Revisiting Microbial Diversity in Hypersaline Microbial Mats from Guerrero Negro for a Better Understanding of Methanogenic Archaeal Communities.

Knowledge regarding the diversity of methanogenic archaeal communities in hypersaline environments is limited because of the lack of efficient cultivation efforts as well as their low abundance and metabolic activities. In this study, we explored the microbial communities in hypersaline microbial mats. Bioinformatic analyses showed significant differences among the archaeal community structures for each studied site. Taxonomic assignment based on 16S rRNA and methyl coenzyme-M reductase (mcrA) gene sequences, as well as metagenomic analysis, corroborated the presence of Methanosarcinales. Furthermore, this study also provided evidence for the presence of Methanobacteriales, Methanomicrobiales, Methanomassiliicoccales, Candidatus Methanofastidiosales, Methanocellales, Methanococcales and Methanopyrales, although some of these were found in extremely low relative abundances. Several mcrA environmental sequences were significantly different from those previously reported and did not match with any known methanogenic archaea, suggesting the presence of specific environmental clusters of methanogenic archaea in Guerrero Negro. Based on functional inference and the detection of specific genes in the metagenome, we hypothesised that all four methanogenic pathways were able to occur in these environments. This study allowed the detection of extremely low-abundance methanogenic archaea, which were highly diverse and with unknown physiology, evidencing the presence of all methanogenic metabolic pathways rather than the sheer existence of exclusively methylotrophic methanogenic archaea in hypersaline environments.

Draft Genome Sequence of Pediococcus pentosaceus Strain PP16CC, Isolated from Oyster Crassostrea corteziensis.

Pediococcus pentosaceus strain PP16CC comes from the intestine of Crassostrea corteziensis. A 1.82-Mbp draft genome of this strain was assembled using A5-miseq from illumina reads, resulting in 4 contigs and 1,856 predicted protein coding genes. Additionally, 23 proteins belonging to various glycosyl hydrolase families and 6 prophage regions were identified.

Screening of xylose utilizing and high lipid producing yeast strains as a potential candidate for industrial application.

BACKGROUND: Sustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose utilizing oleaginous yeasts as cell factories for lipid production, since pentose sugar catabolism plays a major role in efficient utilization of lignocellulosic feedstocks. Glycerol utilization, which is also beneficial in yeast-based oil production as glycerol is a common by-product of biodiesel production, was investigated as well. Natural yeast isolates were studied for lipid accumulation on a variety of substrates, and the highest lipid accumulating strains were further investigated in shake flask cultivations and fermenter studies on xylose and hydrolysate. RESULTS: By collecting leaves from exotic plants in greenhouses and selective cultivation on xylose, a high frequency of oleaginous yeasts was obtained (> 40%). Different cultivation conditions lead to differences in fatty acid contents and compositions, resulting in a set of strains that can be used to select candidate production strains for different purposes. In this study, the most prominent strains were identified as Pseudozyma hubeiensis BOT-O and Rhodosporidium toruloides BOT-A2. The fatty acid levels per cell dry weight after cultivation in a nitrogen limited medium with either glucose, xylose or glycerol as carbon source, respectively, were 46.8, 43.2 and 38.9% for P. hubeiensis BOT-O, and 40.4, 27.3 and 42.1% for BOT-A2. Furthermore, BOT-A2 accumulated 45.1% fatty acids per cell dry weight in a natural plant hydrolysate, and P. hubeiensis BOT-O showed simultaneous glucose and xylose consumption with similar growth rates on both carbon sources. The fatty acid analysis demonstrated both long chain and poly-unsaturated fatty acids, depending on strain and medium. CONCLUSIONS: We found various natural yeast isolates with high lipid production capabilities and the ability to grow not only on glucose, but also xylose, glycerol and natural plant hydrolysate. R. toruloides BOT-A2 and P. hubeiensis BOT-O specifically showed great potential as production strains with high levels of storage lipids and comparable growth to that on glucose on various other substrates, especially compared to currently used lipid production strains. In BOT-O, glucose repression was not detected, making it particularly desirable for utilization of plant waste hydrolysates. Furthermore, the isolated strains were shown to produce oils with fatty acid profiles similar to that of various plant oils, making them interesting for future applications in fuel, food or feed production.

