Identification and mechanism determination of the efflux pump subunit amrB gene mutations linked to gentamicin susceptibility in clinical Burkholderia pseudomallei from Malaysian Borneo.

The bacterium Burkholderia pseudomallei is typically resistant to gentamicin but rare susceptible strains have been isolated in certain regions, such as Thailand and Sarawak, Malaysia. Recently, several amino acid substitutions have been reported in the amrB gene (a subunit of the amrAB-oprA efflux pump gene) that confer gentamicin susceptibility. However, information regarding the mechanism of the substitutions conferring the susceptibility is lacking. To understand the mechanism of amino acid substitution that confers susceptibility, this study identifies the corresponding mutations in clinical gentamicin-susceptible B. pseudomallei isolates from the Malaysian Borneo (n = 46; Sarawak: 5; Sabah: 41). Three phenotypically confirmed gentamicin-susceptible (GENs) strains from Sarawak, Malaysia, were screened for mutations in the amrB gene using gene sequences of gentamicin-resistant (GENr) strains (QEH 56, QEH 57, QEH20, and QEH26) and publicly available sequences (AF072887.1 and BX571965.1) as the comparator. The effect of missense mutations on the stability of the AmrB protein was determined by calculating the average energy change value (ΔΔG). Mutagenesis analysis identified a polymorphism-associated mutation, g.1056 T > G, a possible susceptible-associated in-frame deletion, Delta V412, and a previously confirmed susceptible-associated amino acid substitution, T368R, in each of the three GENs isolates. The contribution of Delta V412 needs further confirmation by experimental mutagenesis analysis. The mechanism by which T368R confers susceptibility, as elucidated by in silico mutagenesis analysis using AmrB-modeled protein structures, is proposed to be due to the location of T368R in a highly conserved region, rather than destabilization of the AmrB protein structure.

Identification of Sutcliffiella horikoshii strains in an immunobiological pharmaceutical industry facility.

The pharmaceutical industry must comply with the requirements for good manufacturing practices to reduce inherent contamination risks in the production process. Bacillus and related genera are among the main bacterial isolated from clean areas, raw material, and products in the pharmaceutical industries, but the correct identification of these species is still a challenge. The aim of this study was to characterize by phenotyping, protein profiling, and 16S rRNA gene sequencing Sutcliffiellahorikoshii strains (n = 6) isolated from an immunobiological pharmaceutical facility, and to propose the reclassification of Bacillus tianshenii to the genus Sutcliffiella, and Sutcliffiella tianshenii sp. nov. The strains were characterized by VITEK®2, matrix-assisted laser desorption ionization-time of flight/mass spectrometry (MALDI-TOF/MS) using VITEK®MS, and 16S rRNA gene sequencing analysis. MALDI-TOF/MS did not identify any strains that were identified by 16S rRNA as S. horikoshii. VITEK®2 showed false-positive results, with misidentification as B. sporothermodurans (reclassified as Heyndrickxia sporothermodurans) and Geobacillus thermoleovorans. After MALDI-TOF/MS database expansion, with the creation of SuperSpectrum, the strains were correctly identified as S. horikoshii. This study is the first report of isolation of S. horikoshii strains from a pharmaceutical industry. More studies are necessary to better understand the ability of S. horikoshii to contaminate the environment and products.

Characterization of a unique Sikkimese yak population of India: a multivariate approach.

Native Sikkimese yak in Sikkim state of India is a pastoral treasure being raised through centuries-old transhumance practices and has evolved in response to natural and man-made selection. Currently, the population of Sikkimese yak is at risk with about five thousand total headcounts. Characterization is essential for taking appropriate decisions for conservation of any endangered population. In an attempt to phenotypically characterize the Sikkimese yaks, this study recorded phenotypic morphometric traits information, viz., body length (LG), height at withers (HT), heart girth (HG), paunch girth (PG), horn length (HL), horn circumference (HC), distance between horns (DbH), ear length (EL), face length (FL), face width (FW), and tail length with switch (TL), on 2154 yaks of both sexes. Multiple correlation estimation highlighted that HG and PG, DbH and FW, and EL and FW were highly correlated. Using principal component analysis, LG, HT, HG, PG, and HL were found to be the most important traits for phenotypic characterization of Sikkimese yak animals. Discriminant analysis based on different locations of Sikkim hinted at the existence of two separate clusters, however, broadly, phenotypic uniformity could be observed. Subsequent genetic characterization can offer greater insights and can pave the way for future breed registration and conservation of the population.

