Phylogenetic and Associated Phenotypic Analysis of Salmonella Enterica Serovar Mbandaka

Food borne salmonellosis is a global public health concern caused by Salmonella, that causes enteric disease both in humans and animals. Most of the pathogenic Salmonella serovars fall under Salmonella enterica subspecies enterica, a major subspecies group that includes more than 50% of total identified Salmonella serovars. New serovars are identified each year and overall incidence of salmonellosis may mask the outbreak incidences caused by individual serovars. Infrequently reported serovar outbreaks can be a significant threat to public health. Salmonella enterica serovar Mbandaka is one of the infrequently reported causative agents of non-typhoidal salmonellosis in USA. But it has been considered as one of the frequent human Salmonella serovar in other countries such as European countries, Israel, Africa as well as in New Zealand. Published researches about this serovar were very limited and no prior studies have been reported about S. Mbandaka isolates from USA especially at the genomic level. Knowledge about the population structure and intra serovar genetic diversity that exists within the S. Mbandaka isolates in a global context remains a mystery. This research was aimed to understand the population structure of globally distributed S. Mbandaka isolates with a hypothesis that isolates of this serovar from different geographical area may be genetically close and form specific clonal groups. Genome sequence data of 465 isolates from different parts of the world were collected from NCBI database and were used to analyze diversity at single nucleotide level. Phylogenetic tree, created based on SNP analysis, revealed partitioning of isolates into two major clusters and six sub clusters. Cluster formation was validated by further analysis with core genome MLST. This research was also aimed to understand the antimicrobial resistance gene pattern and distribution of virulence factors in S. Mbandaka isolates from different isolation sources. Analysis of ability to invade host cells and resistance to low pH environment in 76 USA isolates showed no major difference in these phenotypic properties irrespective of isolation source. Overall this research provides a solid platform for the epidemiological investigation of future Salmonella outbreaks caused by serovar S. Mbandaka

Exploration of Host Health Benefits by a Defined Consortium of Butyrate-Producing Human Gut Bacteria In Gnotobiotic Mouse Model

Aberrant gut microbiota composition is found to be associated with several human diseases such as inflammatory bowel diseases (IBD). Reduction in butyrate producing bacteria is one of the characteristic features of such dysbiotic bacterial community in the gut. Modulation of gut microbiota to bring the dysbiotic state back to normal healthy state is a promising therapeutic strategy to cure several diseases like recurrent Clostridium difficile infection (rCDI) and IBD where traditional therapies using pharmacological substances fails to make a difference. Restoration of butyrate producers is found to be an effective method of such gut microbiota modulation. The potential to produce butyrate is phylogenetically diverse and not even present in all members of the same family. Hence the number of identified bacteria that can produce butyrate is not vast and most of them were not explored for their functional roles towards host health benefits. Low abundance and the difficulty to culture these strictly anaerobic organisms are the few reasons of their under exploration. Here we examine the immunomodulatory properties of four butyrate producing human gut bacterial species as a defined mix using gnotobiotic mouse as a model. Our study shows, these allochthonous bacterial strains assemble in the germ-free mouse gut and produce butyrate as one of the short chain fatty acids. Without causing any pathological changes, successful colonization of these bacteria fortifies the innate immune defense system by enhancing the expression of regenerating islet-derived protein 3 beta (Reg3b), regenerating islet-derived protein 3 gamma (Reg3g), mucin 2 (Muc2) and defensin beta (Defb) genes in the colon and modulating the adaptive immune cell populations at the systemic levels. However, precolonization of these bacteria did not show any significant changes in pro and antiinflammatory responses in gnotobiotic mice compared to germ free mice under colitis induced by Dextran sulfate sodium (DSS). But a better gross and histopathological appearance of colon in gnotobiotic mice indicates that these bacteria have some role in attenuating colitis rather aggravating it and thus can be considered as candidates of bacterial therapeutics

The microbial nitrogen cycling, bacterial community composition, and functional potential in a natural grassland are stable from breaking dormancy to being dormant again