Genomic analysis of virulence factors and antimicrobial resistance of group B Streptococcus isolated from pregnant women in northeastern Mexico.

INTRODUCTION: Group B Streptococcus (GBS) causes infections in women during pregnancy and puerperium and invasive infections in newborns. The genes lmb, cylE, scpB, and hvgA are involved with increased virulence of GBS, and hypervirulent clones have been identified in different regions. In addition, increasing resistance of GBS to macrolides and lincosamides has been reported, so knowing the patterns of antibiotic resistance may be necessary to prevent and treat GBS infections. This study aimed to identify virulence genes and antibiotic resistance associated with GBS colonization in pregnant women from northeastern Mexico. METHODS: Pregnant women with 35-37 weeks of gestation underwent recto-vaginal swabbing. One swab was inoculated into Todd-Hewitt broth supplemented with gentamicin and nalidixic acid, a second swab was inoculated into LIM enrichment broth, and a third swab was submerged into a transport medium. All samples were subcultured onto blood agar. After overnight incubation, suggestive colonies with or without hemolysis were analyzed to confirm GBS identification by Gram staining, catalase test, hippurate hydrolysis, CAMP test, and incubation in a chromogenic medium. We used latex agglutination to confirm and serotype GBS isolates. Antibiotic resistance patterns were assessed by Vitek 2 and disk diffusion. Periumbilical, rectal and nasopharyngeal swabs were collected from some newborns of colonized mothers. All colonized women and their newborns were followed up for three months to assess the development of disease attributable to GBS. Draft genomes of all GBS isolates were obtained by whole-genome sequencing. In addition, bioinformatic analysis to identify genes encoding capsular polysaccharides and virulence factors was performed using BRIG, while antibiotic resistance genes were identified using the CARD database. RESULTS: We found 17 GBS colonized women out of 1154 pregnant women (1.47%). None of the six newborns sampled were colonized, and no complications due to GBS were detected in pregnant women or newborns. Three isolates were serotype I, 5 serotype II, 3 serotype III, 4 serotype IV, and 2 serotype V. Ten distinct virulence gene profiles were identified, being scpB, lmb, fbsA, acp, PI-1, PI-2a, cylE the most common (3/14, 21%). The virulence genes identified were scpB, lmb, cylE, PI-1, fbsA, PI-2a, acp, fbsB, PI-2b, and hvgA. We identified resistance to tetracycline in 65% (11/17) of the isolates, intermediate susceptibility to clindamycin in 41% (7/17), and reduced susceptibility to ampicillin in 23.5% (4/17). The tetM gene associated to tetracyclines resistance was found in 79% (11/14) and the mel and mefA genes associated to macrolides resistance in 7% (1/14). CONCLUSIONS: The low prevalence of colonization and the non-occurrence of mother-to-child transmission suggest that the intentional search for GBS colonization in this population is not justified. Our results also suggest that risk factors should guide the use of intrapartum antibiotic prophylaxis. The detection of strains with genes coding virulence factors means that clones with pathogenic potential circulates in this region. On the other hand, the identification of decreased susceptibility to antibiotics from different antimicrobial categories shows the importance of adequately knowing the resistance patterns to prevent and to treat GBS perinatal infection.

Molecular variability and genetic structure of white spot syndrome virus strains from northwest Mexico based on the analysis of genomes.

White spot syndrome virus (WSSV) has a ∼300 kb double-stranded DNA genome. It originated in China, spread rapidly through shrimp farms in Asia, and subsequently to America. This study determined complete genome sequences for nine historic WSSV strains isolated from Pacific white shrimp (Litopenaeus vannamei) captured in farm ponds in northwest Mexico (Sinaloa and Nayarit). Genomic DNA was captured by an amplification method using overlapping long-range PCR and sequencing by Ion Torrent-PGM. Complete genome sequences were assembled (length range 255-290 kb) and comparative genome analysis with WSSV strains revealed substantial deletions (3 and 10 kb in two regions) in seven strains, with two strains differing from the rest. Phylogenetic analysis identified that the WSSV strains from the northern area of the state of Sinaloa clustered with strains from China (LC1, LC10, DVI) and Korea (ACF2, ACF4), while those from the southern region of the state of Nayarit (AC1 and JP) differed from both of those and from strains found in Taiwan and Thailand. Our data offer insights into the diversity of the WSSV genome in one country and their divergent origin, suggest that it entered Mexico via multiple routes and that specific genome regions can accommodate substantial deletions without compromising viability.