Genomic evidence for two pathways of formaldehyde oxidation and denitrification capabilities of the species Paracoccus methylovorus sp. nov.

Three strains (H4-D09T, S2-D11 and S9-F39) of a member of the genus Paracoccus attributed to a novel species were isolated from topsoil of temperate grasslands. The genome sequence of the type strain H4-D09T exhibited a complete set of genes required for denitrification as well as methylotrophy. The genome of H4-D09T included genes for two alternative pathways of formaldehyde oxidation. Besides the genes for the canonical glutathione (GSH)-dependent formaldehyde oxidation pathway, all genes for the tetrahydrofolate-formaldehyde oxidation pathway were identified. The strain has the potential to utilize methanol and/or methylamine as a single carbon source as evidenced by the presence of methanol dehydrogenase (mxaFI) and methylamine dehydrogenase (mau) genes. Apart from dissimilatory denitrification genes (narA, nirS, norBC and nosZ), genes for assimilatory nitrate (nasA) and nitrite reductases (nirBD) were also identified. The results of phylogenetic analysis based on 16S rRNA genes coupled with riboprinting revealed that all three strains represented the same species of genus Paracoccus. Core genome phylogeny of the type strain H4-D09T indicated that Paracoccus thiocyanatus and Paracoccus denitrificans are the closest phylogenetic neighbours. The average nucleotide index (ANI) and digital DNA-DNA hybridization (dDDH) with the closest phylogenetic neighbours revealed genetic differences at the species level, which were further substantiated by differences in several physiological characteristics. The major respiratory quinone is Q-10, and the predominant cellular fatty acids are C18 : 1ω7c, C19 : 0cyclo ω7c, and C16 : 0, which correspond to those detected in other members of the genus. The polar lipid profile consists of a diphosphatidylglycerol (DPG), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylcholine (PC), aminolipid (AL), glycolipid (GL) and an unidentified lipid (L).On the basis of our results, we concluded that the investigated isolates represent a novel species of the genus Paracoccus, for which the name Paracoccus methylovorus sp. nov. (type strain H4-D09T=LMG 31941T= DSM 111585T) is proposed.

ToxR modulates biofilm formation in fish pathogen Vibrio harveyi.

Vibrio harveyi is a common aquaculture pathogen causing diseases in a variety of aquatic animals. toxR, a conserved virulence-associated gene in vibrios, is identified in V. harveyi 345, a pathogenic strain isolated from diseased fish. In this study, to gain insight into function of ToxR in V. harveyi, an in-frame deletion of the toxR gene was constructed to reveal the role of ToxR in the physiology and virulence of V. harveyi. The statistical analysis showed no significant differences in the growth ability, motility, extracellular protease secretion, antibiotic susceptibility, virulence by intraperitoneal injection and the ability of V. harveyi to colonize the spleen and liver tissues of the pearl gentian grouper between the wild-type (WT) and the toxR mutant. However, the deletion of toxR increased the biofilm formation. The structure of the V. harveyi biofilm was further analysed by using scanning electron microscopy (SEM) and confocal laser scanning microscopy, and the results showed that deletion of toxR increased the number and density of V. harveyi biofilm. Since biofilm production is flagella, exopolysaccharide (EPS) and lipopolysaccharide dependent, 16 of V. harveyi biofilm-related genes were selected for further analysis. Based on quantitative real-time reverse transcription-PCR, the expression levels of these genes, including genes flrB, motY and mshA, flaE, flrA and gmhD, were significantly up-regulated in the ΔtoxR+ strain as compared with the WT+ and C-ΔtoxR strains during the early and mid-exponential, while epsG, flaA, flaE, flgD, flgE, flrB, flrC, lpxB, motY, mshA and scrG genes were inhibited because of deletion of the toxR gene in the stationary growth phase. Our results indicate that ToxR plays an important role in controlling the biofilm in V. harveyi.

