Empirical phenotyping and genome-wide association study reveal the association of panicle architecture with yield in Chenopodium quinoa.

Chenopodium quinoa manifests adaptability to grow under varying agro-climatic scenarios. Assessing quinoa germplasm's phenotypic and genetic variability is a prerequisite for introducing it as a potential candidate in cropping systems. Adaptability is the basic outcome of ecological genomics of crop plants. Adaptive variation predicted with a genome-wide association study provides a valuable basis for marker-assisted breeding. Hence, a panel of 72 quinoa plants was phenotyped for agro morphological attributes and association-mapping for distinct imperative agronomic traits. Inter simple sequence repeat (ISSR) markers were employed to assess genetic relatedness and population structure. Heatmap analysis showed three genotypes were early maturing, and six genotypes were attributed for highest yield. The SD-121-07 exhibited highest yield per plant possessing green, glomerulate shaped, compact density panicle with less leaves. However, SJrecm-03 yielded less exhibiting pink, intermediate shape, intermediate density panicles with less leaves. The phenotyping revealed strong correlation of panicle architecture with yield in quinoa. A genome-wide association study unraveled the associations between ISSR makers and agro-morphological traits. Mixed linear modes analysis yielded nine markers associated with eight traits at p ≤ 0.01. Moreover, ISSR markers significantly associated with panicle shape and leafiness were also associated with yield per plant. These findings contribute to the provision of authenticity for marker-assisted selection that ultimately would support quinoa breeding programs.

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.

Genetic and Phenotypic Virulence Potential of Non-O1/Non-O139 Vibrio cholerae Isolated from German Retail Seafood.

Non-O1 and non-O139 Vibrio cholerae (NOVC) can cause gastrointestinal infections in humans. Contaminated food, especially seafood, is an important source of human infections. In this study, the virulence potential of 63 NOVC strains isolated from retail seafood were characterized at the genotypic and phenotypic levels. Although no strain encoded the cholera toxin (CTX) and the toxin-coregulated pilus (TCP), several virulence factors, including the HlyA hemolysin, the cholix toxin ChxA, the heat-stable enterotoxin Stn, and genes coding for the type 3 and type 6 secretion systems, were detected. All strains showed hemolytic activity against human and sheep erythrocytes: 90% (n = 57) formed a strong biofilm, 52% (n = 33) were highly motile at 37 °C, and only 8% (n = 5) and 14% (n = 9) could resist ≥60% and ≥40% human serum, respectively. Biofilm formation and toxin regulation genes were also detected. cgMLST analysis demonstrated that NOVC strains from seafood cluster with clinical NOVC strains. Antimicrobial susceptibility testing (AST) results in the identification of five strains that developed non-wildtype phenotypes (medium and resistant) against the substances of the classes of beta-lactams (including penicillin, carbapenem, and cephalosporin), polymyxins, and sulphonamides. The phenotypic resistance pattern could be partially attributed to the acquired resistance determinants identified via in silico analysis. Our results showed differences in the virulence potential of the analyzed NOVC isolated from retail seafood products, which may be considered for further pathogenicity evaluation and the risk assessment of NOVC isolates in future seafood monitoring.

Characterization of avian pathogenic Escherichia coli isolates based on biofilm formation, ESBL production, virulence-associated genes, and phylogenetic groups.

Escherichia coli is a part of both animal and human commensal microbiota. Avian pathogenic E. coli (APEC) is responsible for colibacillosis in poultry, an economically important disease. However, the close similarities among APEC isolates make it difficult to differentiate between pathogenic and commensal bacteria. The aim of this study was to determine phenotypic and molecular characteristics of APEC isolates and to compare them with their in vivo pathogenicity indices. A total of 198 APEC isolates were evaluated for their biofilm-producing ability and extended-spectrum ß-lactamase (ESBL) production phenotypes. In addition, 36 virulence-associated genes were detected, and the isolates were classified into seven phylogenetic groups using polymerase chain reaction. The sources of the isolates were not associated with biofilms, ESBL, genes, or phylogroups. Biofilm and ESBL production were not associated with pathogenicity. Group B2 had the highest pathogenicity index. Groups B2 and E were positively associated with high-pathogenicity isolates and negatively associated with low-pathogenicity isolates. In contrast, groups A and C were positively associated with apathogenic isolates, and group B1 was positively associated with low-pathogenicity isolates. Some virulence-associated genes showed positive or negative associations with specific phylogenetic groups. None of the individual techniques produced results that correlated with the in vivo pathogenicity index. However, the combination of two techniques, namely, detection of virulence-associated genes and the phylogenetic groups, could help the classification of the isolates as pathogenic or commensal.