DATA AVAILABILITY STATEMENT : The raw data is available on NCBI’s Sequence Read Archive (SRA) database under BioProject: PRJNA803487.SUPPLEMENTARY MATERIALS : FIGURE S1: Soil textures of the sampling sites; FIGURE S2: Distribution of plant groups across the six study sites; FIGURE S3: Plant species distribution across the study sites. The legume species are represented by asterisk (*) on the legend. The abbreviations represent: BRIN, Bromus inermis; POPR, Poa pratensis; PHAR, Phalaris arundinacea; CA, Carex sp.; NA, Nassella sp.; ANGE, Andropogon gerardii; SCSC, Schizachyrium scoparium; BOCU, Bouteloua curtipendula; DIOL, Dichanthelium oligosanthes; CIFL, Cirsium flodmanii; ASSP, Asclepias speciose; SOMI, Solidago missouriensis; HEMA, Helianthus maximiliani; ANCA, Anemone canadensis; SOCA, Solidago canadensis; GLLE, Glycyrrhiza lepidota; PH, Physalis sp.; AMCA*, Amorpha canescens; DAPU*, Dalea purpurea; PE*, Pediomelum sp.; RO, Rosa sp.; FIGURE S4: Soil chemical properties across sampling sites within the sampling time points with Kruskal–Wallis test results; FIGURE S5: Alpha-diversity of the bacterial communities across 6 months. (a) Shannon diversity and (b) Pielou Evenness.; FIGURE S6: Taxa differences across at least one time point across the seasons (p < 0.01). The names of the phyla shown here are based on the taxonomic profile downloaded from the Greengenes database, however, some of the phylum names have recently been changed [38]; TABLE S1. G-Block solutions; TABLE S2. Primers used for nitrogen cycle genes. SUPPLEMENTARY DATA S3. R 2 values of the qPCR assays. SUPPLEMENTARY DATA S4. OTU table.The quantity of grass-root exudates varies by season, suggesting temporal shifts in soil microbial community composition and activity across a growing season. We hypothesized that bacterial community and nitrogen cycle-associated prokaryotic gene expressions shift across three phases of the growing season. To test this hypothesis, we quantified gene and transcript copy number of nitrogen fixation (nifH), ammonia oxidation (amoA, hao, nxrB), denitrification (narG, napA, nirK, nirS, norB, nosZ), dissimilatory nitrate reduction to ammonia (nrfA), and anaerobic ammonium oxidation (hzs, hdh) using the pre-optimized Nitrogen Cycle Evaluation (NiCE) chip. Bacterial community composition was characterized using V3-V4 of the 16S rRNA gene, and PICRUSt2 was used to draw out functional inferences. Surprisingly, the nitrogen cycle genes and transcript quantities were largely stable and unresponsive to seasonal changes. We found that genes and transcripts related to ammonia oxidation and denitrification were different for only one or two time points across the seasons (p < 0.05). However, overall, the nitrogen cycling genes did not show drastic variations. Similarly, the bacterial community also did not vary across the seasons. In contrast, the predicted functional potential was slightly low for May and remained constant for other months. Moreover, soil chemical properties showed a seasonal pattern only for nitrate and ammonium concentrations, while ammonia oxidation and denitrification transcripts were strongly correlated with each other. Hence, the results refuted our assumptions, showing stability in N cycling and bacterial community across growing seasons in a natural grassland.The South Dakota Agricultural Experiment Station.https://www.mdpi.com/journal/microorganismsam2023BiochemistryGeneticsMicrobiology and Plant Patholog

Whole genome sequencing-based detection of antimicrobial resistance and virulence in non-typhoidal Salmonella enterica isolated from wildlife

The aim of this study was to generate a reference set of Salmonella enterica genomes isolated from wildlife from the United States and to determine the antimicrobial resistance and virulence gene profile of the isolates from the genome sequence data. We sequenced the whole genomes of 103 Salmonella isolates sampled between 1988 and 2003 from wildlife and exotic pet cases that were submitted to the Oklahoma Animal Disease Diagnostic Laboratory, Stillwater, Oklahoma. Among 103 isolates, 50.48% were from wild birds, 0.9% was from fish, 24.27% each were from reptiles and mammals. 50.48% isolates showed resistance to at least one antibiotic. Resistance against the aminoglycoside streptomycin was most common while 9 isolates were found to be multi-drug resistant having resistance against more than three antibiotics. Determination of virulence gene profile revealed that the genes belonging to csg operons, the fim genes that encode for type 1 fimbriae and the genes belonging to type III secretion system were predominant among the isolates. The universal presence of fimbrial genes and the genes encoded by pathogenicity islands 1-2 among the isolates we report here indicates that these isolates could potentially cause disease in humans. Therefore, the genomes we report here could be a valuable reference point for future traceback investigations when wildlife is considered to be the potential source of human Salmonellosis.Peer reviewedOklahoma Animal Disease Diagnostic Laborator