Typology and characteristics of indigenous goats and production systems in different agro-ecological zones of Tanzania.

Tanzania has a goat population of about 24.8 million most of which belong to the Small East African breed distributed in almost all agro-ecological zones. The different goat populations and the production system in which they are raised are not well characterized depriving animal breeders useful information in designing and running improvement and conservation programs. Therefore, the study was conducted in all agro-ecological zones in Tanzania to characterize the indigenous goats and the production system in which they are raised. Data on animals were collected from 688 randomly selected adult female goats and for production system description; 220 households were interviewed. Analysis of variance and discriminant analysis were used on quantitative data, while frequency analysis was used on qualitative data. Income generation and meat production were the primary goat rearing objectives. More than 55% of respondents grazed their animals freely in communal lands where natural pasture was the chief feed resource. Mating was mainly uncontrolled with apron and castration being used by goat keepers as mating control methods. Common diseases were contagious caprine pleural pneumonia and helminthiasis. Feed shortage, prevalence of diseases, and water scarcity were the major goat production constraints. There were morphological variations between and within these goat populations, and based on quantitative data, the goats were categorized into two groups. High twinning was observed in Ujiji and Lindi goats and low for Sukuma. The dominant coat color was plain white in Pare, Gogo, Maasai, and Tanga. Other coat color patterns were mixed black and white for Sukuma, reddish-brown for Lindi, black and reddish-brown for Ujiji, and white and reddish-brown for Pwani and Maasai. High within population variation is observed which is important as it can be used as a basis for genetic improvement through selection.

Human adipose-derived stromal/stem cells are distinct from dermal fibroblasts as evaluated by biological characterization and RNA sequencing.

Human adipose-derived stromal/stem cells (ASC) have immunomodulatory properties and the potential to differentiate into several cell lines, important for application in regenerative medicine. However, the contamination with dermal fibroblasts (FIB) can impair the beneficial effects of ASC in cell therapy. It is then essential to develop new strategies that contribute to the distinction between these two cell types. In this study, we performed functional assays, high-throughput RNA sequencing (RNA-Seq) and quantitative PCR (qPCR) to find new markers that can distinguish ASC and FIB. We showed that ASC have adipogenic and osteogenic differentiation capacity and alkaline phosphatase activity, not observed in FIB. Gene expression variation analysis identified more than 2000 differentially expressed genes (DEG) between these two cell types. We validated 16 genes present in the list of DEG, including the alkaline phosphatase gene (ALPL). In conclusion, we showed that ASC and FIB have distinct biological properties as demonstrated by alkaline phosphatase activity and differentiation capacity, besides having different gene expression profiles. SIGNIFICANCE OF THE STUDY: Although many differences between stromal stem cells derived from human adipose tissue (ASC) and human dermal fibroblasts (FIB) are described, it is still difficult to find specific markers to differentiate them. This problem can interfere with the therapeutic use of ASC. This work aimed to find new markers to differentiate these two cell populations. Our findings suggest that these cells can be distinguished by biological and molecular characteristics, such as adipogenic and osteogenic differentiation, alkaline phosphatase activity and differential gene expression profiles. The DEG were related to the regulation of the cell cycle, development process, structural organization of the cell and synthesis of the extracellular matrix. This study helps to find new cellular markers to distinguish the two populations and to better understand the properties of these cells, which can improve cell therapy.

R-SNARE FgSec22 is essential for growth, pathogenicity and DON production of Fusarium graminearum.