Developmental and epileptic encephalopathy in a young Italian woman with a de novo missense variant in the CLCN4 gene: A case report.

INTRODUCTION: Raynaud-Claes syndrome is a very rare X-linked condition, characterized by intellectual disability, impaired language development, brain abnormalities, facial dysmorphisms and drug-resistant epilepsy. It is caused by loss-of-function variants in the CLCN4 gene, which encodes the 2Cl-/H + exchanger ClC-4, prominently expressed in the hippocampus and cerebellum. Different genotypic variants have been described, each exhibiting specific phenotypic characteristics. The loss-of-function variant p.Gly544Arg in the CLCN4 gene has been described in only two male probands, but there are no reports on phenotypic characterization in females. CASE PRESENTATION: We present a 30-year-old Italian woman with early-onset drug-resistant epilepsy, developmental and epileptic encephalopathy, developmental delay, absence of verbal language development, behavioral impairment with autistic features, and clusters of seizures during catamenial periods. The interictal EEG showed slight inconstant slowing of the background rhythm, with abnormal frontal predominant mu like rhythm and generalized spike and polyspike wave discharges, which increased in frequency during drowsiness. A brain MRI showed slight cranio-encephalic asymmetry and a smaller size of the left hippocampus. The whole exome sequencing (WES) revealed a de novo heterozygous c.1630G > A variant in the CLCN4 gene, resulting in the amino acid substitution p.Gly544Arg (rs587777161), consistent with Raynaud-Claes syndrome. DISCUSSION AND CONCLUSION: Our patient is the first case of a de novo p.Gly544Arg variant of the CLCN4 gene in a female proband, confirming that female patients with Raynaud-Claes syndrome can be as severely affected as the male counterparts. Our case expands the phenotypic characterization of different genotypic CLCN4 variants, which can become crucial in the future for early diagnosis if targeted therapy becomes available.

Phenotypic and Genotypic Characterization and Antifungal Susceptibility of Sporothrix schenckii sensu stricto Isolated from a Feline Sporotrichosis Outbreak in Bangkok, Thailand.

Sporotrichosis, an invasive fungal infection caused by Sporothrix schenckii, has emerged in Southeast Asia, affecting cats and posing a potential zoonotic risk to humans. We evaluated 38 feline sporotrichosis cases in and around Bangkok, Thailand, from 2017 to 2021. The isolates were phenotypically and genotypically characterized. The cats infected with sporotrichosis were mainly young adults, males, and domestic short hairs with uncontrolled outdoor access, and they lived in Bangkok. All isolates showed low thermotolerance and converted to the yeast phase at 35 °C. Based on the internal transcribed spacer region of rDNA sequences, our strains belonged to S. schenckii sensu stricto and clustered with clinical clade D. Based on the concatenated tree of calmodulin and beta-tubulin genes, five groups of S. schenckii were generated, and the monophyletic clade, Group II, of Thai strains was recognized. In vitro antifungal susceptibility testing demonstrated that the MIC50 of our isolates to amphotericin B, itraconazole, and posaconazole were within the limit of the species-specific epidemiological cutoff values, suggesting that the organisms were the wild type. Addressing the outbreak of feline sporotrichosis in Thailand by providing guidelines for diagnosis and effective treatment may help control the spread of disease and reduce the risk of cat-transmitted sporotrichosis to humans.

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.

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.

Soil samples from sporotrichosis transmission belt area: Searching for fungal species and their antagonistic activity against Sporothrix brasiliensis.

Since 1998, the state of Rio de Janeiro, Brazil, has become a public health problem regarding sporotrichosis, a disease caused by Sporothrix spp. involving contact with infected cats. Efforts to isolate these species from environmental sources are not always successful. In our study, soil from residences situated in cities of Rio de Janeiro where cats with sporotrichosis live was collected and cultured an attempt to isolate Sporothrix spp. but it was not successful. However, other saprophytic fungal species were isolated from soil and identified and among them Purpureocillium lilacinum was the most frequent. From there, we decided to study the in vitro interaction of this species with S. brasiliensis, the principal agent that causes sporotrichosis in this state. The results showed that ten isolates of P. lilacinum inhibited the radial mycelial growth of S. brasiliensis with different percentage of inhibition. The interaction between them revealed the pattern described as overgrowth by antagonist. In conclusion, our data suggest that fungal species with very fast growth and capable of producing metabolites could hinder the growth of Sporothrix spp., it also opens the way for the identification of secondary metabolites with biological activity that could be tested against pathogenic fungi.