Administration of Probiotics Improves the Brine Shrimp Production and Prevents Detrimental Effects of Pathogenic Vibrio Species.

In this study, we evaluated a consortium of probiotic bacteria as an environmentally-friendly strategy for controlling pathogenic Vibrio species during the brine shrimp incubation period. Probiotic strains were initially selected on basis of (i) their ability to colonize the cyst surfaces, (ii) their absence of cross-inhibitory effects, and (iii) no detrimental effect on cyst hatching. The cysts and nauplius surfaces were immediately colonized after the application of selected probiotic strains, without detrimental effects on survival. Ten probiotic strains were mixed at similar proportions (probiotic consortium) and evaluated at different concentrations into brine shrimp cultures during incubation and early stages of development. Subsequently, these cultures were challenged with Vibrio parahaemolyticus and Vibrio harveyi. The probiotic consortium was effective to reduce the abundance of pathogenic Vibrio species and to prevent the mortality during Vibrio challenges; however, its effect was concentration-dependent and was successful at a starting concentration of 1.8 × 106 CFU/ml. Our results suggest that this probiotic consortium offers an alternative to antimicrobial agents routinely used to reduce the incidence and prevalence of pathogenic Vibrio species in brine shrimp production.

Genotyping WSSV isolates from northwestern Mexican shrimp farms affected by white spot disease outbreaks in 2010-2012.

White spot disease (WSD) causes high mortality in cultured shrimp throughout the world. Its etiologic agent is the white spot syndrome virus (WSSV). The genomic repeat regions ORF 75, ORF 94, and ORF 125 have been used to classify WSSV isolates in epidemiological studies using PCR with specific primers and sequencing. The present study investigated the variation in nucleotide sequences from 107, 150, and 143 isolates of WSSV collected from Litopenaeus vannamei shrimp ponds with WSD outbreaks in northwestern Mexico during the period 2010-2012, in the genomic repeat regions ORFs 75, 94, and 125, respectively. The haplotypic nomenclature for each isolate was based on the number of repeat units and the position of single nucleotide polymorphisms on each ORF. We report finding 17, 43, and 66 haplotypes of ORFs 75, 94, and 125, respectively. The study found high haplotypic diversity in WSSV using the complete sequences of ORFs 94 and 125 as independent variables, but low haplotypic diversity for ORF 75. Different haplotypes of WSSV were found from region-to-region and year-to-year, though some individual haplotypes were found in different places and in more than one growing cycle. While these results suggest a high rate of mutation of the viral genome at these loci, or perhaps the introduction of new viral strains into the area, they are useful as a tool for epidemiological surveys. Two haplotypes from some of the ORFs in the same shrimp were encountered, suggesting the possibility of multiple infections.

Genetic structure of populations of whale sharks among ocean basins and evidence for their historic rise and recent decline.

This study presents genetic evidence that whale sharks, Rhincodon typus, are comprised of at least two populations that rarely mix and is the first to document a population expansion. Relatively high genetic structure is found when comparing sharks from the Gulf of Mexico with sharks from the Indo-Pacific. If mixing occurs between the Indian and Atlantic Oceans, it is not sufficient to counter genetic drift. This suggests whale sharks are not all part of a single global metapopulation. The significant population expansion we found was indicated by both microsatellite and mitochondrial DNA. The expansion may have happened during the Holocene, when tropical species could expand their range due to sea-level rise, eliminating dispersal barriers and increasing plankton productivity. However, the historic trend of population increase may have reversed recently. Declines in genetic diversity are found for 6 consecutive years at Ningaloo Reef in Australia. The declines in genetic diversity being seen now in Australia may be due to commercial-scale harvesting of whale sharks and collision with boats in past decades in other countries in the Indo-Pacific. The study findings have implications for models of population connectivity for whale sharks and advocate for continued focus on effective protection of the world's largest fish at multiple spatial scales.

Changes in the composition and diversity of the bacterial microbiota associated with oysters (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) during commercial production.

