samplings. However, an increase in TABC in HP-treated oysters was observed at days 7, 14, and 21 indicating that some bacteria survived the treatment and were able to proliferate during refrigeration conditions. Surprisingly, TABC in HP-treated oysters reached 10(8) CFU/g at 14 days of storage in all samplings (higher than TABC from raw oysters in two of the samplings performed). Analysis of the bacterial flora by 16S rDNA sequencing, revealed six different classes within the bacterial communities. The majority were Gram-negative bacteria, with the Gammaproteobacteria class representing between 56% and 92%. The most common bacterial genera found in this study were Shewanella, Vibrio and Psychrobacter. Four species of human pathogenic bacteria were also identified: V. vulnificus, V. parahaemolyticus, V. alginolyticus, and A. hydrophila although V. vulnificus was detected only in raw oysters.

Transcriptional regulation of hypoxia inducible factors alpha (HIF-α) and their inhibiting factor (FIH-1) of channel catfish (Ictalurus punctatus) under hypoxia

Hypoxia inducible factors (HIFs) are considered to be the master switch of oxygen-dependent gene expression with mammalian species. In most cases, regulation of HIF has been believed at posttranslational levels. However, little is known of HIF regulation in channel catfish, a species highly tolerant to low oxygen condition. Here we report the identification and characterization of HIF-1α, HIF-2αa, HIF-2αb, HIF-3α, and FIH-1 genes, and their mRNA expression under hypoxia conditions. The transcripts of the five genes were found to be regulated temporally and spatially after low oxygen challenge, suggesting regulation of HIF-α genes at pre-translational levels. In most tissues, the HIF-α mRNAs were down-regulated 1.5h but up-regulated 5h after hypoxia treatment. Of these HIF-α mRNAs, the expression of HIF-3α mRNA was induced in the most dramatic fashion, both in the speed of induction and the extent of induction, compared to HIF-1α and HIF-2α genes, suggesting its importance in responses to hypoxia.

Combined use of 16S ribosomal DNA and automated ribosomal intergenic spacer analysis to study the bacterial community in catfish ponds

Aims:  To apply culture‐independent techniques to explore the bacterial community composition in catfish pond water.

High Numbers of Vibrio vulnificus in Tar Balls Collected from Oiled Areas of the North-Central Gulf of Mexico Following the 2010 BP Deepwater Horizon Oil Spill

The Deepwater Horizon Oil Spill was the largest oil spill in USA history releasing approximately 4.9 million barrels of crude oil into the Gulf of Mexico. Soon after the spill started, tar balls and other forms of weathered oil appeared in large numbers on beaches in Mississippi and Alabama. In this study, we analyzed tar balls for total aerobic bacterial (TAB) counts and also for the presence of Vibrio vulnificus, a human pathogen known to be abundant in the Gulf Coast environment and capable of causing severe wound infections by contact with contaminated surfaces. Our results showed that TAB counts were significantly higher in tar balls than in sand and seawater collected at the same location. In addition, V. vulnificus numbers were 10× higher in tar balls than in sand and up to 100× higher than in seawater. Densities of V. vulnificus were higher than 105 colony forming units/g of tar ball in all samples analyzed. Our data suggest that tar balls can act as reservoirs for bacteria including human pathogens.

A Polyphasic Approach to Study the Intraspecif ic Diversity Amongst Vibrio vulnificus Isolates

Summary A polyphasic taxonomic approach using phenotypic and molecular genetic techniques, was carried out on the species Vibrio vulnificus in order to study its intraspecific diversity. Seven techniques, including phenotypic (API 20E, BIOLOG, total protein profiles, serotyping, ELISA), and genotypic methods (ribotyping and AFLP), were employed on 80 V. vulnificus strains of biotypes 1 and 2, including 9 reference cultures. The isolates came from different geographic origins (USA, Spain, Belgium, Sweden, Norway, Japan, Thailand) and types of samples (clinical, health/diseased fish, seafood, water). Diversity indexes calculated for strains of both biotypes revealed a higher phenotypic and genetic diversity of biotype 1 strains in contrast with the homogeneity shown by biotype 2 strains. A species profile was established for the species V. vulnificus by BIOLOG and SDS-PAGE. Serotyping allowed a clear separation within species level, and grouped typical and atypical biotype 2 strains in one serovar. Genetic fingerprinting techniques, especially AFLP, were able to differentiate within strains belonging to the same serovar, allowing the tracking of specific clones in epidemiologic or ecological studies. The techniques employed do not support the maintenance of the two biotypes established for the species.