Torulaspora delbrueckii Phenotypic and Metabolic Profiling towards Its Biotechnological Exploitation.

Wine is a particularly complex beverage resulting from the combination of several factors, with yeasts being highlighted due to their fundamental role in its development. For many years, non-Saccharomyces yeasts were believed to be sources of spoilage and contamination, but this idea was challenged, and many of these yeasts are starting to be explored for their beneficial input to wine character. Among this group, Torulaspora delbrueckii is gaining relevance within the wine industry, owing to its low volatile acidity production, increased release of aromatic compounds and enhanced color intensity. In addition, this yeast was also attracting interest in other biotechnological areas, such as bread and beer fermentation. In this work, a set of 40 T. delbrueckii strains, of varied geographical and technological origins, was gathered in order to characterize the phenotypic behavior of this species, focusing on different parameters of biotechnological interest. The fermentative performance of the strains was also evaluated through individual fermentations in synthetic grape must with the isolates' metabolic profile being assessed by HPLC. Data analysis revealed that T. delbrueckii growth is significantly affected by high temperature (37 °C) and ethanol concentrations (up to 18%), alongside 1.5 mM SO2, showing variable fermentative power and yields. Our computation models suggest that the technological origin of the strains seems to prevail over the geographical origin as regards the influence on yeast properties. The inter-strain variability and profile of the products through the fermentative processes reinforce the potential of T. delbrueckii from a biotechnological point of view.

Phenotypic Characterization of a Virulent PRRSV-1 Isolate in a Reproductive Model With and Without Prior Heterologous Modified Live PRRSV-1 Vaccination.

Reproductive disorders induced by porcine reproductive and respiratory syndrome virus (PRRSV) cause high economic losses in the pig industry worldwide. In this study, we aimed to phenotypically characterize a virulent PRRSV-1 subtype 1 isolate (AUT15-33) in a reproductive model. Furthermore, the protective effect of a heterologous modified live virus vaccine (ReproCyc® PRRS EU) was evaluated. In addition, PRRSV AUT15-33 was genotypically compared to other well-characterized isolates. Sixteen gilts were equally divided into four groups: a vaccinated and infected group (V-I), a vaccinated and non-infected group (V-NI), a non-vaccinated and infected group (NV-I), and a non-vaccinated and non-infected (NV-NI) group. After PRRSV infection on gestation day 84, all gilts were clinically examined on a daily basis, and blood samples were taken at five timepoints. Necropsy was performed 3 weeks after infection. The fetal preservation status was assessed, and PRRSV RNA concentrations were measured in the blood and tissue samples from all gilts and fetuses. After infection, all four gilts in the NV-I group were viremic throughout 17 days post-infection (dpi), whereas two gilts in the V-I group were viremic at only one timepoint at 6 dpi. The viral load was significantly higher in gilt serum, tracheobronchial lymph nodes, uterine lymph nodes, maternal endometrium, and fetal placenta of NV-I gilts compared to the V-I ones (p < 0.05). Moreover, the preservation status of the fetuses derived from NV-I gilts was significantly impaired (55.9% of viable fetuses) compared to the other groups (p < 0.001). Upon comparison with other known isolates, the phylogenetic analyses revealed the closest relation to a well-characterized PRRSV-1 subtype 1 field isolate from Belgium. In conclusion, the high virulence of AUT15-33 was phenotypically confirmed in an experimental reproductive model. The vaccination of the gilts showed promising results in reducing viremia, fetal damage, and transplacental transmission of the PRRSV-1 strain characterized in this study.

Development of a Genomics-Based Approach To Identify Putative Hypervirulent Nontyphoidal Salmonella Isolates: Salmonella enterica Serovar Saintpaul as a Model.