Chemical immobilisation of dhole (Cuon alpinus), Indian jackal (Canis aureus indicus) and Indian wolf (Canis lupus pallipes) with ketamine hydrochloride–xylazine hydrochloride

Maintaining wild animals in captivity often requires chemical immobilisation to achieve various diagnostic, surgical and management interventions. Four dholes, two Indian grey wolves and four Indian jackals were immobilised using ketamine–xylazine combination for either medical or management interventions. Based on the estimated body weight, canids were darted upon with 6–8 mg kg−1 ketamine and 0.7–1.14 mg kg−1 xylazine. Initial signs of drug effect included decreased mentation and progressive ataxia followed by recumbency. The mean ± SD of induction time was 14.25 ± 2.75 (range: 11–17 min), 11 ± 3.16 (range: 8–15 min) and 15.5 ± 3.54 (range: 13–18 min) in dhole, Indian jackal and Indian wolf, respectively. Hyperthermia was initially observed in all the jackals and dholes, whereas rectal temperature in wolves remained well within the normal range for canids. The mean duration of anaesthesia was 31 ± 8.83 (range: 23–43 min), 32.5 ± 5.32 (range: 26–39 min) and 30.5 ± 7.78 (range: 25–36 min) in dhole, Indian jackal and Indian wolf, respectively, with subsequent smooth and uneventful recovery in all the cases. The observations made during immobilisation procedures in this work suggest that chemical immobilisation of captive dhole, Indian wolf and Indian jackal with 6–8 mg kg−1 ketamine and 1 mg kg−1 xylazine is effective and safe for routine management and medical interventions in these species provided body temperature is closely monitored and corrected as appropriate

The Microbial Nitrogen Cycling, Bacterial Community Composition, and Functional Potential in a Natural Grassland Are Stable from Breaking Dormancy to Being Dormant Again

The quantity of grass-root exudates varies by season, suggesting temporal shifts in soil microbial community composition and activity across a growing season. We hypothesized that bacterial community and nitrogen cycle-associated prokaryotic gene expressions shift across three phases of the growing season. To test this hypothesis, we quantified gene and transcript copy number of nitrogen fixation (nifH), ammonia oxidation (amoA, hao, nxrB), denitrification (narG, napA, nirK, nirS, norB, nosZ), dissimilatory nitrate reduction to ammonia (nrfA), and anaerobic ammonium oxidation (hzs, hdh) using the pre-optimized Nitrogen Cycle Evaluation (NiCE) chip. Bacterial community composition was characterized using V3-V4 of the 16S rRNA gene, and PICRUSt2 was used to draw out functional inferences. Surprisingly, the nitrogen cycle genes and transcript quantities were largely stable and unresponsive to seasonal changes. We found that genes and transcripts related to ammonia oxidation and denitrification were different for only one or two time points across the seasons (p &lt; 0.05). However, overall, the nitrogen cycling genes did not show drastic variations. Similarly, the bacterial community also did not vary across the seasons. In contrast, the predicted functional potential was slightly low for May and remained constant for other months. Moreover, soil chemical properties showed a seasonal pattern only for nitrate and ammonium concentrations, while ammonia oxidation and denitrification transcripts were strongly correlated with each other. Hence, the results refuted our assumptions, showing stability in N cycling and bacterial community across growing seasons in a natural grassland

Genome divergence and increased virulence of outbreak associated Salmonella enterica subspecies enterica serovar Heidelberg