The resident microbiota of three oyster species (Crassostrea corteziensis, Crassostrea gigas and Crassostrea sikamea) was characterised using a high-throughput sequencing approach (pyrosequencing) that was based on the V3-V5 regions of the 16S rRNA gene. We analysed the changes in the bacterial community beginning with the postlarvae produced in a hatchery, which were later planted at two grow-out cultivation sites until they reached the adult stage. DNA samples from the oysters were amplified, and 31 008 sequences belonging to 13 phyla (including Proteobacteria, Bacteroidetes, Actinobacteria and Firmicutes) and 243 genera were generated. Considering all life stages, Proteobacteria was the most abundant phylum, but it showed variations at the genus level between the postlarvae and the adult oysters. Bacteroidetes was the second most common phylum, but it was found in higher abundance in the postlarvae than in adults. The relative abundance showed that the microbiota that was associated with the postlarvae and adults differed substantially, and higher diversity and richness were evident in the postlarvae in comparison with adults of the same species. The site of rearing influenced the bacterial community composition of C. corteziensis and C. sikamea adults. The bacterial groups that were found in these oysters were complex and metabolically versatile, making it difficult to understand the host-bacteria symbiotic relationships; therefore, the physiological and ecological significances of the resident microbiota remain uncertain.

Molecular analysis of bacterial microbiota associated with oysters (Crassostrea gigas and Crassostrea corteziensis) in different growth phases at two cultivation sites.

Microbiota presumably plays an essential role in inhibiting pathogen colonization and in the maintenance of health in oysters, but limited data exist concerning their different growth phases and conditions. We analyzed the bacterial microbiota composition of two commercial oysters: Crassostrea gigas and Crassostrea corteziensis. Differences in microbiota were assayed in three growth phases: post-larvae at the hatchery, juvenile, and adult at two grow-out cultivation sites. Variations in the microbiota were assessed by PCR analysis of the 16S rRNA gene in DNA extracted from depurated oysters. Restriction fragment length polymorphism (RFLP) profiles were studied using Dice's similarity coefficient (Cs) and statistical principal component analysis (PCA). The microbiota composition was determined by sequencing temperature gradient gel electrophoresis (TGGE) bands. The RFLP analysis of post-larvae revealed homology in the microbiota of both oyster species (Cs > 88 %). Dice and PCA analyses of C. corteziensis but not C. gigas showed differences in the microbiota according to the cultivation sites. The sequencing analysis revealed low bacterial diversity (primarily ß-Proteobacteria, Firmicutes, and Spirochaetes), with Burkholderia cepacia being the most abundant bacteria in both oyster species. This study provides the first description of the microbiota in C. corteziensis, which was shown to be influenced by cultivation site conditions. During early growth, we observed that B. cepacia colonized and remained strongly associated with the two oysters, probably in a symbiotic host-bacteria relationship. This association was maintained in the three growth phases and was not altered by environmental conditions or the management of the oysters at the grow-out site.

Isolation and characterization of microsatellite loci in the whale shark (Rhincodon typus).

In preparation for a study on population structure of the whale shark (Rhincodon typus), nine species-specific polymorphic microsatellite DNA markers were developed. An initial screening of 50 individuals from Holbox Island, Mexico found all nine loci to be polymorphic, with two to 17 alleles observed per locus. Observed and expected heterozygosity per locus ranged from 0.200 to 0.826 and from 0.213 to 0.857, respectively. Neither statistically significant deviations from Hardy-Weinberg expectations nor statistically significant linkage disequilibrium between loci were observed. These microsatellite loci appear suitable for examining population structure, kinship assessment and other applications.

MHC DQB-1 polymorphism in the Gulf of California fin whale (Balaenoptera physalus) population.

One of the most isolated populations of fin whales occurs in the Gulf of California (GOC) with 400-800 individuals. This population shows reduced neutral genetic variation in comparison to the North Pacific population and thus might also display limited adaptive polymorphism. We sampled 36 fin whales from the GOC and assessed genetic variation at exon 2 of the major histocompatibility complex class II DQB-1 genes responsible for eliciting immune responses. Three divergent alleles were found with higher nonsynonymous than synonymous substitution rates within the peptide-binding region positions as well as the likely retention of ancient alleles, indicating that positive selection has shaped diversity in this species. Limited levels of nonneutral polymorphism, in addition to previously described low levels of neutral polymorphism, are consistent with the results of previous studies on vertebrate populations that have remained small and demographically stable for a very long time. Such low genetic variation in the GOC fin whales could be explained by 2 demographic scenarios: an ancient isolated population with limited gene flow or a more recent founder event after the last glacial maximum with very restricted gene flow.