While differences in human virulence have been reported across nontyphoidal Salmonella (NTS) serovars and associated subtypes, a rational and scalable approach to identify Salmonella subtypes with differential ability to cause human diseases is not available. Here, we used NTS serovar Saintpaul (S. Saintpaul) as a model to determine if metadata and associated whole-genome sequence (WGS) data in the NCBI Pathogen Detection (PD) database can be used to identify (i) subtypes with differential likelihoods of causing human diseases and (ii) genes and single nucleotide polymorphisms (SNPs) potentially responsible for such differences. S. Saintpaul SNP clusters (n = 211) were assigned different epidemiology types (epi-types) based on statistically significant over- or underrepresentation of human clinical isolates, including human associated (HA; n = 29), non-human associated (NHA; n = 23), and other (n = 159). Comparative genomic analyses identified 384 and 619 genes overrepresented among isolates in 5 HA and 4 NHA SNP clusters most significantly associated with the respective isolation source. These genes included 5 HA-associated virulence genes previously reported to be present on Gifsy-1/Gifsy-2 prophages. Additionally, premature stop codons in 3 and 7 genes were overrepresented among the selected HA and NHA SNP clusters, respectively. Tissue culture experiments with strains representing 4 HA and 3 NHA SNP clusters did not reveal evidence for enhanced invasion or intracellular survival for HA strains. However, the presence of sodCI (encoding a superoxide dismutase), found in 4 HA and 1 NHA SNP clusters, was positively correlated with intracellular survival in macrophage-like cells. Post hoc analyses also suggested a possible difference in intracellular survival among S. Saintpaul lineages. IMPORTANCE Not all Salmonella isolates are equally likely to cause human disease, and Salmonella control strategies may unintentionally focus on serovars and subtypes with high prevalence in source populations but are rarely associated with human clinical illness. We describe a framework leveraging WGS data in the NCBI PD database to identify Salmonella subtypes over- and underrepresented among human clinical cases. While we identified genomic signatures associated with HA/NHA SNP clusters, tissue culture experiments failed to identify consistent phenotypic characteristics indicative of enhanced human virulence of HA strains. Our findings illustrate the challenges of defining hypo- and hypervirulent S. Saintpaul and potential limitations of phenotypic assays when evaluating human virulence, for which in vivo experiments are essential. Identification of sodCI, an HA-associated virulence gene associated with enhanced intracellular survival, however, illustrates the potential of the framework and is consistent with prior work identifying specific genomic features responsible for enhanced or reduced virulence of nontyphoidal Salmonella.

Assessment of VITEK® 2, MALDI-TOF MS and full gene 16S rRNA sequencing for aerobic endospore-forming bacteria isolated from a pharmaceutical facility.

VITEK®2, MALDI-TOF MS and 16S rRNA sequencing were evaluated for the identification of aerobic endospore-forming bacteria (AEB) from a pharmaceutical facility. MALDI-TOF MS demonstrated higher accuracy compared to VITEK®2, although both databases were insufficient to identify AEB species. Sequencing was the best methodology, but unable to identify closely related species.

Epidemiology of Pityriasis versicolor in Tunisia: Clinical features and characterization of Malassezia species.

Malassezia (M.) genus includes commensal yeasts of increasing medical importance, as they result in many diseases, ranging from pityriasis versicolor (PV) to systemic infections. Previous studies reported geographical variations in distribution of Malassezia species in PV lesions. The aims of the current study were to define the clinico-demographic features of PV in Tunisia, to characterize Malassezia isolates using phenotypic and molecular techniques and to find out any association between species and clinico-demographic parameters. In total, 120 PV patients were enrolled in this study. Skin scrapings were collected and inoculated on Sabouraud agar and modified Dixon medium. Malassezia species were identified using conventional phenotypic methods and 26 s rDNA PCR-RFLP. The highest prevalence of PV was observed among young adults' group. The most affected body areas were the back and neck. In overall, 50.8% and 35% of PV cases had pruritus and history of recurrence respectively. The overall concordance between phenotypic and molecular methods was high (80.95%). The discordant results are rather due to the presence of multiple species in a single culture than true misidentification. Using PCR-RFLP, M. furfur was the most isolated species (38.7%) followed by M. globosa (37.7%), M. restricta and M. sympodialis. No statistically significant association was noted between Malassezia spp. and clinico-demographic characteristics. Unlike many reports from temperate climate countries, M. furfur and M. globosa along together were the most frequently isolated species in Tunisian PV patients. Although phenotypic methods remain simple and cost-effective, molecular techniques are considered as fast and accurate methods for diagnosis purposes.

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.

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.