Abstract Salmonella enterica serotype Heidelberg is primarily a poultry adapted serotype of Salmonella that can also colonize other hosts and cause human disease. In this study, we compared the genomes of outbreak associated non-outbreak causing Salmonella ser. Heidelberg strains from diverse hosts and geographical regions. Human outbreak associated strains in this study were from a 2015 multistate outbreak of Salmonella ser. Heidelberg involving 15 states in the United States which originated from bull calves. Our clinicopathologic examination revealed that cases involving Salmonella ser. Heidelberg strains were predominantly young, less than weeks-old, dairy calves. Pre-existing or concurrent disease was found in the majority of the calves. Detection of Salmonella ser. Heidelberg correlated with markedly increased death losses clinically comparable to those seen in herds infected with S. Dublin, a known serious pathogen of cattle. Whole genome based single nucleotide polymorphism based analysis revealed that these calf isolates formed a distinct cluster along with outbreak associated human isolates. The defining feature of the outbreak associated strains, when compared to older isolates of S. Heidelberg, is that all isolates in this cluster contained Saf fimbrial genes which are generally absent in S. Heidelberg. The acquisition of several single nucleotide polymorphisms and the gain of Saf fimbrial genes may have contributed to the increased disease severity of these Salmonella ser. Heidelberg strains

Gut Microbial Dynamics during Conventionalization of Germfree Chicken

The domestic chicken is the cornerstone of animal agriculture worldwide, with a flock population exceeding 40 billion birds/year. It serves as an economically valuable source of protein globally. The microbiome of poultry has important effects on chicken growth, feed conversion, immune status, and pathogen resistance. The aim of our research was to develop a gnotobiotic chicken model appropriate for the study chicken gut microbiota function. Our experimental model shows that young germfree chicks are able to colonize diverse sets of gut bacteria. Therefore, besides the use of this model to study mechanisms of gut microbiota interactions in the chicken gut, it could be also used for applied aspects such as determining the safety and efficacy of new probiotic strains derived from chicken gut microbiota.A gnotobiotic Gallus gallus (chicken) model was developed to study the dynamics of intestinal microflora from hatching to 18 days of age employing metagenomics. Intestinal samples were collected from a local population of feral chickens and administered orally to germfree 3-day-old chicks. Animals were euthanized on days 9 and 18 postinoculation, and intestinal samples were collected and subjected to metagenomic analysis. On day 18, the five most prevalent phyla were Bacteroidetes (43.03 ± 3.19%), Firmicutes (38.51 ± 2.67%), Actinobacteria (6.77 ± 0.7%), Proteobacteria (6.38 ± 0.7%), and Spirochaetes (2.71 ± 0.55%). Principal-coordinate analysis showed that the day 18 variables clustered more closely than the day 9 variables, suggesting that the microbial communities had changed temporally. The Morista-Horn index values ranged from 0.7 to 1, indicating that the communities in the inoculum and in the day 9 and day 18 samples were more similar than dissimilar. The predicted functional profiles of the microbiomes of the inoculum and the day 9 and day 18 samples were also similar (values of 0.98 to 1). These results indicate that the gnotobiotic chicks stably maintained the phylogenetic diversity and predicted metabolic functionality of the inoculum community

Antimicrobial-Resistant Escherichia coli from Environmental Waters in Northern Colorado

Waterborne Escherichia coli are a major reservoir of antimicrobial resistance (AMR), including but not limited to extended-spectrum beta-lactamase (ESBL) and Klebsiella pneumoniae carbapenemase (KPC) mechanisms. This study quantified and described ESBL- and KPC-producing E. coli in Northern Colorado from sewer water, surface water, and influent and effluent wastewater treatment sources. Total detected bacteria and E. coli abundances, and the percentages that contain ESBL and/or KPC, were compared between water sources. Seventy E. coli isolates from the various waters had drug resistance validated with a panel of 17 antibiotics using a broth microdilution assay. The diverse drug resistance observed across E. coli isolates was further documented by polymerase chain reaction of common ESBL genes and functional relatedness by PhenePlate assay-generated dendrograms (n=70). The total E. coli abundance decreased through the water treatment process as expected, yet the percentages of E. coli harboring ESBL resistance were increased (1.70%) in surface water. Whole-genome sequencing analysis was completed for 185 AMR genes in wastewater E. coli isolates and confirmed the presence of diverse AMR gene classes (e.g., beta-lactams and efflux pumps) in isolate genomes. This study completed surveillance of AMR patterns in E. coli that reside in environmental water systems and suggests a role for integrating both phenotypic and genotypic profiling beyond ESBL and KPC mechanisms. AMR screening via multiple approaches may assist in the prevention of drug-resistant E. coli spread from waters to animals and humans