Genetic drift vs. natural selection in a long-term small isolated population: major histocompatibility complex class II variation in the Gulf of California endemic porpoise (Phocoena sinus).

Although many studies confirm long-term small isolated populations (e.g. island endemics) commonly sustain low neutral genetic variation as a result of genetic drift, it is less clear how selection on adaptive or detrimental genes interplay with random forces. We investigated sequence variation at two major histocompatibility complex (Mhc) class II loci on a porpoise endemic to the upper Gulf of California, México (Phocoena sinus, or vaquita). Its unique declining population is estimated around 500 individuals. Single-strand conformation polymorphism analysis revealed one putative functional allele fixed at the locus DQB (n = 25). At the DRB locus, we found two presumed functional alleles (n = 29), differing by a single nonsynonymous nucleotide substitution that could increase the stability at the dimer interface of alphabeta-heterodimers on heterozygous individuals. Identical trans-specific DQB1 and DRB1 alleles were identified between P. sinus and its closest relative, the Burmeister's porpoise (Phocoena spinipinnis). Comparison with studies on four island endemic mammals suggests fixation of one allele, due to genetic drift, commonly occurs at the DQA or DQB loci (effectively neutral). Similarly, deleterious alleles of small effect are also effectively neutral and can become fixed; a high frequency of anatomical malformations on vaquita gave empirical support to this prediction. In contrast, retention of low but functional polymorphism at the DRB locus was consistent with higher selection intensity. These observations indicated natural selection could maintain (and likely also purge) some crucial alleles even in the face of strong and prolonged genetic drift and inbreeding, suggesting long-term small populations should display low inbreeding depression. Low levels of Mhc variation warn about a high susceptibility to novel pathogens and diseases in vaquita.

Molecular identification of Vibrio harveyi-related isolates associated with diseased aquatic organisms.

Fifty strains belonging to Vibrio harveyi, Vibrio campbellii, and the recently described Vibrio rotiferianus, were analysed using phenotypic and genomic techniques with the aim of analysing the usefulness of the different techniques for the identification of V. harveyi-related species. The species V. harveyi and V. campbellii were phenotypically indistinguishable by more than 100 phenotypic features. Thirty-nine experimental strains were phenotypically identified as V. harveyi, but FAFLP, REP-PCR, IGS-PCR and DNA-DNA hybridization proved that they in fact belong to the species V. campbellii. Similar groupings were found among all fingerprinting methodologies (except IGS-PCR). Thirty-two experimental strains clustered with the V. campbellii type and one reference strain; seven strains clustered with the V. harveyi type and three reference strains; and the type and four reference strains of V. rotiferianus grouped together. The correlations between DNA-DNA hybridization and the genomic fingerprinting by FAFLP and (GTG)(5)-PCR were found to be above 0.68 and statistically significant, suggesting the value of the latter techniques for the reliable identification of V. harveyi-related species. The results presented indicate that strains phenotypically identified as V. harveyi are in fact V. campbellii; these findings position V. campbellii as an important species involved in diseases of reared aquatic organisms.

Cr(VI) reduction in a chromate-resistant strain of Candida maltosa isolated from the leather industry.

A Cr(VI)-resistant yeast was isolated from tanning liquors from a leather factory in Leon, Guanajuato, Mexico. Based on morphological and physiological analyses and the D1/D2 domain sequence of the 26S rDNA, the yeast was identified as Candida maltosa. Resistance of the strain to high Cr(VI) concentrations and its ability to chemically reduce chromium was studied. When compared to the three laboratory yeasts Candida albicans, Saccharomyces cerevisiae and Yarrowia lipolytica, the C. maltosa strain was found to tolerate chromate concentrations as high as 100 micro g/ml. In addition to this phenotypic trait, the C. maltosa strain showed ability to reduce Cr(VI). Chromate reduction occurred both in intact cells (grown in culture medium or in soil containing chromate) as well as in cell-free extracts. NADH-dependent chromate reductase activity was found associated with soluble protein and, to a lesser extent, with the membrane fraction.