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) facilitate intracellular vesicle trafficking and membrane fusion in eukaryotic cells, and play a vital role in growth, development and pathogenicity of phytopathogens. Fusarium head blight (FHB) caused by F. graminearum is one of the most devastating diseases of wheat and barley worldwide. Sec22 is a member of the SNARE family of proteins and its homologues have been shown to have diverse biological roles in different organisms. However, the functions of this protein in the development and pathogenesis of F. graminearum are currently unknown. In this study, we employed integrated biochemical, microbiological and molecular genetic approaches to investigate the roles of FgSec22 in F. graminearum. Our data reveal that this SNARE protein is localized to endoplasmic reticulum (ER) and is indispensable for normal conidiation, conidial morphology and pathogenesis of this phytopathogenic fungus. Our biochemical assay of deoxynivalenol (DON) reveals the active involvement of this protein in the production of this mycotoxin in F. graminearum. This has further been confirmed by qRT-PCR analyses of trichothecene (TRI) genes' expression where the ΔFgsec22 deletion mutant demonstrated a significant down-regulation of these genes in comparison to the wild-type PH-1. Unlike the wild-type and the complemented strain, the mutant strain presents a remarkable defect in colony formation which reflects the critical role it plays in vegetative growth. Collectively, our data support that the SNARE protein FgSec22 is required for vegetative growth, pathogenesis and DON biosynthesis in F. graminearum.

New insights into halophilic prokaryotes isolated from salting-ripening anchovies (Engraulis anchoita) process focused on histamine-degrading strains.

Salted and ripened fish foods are susceptible to cause histamine poisoning. The present study focuses on microbial histamine degradation from high salted fermented fishery products to deepen our understanding about this new and growing field of research. As a result of this first study related to salted-ripened anchovies (Engraulis anchoita), fifty seven moderate and extreme halophilic microbial isolates from salt and salted-ripened anchovy processes were characterized in terms of their phenotype and histamine-degrading capacity. Only 7%-4 isolates-were able to degrade histamine. None of the histamine-degrading isolates presented proteolytic and/or lipolytic activity. One of them designated A18 was chemotactic toward histamine, an interesting property not previously reported for that chemoattractant. However, the S18 and A18 isolates, genotypically identified as Halobacterium sp. and Halomonas sp. respectively, produced indole and/or H2S, both undesirable characteristics associated to off-flavors occurrence. On the other hand, A28 and S20, identified as Halovibrio sp. and Halobacterium sp. respectively, presented desirable properties, such as cytochrome oxidase and catalase activity, and non-production of H2S and indole. These strains also showed characteristics previously reported as dominant in the ripened stage. The results are promising, and A28 and S20 may have the desirable features to improve the anchovy salting-ripening process.

Phenotypic and Genotypic Characterization of Enterobacteriaceae Producing Oxacillinase-48-Like Carbapenemases, United States.

Oxacillinase (OXA)-48-like carbapenemases remain relatively uncommon in the United States. We performed phenotypic and genotypic characterization of 30 Enterobacteriaceae producing OXA-48-like carbapenemases that were recovered from patients during 2010-2014. Isolates were collected from 12 states and not associated with outbreaks, although we could not exclude limited local transmission. The alleles ß-lactamase OXA-181 (blaOXA-181) (43%), blaOXA-232 (33%), and blaOXA-48 (23%) were found. All isolates were resistant to ertapenem and showed positive results for the ertapenem and meropenem modified Hodge test and the modified carbapenem inactivation method; 73% showed a positive result for the Carba Nordmann-Poirel test. Whole-genome sequencing identified extended-spectrum ß-lactamase genes in 93% of isolates. In all blaOXA-232 isolates, the gene was on a ColKP3 plasmid. A total of 12 of 13 isolates harboring blaOXA-181 contained the insertion sequence ΔISEcp1. In all isolates with blaOXA-48, the gene was located on a TN1999 transposon; these isolates also carried IncL/M plasmids.

Within-a-Day Detection and Rapid Characterization of Carbapenemase by Use of a New Carbapenem Inactivation Method-Based Test, CIMplus.

The dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major threat to public health. Rapid and accurate detection of CPE is essential for initiating appropriate antimicrobial treatment and establishing infection control measures. The carbapenem inactivation method (CIM), which has good sensitivity and specificity but a detection time of 20 h, was recently described. In this study, we evaluated the performances of a new version, the CIMplus test, which allows detection of carbapenemases in 8 h and characterization of carbapenemase classes, according to the Ambler classification, in 20 h. A panel of 110 carbapenem-resistant Enterobacteriaceae strains, including 92 CPE strains (with NDM, VIM, IMP, KPC, GES, OXA-48, and OXA-48-like enzymes), was used to evaluate test performance. Carbapenemase activity was detected at 8 h and 20 h. Characterization of carbapenemase classes, using specific inhibitors, was possible in 20 h. The CIMplus test had sensitivities of 95.7% and 97.8% at 8 h and 20 h, respectively, and a specificity of 94.4%, independent of the culture duration. Using a decision algorithm, this test was successful in identifying the carbapenemase class for 98.9% of tested CPE isolates (87/88 isolates). In total, the characterization was correct for 100%, 96.9%, and 100% of Ambler class A, B, and D isolates, respectively. Therefore, this test allows detection of carbapenemase activity in 8 h and characterization of carbapenemase classes, according to the Ambler classification, in 20 h. The CIMplus test represents a simple, affordable, easy-to-read, and accurate tool that can be used without any specific equipment.

Phenotypic characterization of pathogenic Cronobacter spp. strains.

BACKGROUND: Cronobacter species are Gram-negative opportunistic foodborne pathogens that may cause enterocolitis, bacteremia and meningitis in neonates and premature neonates. Lipopolysaccharide (LPS) serves as the major component of the outer membrane of cell, is a potential virulence factor for Cronobacter. METHODS: Given the potential importance of this molecule in infection and virulence, SDS-PAGE of LPS, MS and TLC characterization of phospholipids and phenotypic characterization of Cronobacter spp. strains were carried out. RESULT: The phospholipids from Cronobacter yielded four major peaks at m/z 719.9, 733.9, 747.9 and 773.9 in the spectrum. All Cronobacter showed O-antigen bands except C. muytjensii ATCC 51329. When Cronobacter defect O-antigen, the outer membrane permeability and cell surface hydrophobicities are increased. All Cronobacter are able to grow under pH 5.0 condition and able to grow under 6% NaCl concentration. C. dublinensis DSM 18705 has a higher infection rate to Caco-2 cells than other Cronobacter. CONCLUSION: Invasion of pathogens into a host cell is critical component to an infectious case. And C. dublinensis DSM 18705 has a higher infection rate to Caco-2 cells than other Cronobacter.

Xanthomonas prunicola sp. nov., a novel pathogen that affects nectarine (Prunus persica var. nectarina) trees.

Three isolates obtained from symptomatic nectarine trees (Prunus persica var. nectarina) cultivated in Murcia, Spain, which showed yellow and mucoid colonies similar to Xanthomonas arboricola pv. pruni, were negative after serological and real-time PCR analyses for this pathogen. For that reason, these isolates were characterized following a polyphasic approach that included both phenotypic and genomic methods. By sequence analysis of the 16S rRNA gene, these novel strains were identified as members of the genus Xanthomonas, and by multilocus sequence analysis (MLSA) they were clustered together in a distinct group that showed similarity values below 95 % with the rest of the species of this genus. Whole-genome comparisons of the average nucleotide identity (ANI) of genomes of the strains showed less than 91 % average nucleotide identity with all other species of the genus Xanthomonas. Additionally, phenotypic characterization based on API 20 NE, API 50 CH and BIOLOG tests differentiated the strains from the species of the genus Xanthomonas described previously. Moreover, the three strains were confirmed to be pathogenic on peach (Prunus persica), causing necrotic lesions on leaves. On the basis of these results, the novel strains represent a novel species of the genus Xanthomonas, for which the name Xanthomonas prunicola is proposed. The type strain is CFBP 8353 (=CECT 9404=IVIA 3287.1).

Phenotypic characterization of beef cattle breeds and production practices in Liberia.