Phenotypic Characterization and Identification of Potential L-Asparaginase-Producing Thermohalophilic Bacteria from Wawolesea Hot Spring, North Konawe, Southeast Sulawesi, Indonesia.

<b>Background and Objective:</b> L-asparaginase-producing thermohalophilic bacteria have the potential of producing an enzyme tolerant to high heat and salt levels. This enzyme, L-asparaginase, can be used as a biological agent for the cancer therapy of acute lymphoblastic leukemia and melanosarcoma as it has a specific ability to inhibit the formation of nutrients for cancer cells. This enzyme is also used effectively in food industries operating at high temperatures due to its ability to reduce acrylamide, a trigger of cancer cells. This study sought to figure out the phenotypic characters of and identify potential L-asparaginase-producing thermohalophilic bacteria from Wawolesea Hot Spring, North Konawe, Southeast Sulawesi. <b>Materials and Methods:</b> The characterization conducted on potential L-asparaginase-producing thermohalophilic bacterial isolates consisted of the following: Colony morphological characterization, covering the shapes, edges, internal structures, elevations and colours of the colonies, cell morphological characterization, covering gram staining, endospore formation and motility, biochemical characterization, covering catalase, Methyl Red and Voges Proskauer (MR-VP), gelatin hydrolysis, citrate, indole and carbohydrate fermentation tests and physiological characterization, covering pH effect, salinity, oxygen demand and temperature effect tests. Bacterial isolate identification was carried out in two stages, namely phenetic identification based on the phenotypic characterization data determine through a preliminary identification and numeric-phenetic identification. <b>Results:</b> The characterization results showed that the bacterial isolates AAT 1.4, AAT 3.2 and CAT 3.4 were <i>bacillus</i>-shaped, Gram-positive, motile, catalase-positive and aerobic. Based on the numeric-phenetic analysis results, the isolates AAT 1.4 and CAT 3.4 had a 92.9% similarity to <i>Bacillus subtilis</i>, while isolate AAT 3.2 had a 92.9% similarity to <i>Brevibacillus limnophilus</i>. <b>Conclusion:</b> According to the numeric-phenetic analysis results, the isolates AAT 1.4 and CAT 3.4 belong to the species <i>Bacillus subtilis</i>, while isolate AAT 3.2 belongs to the species <i>Brevibacillus limnophilus</i>.

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.

Genotypic and Phenotypic Characterization of Antimicrobial Resistance Profiles in Non-typhoidal Salmonella enterica Strains Isolated From Cambodian Informal Markets.

Non-typhoidal Salmonella enterica is a pathogen of global importance, particularly in low and middle-income countries (LMICs). The presence of antimicrobial resistant (AMR) strains in market environments poses a serious health threat to consumers. In this study we identified and characterized the genotypic and phenotypic AMR profiles of 81 environmental S. enterica strains isolated from samples from informal markets in Cambodia in 2018-2019. AMR genotypes were retrieved from the NCBI Pathogen Detection website (https://www.ncbi.nlm.nih.gov/pathogens/) and using ResFinder (https://cge.cbs.dtu.dk/services/) Salmonella pathogenicity islands (SPIs) were identified with SPIFinder (https://cge.cbs.dtu.dk/services/). Susceptibility testing was performed by broth microdilution according to the Clinical and Laboratory Standards Institute (CLSI) standard guidelines M100-S22 using the National Antimicrobial Resistance Monitoring System (NARMS) Sensititre Gram Negative plate. A total of 17 unique AMR genes were detected in 53% (43/81) of the isolates, including those encoding tetracycline, beta-lactam, sulfonamide, quinolone, aminoglycoside, phenicol, and trimethoprim resistance. A total of 10 SPIs (SPI-1, 3-5, 8, 9, 12-14, and centisome 63 [C63PI]) were detected in 59 isolates. C63PI, an iron transport system in SPI-1, was observed in 56% of the isolates (n = 46). SPI-1, SPI-4, and SPI-9 were present in 13, 2, and 5% of the isolates, respectively. The most common phenotypic resistances were observed to tetracycline (47%; n = 38), ampicillin (37%; n = 30), streptomycin (20%; n = 16), chloramphenicol (17%; n = 14), and trimethoprim-sulfamethoxazole (16%; n = 13). This study contributes to understanding the AMR genes present in S. enterica isolates from informal markets in Cambodia, as well as support domestic epidemiological investigations of multidrug resistance (MDR) profiles.