Phenotypic characterization of animal genetic resources (AnGR) involves identification of distinct breed populations, describing their external and production characteristics in a given environment and management, and taking into account the social and economic factors that affect them. A survey involving 346 livestock farmers was conducted in the 15 counties of Liberia to collect data on production practices and phenotypic characteristics of beef cattle. A pre-tested structured questionnaire, focus group discussions, and in-depth interviews were utilized in data collection. Phenotypic descriptors were measured using a measuring tape and weighing scale. The purpose for keeping beef cattle, preferred cattle traits, and challenges of beef cattle farmers were also assessed. Liberia's beef cattle are predominantly of Ndama (50%), Muturu (38%), and Zebu (11%) breeds. Beef cattle are mainly kept on free range with little investment in housing, feeding, and veterinary care. Beef cattle are raised mainly for income generation, with high cost and low availability of feed (32%), insufficient housing (25%), diseases (21%), and high costs of veterinary medicines (12%) being the main challenges faced by the farmers. Liberian beef cattle were characterized as being docile (53%) or moderate (33%) in temperament, with solid/uniform coat color pattern (61%), black coat color (26%), and non-pigmented skin (84%). The animals had mainly erect or lateral ears, straight back lines, and are sloped rumps. Disease (54%) and drought tolerance (30%) were the main adaptive traits reported. The body weight of beef cattle sampled ranged from 213 to 226 kg, the body length from 119 to 122 cm, and the heart girth from 134 to 140 cm. The government of Liberia should improve policies on local AnGR management and support stakeholder institutions to ensure their sustainable use and conservation.

Preserved dopaminergic homeostasis and dopamine-related behaviour in hemizygous TH-Cre mice.

Cre-driver mouse lines have been extensively used as genetic tools to target and manipulate genetically defined neuronal populations by expression of Cre recombinase under selected gene promoters. This approach has greatly advanced neuroscience but interpretations are hampered by the fact that most Cre-driver lines have not been thoroughly characterized. Thus, a phenotypic characterization is of major importance to reveal potential aberrant phenotypes prior to implementation and usage to selectively inactivate or induce transgene expression. Here, we present a biochemical and behavioural assessment of the dopaminergic system in hemizygous tyrosine hydroxylase (TH)-Cre mice in comparison to wild-type (WT) controls. Our data show that TH-Cre mice display preserved dopaminergic homeostasis with unaltered levels of TH and dopamine as well as unaffected dopamine turnover in striatum. TH-Cre mice also show preserved dopamine transporter expression and function supporting sustained dopaminergic transmission. In addition, TH-Cre mice demonstrate normal responses in basic behavioural paradigms related to dopaminergic signalling including locomotor activity, reward preference and anxiolytic behaviour. Our results suggest that TH-Cre mice represent a valid tool to study the dopamine system, though careful characterization must always be performed to prevent false interpretations following Cre-dependent transgene expression and manipulation of selected neuronal pathways.

Rapid Resolution of Blended or Composite Multigenic Disease in Infants by Whole-Exome Sequencing.

Whole-exome sequencing identified multiple genetic causes in 2 infants with heterogeneous disease. Three gene defects in the first patient explained all symptoms, but manifestations were overlapping (blended phenotype). Two gene defects in the second patient explained nonoverlapping symptoms (composite phenotype). Whole-exome sequencing rapidly and comprehensively resolves heterogeneous genetic disease.

Molecular and phenotypic characterization of endophytic bacteria isolated from sulla nodules.

In the current study, bacterial diversity was investigated in root nodules of Sulla pallida and Sulla capitata. The isolates were analyzed on the basis of their phenotypic and molecular characteristics. The phylogenetic analysis based on 16S rRNA and housekeeping genes (recA and atpD) showed that the isolated bacteria related to Sinorhizobium, Neorhizobium, Phyllobacterium, Arthrobacter, Variovorax and Pseudomonas genera. This is the first report of Neorhizobium genus associated with Hedysarum genus. Phenotypically, all strains tolerate the elevated temperature of 40 °C, and salt stress at a concentration of 2%. In addition, the isolates failed to induce nodulation on their original host; and the symbiotic genes could not be amplified, suggesting that these strains are endophytic bacteria.

Phenotypic diversity, major genes and production potential of local chickens and guinea fowl in Tamale, northern Ghana.

OBJECTIVE: Our study provides information on phenotypes of local chickens and guinea fowl and their body measures as well as on major genes in local chickens in northern Ghana. METHODS: Qualitative and morphometric traits were recorded on 788 local chickens and 394 guinea fowl in urban households in Tamale, Ghana. RESULTS: The results showed considerable variation of color traits and numerous major genes in local chickens, while color variations and related genotypes in guinea fowl were limited. In local chickens, white was preferred for plumage, whereas dark colors were preferred for beak and shanks. More than half of the chickens carried at least one major gene, but the contributions of single gene carriers were low. All calculated allele frequencies were significantly lower than their expected Mendelian allele frequencies. We observed higher mean body weight and larger linear body measures in male as compared to female chickens. In female chickens, we detected a small effect of major genes on body weight and chest circumference. In addition, we found some association between feather type and plumage color. In guinea fowl, seven distinct plumage colors were observed, of which pearl grey pied and pearl grey were the most prevalent. Male pearl grey pied guinea fowl were inferior to pearl grey and white guinea fowl in terms of body weight, body length and chest circumference; their shank length was lower than that of pearl grey fowl. CONCLUSION: Considerable variation in qualitative traits of local chickens may be indicative of genetic diversity within local chicken populations, but major genes were rare. In contrast, phenotypic and genetic diversity in local guinea fowl is limited. Broader genetic diversity studies and evaluation of trait preferences of local poultry producers are required for the design of appropriate breeding programs.

The phenotypic characterization of yeast strains to stresses inherent to wine fermentation in warm climates.

AIMS: Climate change is exerting an increasingly profound effect on grape composition, microbiology, chemistry and the sensory aspects of wine. Identification of autochthonous yeasts tolerant to stress could help to alleviate this effect. METHODS AND RESULTS: Tolerance to osmotic pressure, ethanol and pH of 94 Saccharomyces cerevisiae strains and 29 strains non-Saccharomyces from the warm climate region DO 'Vinos de Madrid' (Spain) using phenotypic microarray and their fermentative behaviour were studied. The screening highlighted 12 strains of S. cerevisiae isolated from organic cellars with improved tolerance to osmotic stress, high ethanol concentrations and suitable fermentative properties. Screening of non-Saccharomyces spp. such as Lanchacea thermotolerans, Torulaspora delbrueckii, Schizosaccharomyces pombe and Mestchnikowia pulcherrima also highlighted tolerance to these stress conditions. CONCLUSIONS: This study confirmed the adaptation of native strains to the climatic conditions in each area of production and correlated these adaptations with good fermentation properties. Screening has revealed that identifying yeast strains adapted to fermentation stresses is an important approach for making quality wines in very warm areas. SIGNIFICANCE AND IMPACT OF THE STUDY: The results have special relevance because it is a pioneering study that has approached the problem of climate change for wines from a microbiological aspect and has analysed the situation in situ in wineries from a warm climate zone. Resistant strains were found with good biological properties; studying these strains for their stress response mechanisms during fermentation will be of interest to the wine making industry.

Intrastrain genomic and phenotypic variability of the commercial Saccharomyces cerevisiae strain Zymaflore VL1 reveals microevolutionary adaptation to vineyard environments.

The maintenance of microbial species in different environmental conditions is associated with adaptive microevolutionary changes that are shown here to occur within the descendants of the same strain in comparison with the commercial reference strain. However, scarce information is available regarding changes that occur among strain descendants during their persistence in nature. Herein we evaluate genome variations among four isolates of the commercial winemaking strain Saccharomyces cerevisiae Zymaflore VL1 that were re-isolated from vineyards surrounding wineries where this strain was applied during several years, in comparison with the commercial reference strain. Comparative genome hybridization showed amplification of 14 genes among the recovered isolates being related with mitosis, meiosis, lysine biosynthesis, galactose and asparagine catabolism, besides 9 Ty elements. The occurrence of microevolutionary changes was supported by DNA sequencing that revealed 339-427 SNPs and 12-62 indels. Phenotypic screening and metabolic profiles also distinguished the recovered isolates from the reference strain. We herein show that the transition from nutrient-rich musts to nutritionally scarce natural environments induces adaptive responses and microevolutionary changes promoted by Ty elements and by nucleotide polymorphisms that were not detected in the reference strain.