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Background: Burkholderia pseudomallei, causative agent of melioidosis, is a One Health concern as it is acquired directly from soil and water and causes disease in humans and agricultural and wild animals. We examined B. pseudomallei in soil and goats at a single farm in the Northern Territory of Australia where >30 goats acquired melioidosis over nine years., Methodology/principal Findings: We cultured 45 B. pseudomallei isolates from 35 goats and sampled soil in and around goat enclosures to isolate and detect B. pseudomallei and evaluate characteristics associated with its occurrence; 33 soil isolates were obtained from 1993-1994 and 116 in 2006. Ninety-two goat and soil isolates were sequenced; mice were challenged with six soil isolates to evaluate virulence. Sampling depth and total N/organic C correlated with B. pseudomallei presence. Twelve sequence types (STs) were identified. Most goat infections (74%) were ST617, some with high similarity to 2006 soil isolates, suggesting ST617 was successful at persisting in soil and infecting goats. ST260 and ST266 isolates were highly virulent in mice but other isolates produced low/intermediate virulence; three of these were ST326 isolates, the most common soil ST in 2006. Thus, virulent and non-virulent lineages can co-occur locally. Three genes associated with virulence were present in ST260 and ST266, absent in most ST326 isolates, and present or variably present in ST617., Conclusions/significance: Agricultural animals can influence B. pseudomallei abundance and diversity in local environments. This effect may persist, as B. pseudomallei was detected more often from soil collected inside and adjacent to goat enclosures years after most goats were removed. Following goat removal, the low virulence ST326, which was not isolated from soil when goats were present, became the predominant ST in soil by 2006. Although multiple diverse lineages of B. pseudomallei may exist in a given location, some may infect mammals more efficiently than others., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Busch et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Escherichia coli is a diverse pathogen, causing a range of disease in humans, from self-limiting diarrhea to urinary tract infections (UTIs). Uropathogenic E. coli (UPEC) is the most frequently observed uropathogen in UTIs, a common disease in high-income countries, incurring billions of dollars yearly in treatment costs. Although E. coli is easily grown and identified in the clinical laboratory, genotyping the pathogen is more complicated, yet critical for reducing the incidence of disease. These goals can be achieved through whole-genome sequencing of E. coli isolates, but this approach is relatively slow and typically requires culturing the pathogen in the laboratory. To genotype E. coli rapidly and inexpensively directly from clinical samples, including but not limited to urine, we developed and validated a multiplex amplicon sequencing assay, called ColiSeq. The assay consists of targets designed for E. coli species confirmation, high resolution genotyping, and mixture deconvolution. To demonstrate its utility, we screened the ColiSeq assay against 230 clinical urine samples collected from a hospital system in Flagstaff, Arizona, USA. A limit of detection analysis demonstrated the ability of ColiSeq to identify E. coli at a concentration of ~2 genomic equivalent (GEs)/mL and to generate high-resolution genotyping at a concentration of 1 × 10 5 GEs/mL. The results of this study suggest that ColiSeq could be a valuable method to understand the source of UPEC strains and guide infection mitigation efforts. As sequence-based diagnostics become accepted in the clinical laboratory, workflows such as ColiSeq will provide actionable information to improve patient outcomes.IMPORTANCEUrinary tract infections (UTIs), caused primarily by Escherichia coli , create an enormous health care burden in the United States and other high-income countries. The early detection of E. coli from clinical samples, including urine, is important to target therapy and prevent further patient complications. Additionally, understanding the source of E. coli exposure will help with future mitigation efforts. In this study, we developed, tested, and validated an amplicon sequencing assay focused on direct detection of E. coli from urine. The resulting sequence data were demonstrated to provide strain level resolution of the pathogen, not only confirming the presence of E. coli , which can focus treatment efforts, but also providing data needed for source attribution and contact tracing. This assay will generate inexpensive, rapid, and reproducible data that can be deployed by public health agencies to track, diagnose, and potentially mitigate future UTIs caused by E. coli ., Competing Interests: The authors declare no conflict of interest.

Although infections caused by Clostridioides difficile have historically been attributed to hospital acquisition, growing evidence supports the role of community acquisition in C. difficile infection (CDI). Symptoms of CDI can range from mild, self-resolving diarrhoea to toxic megacolon, pseudomembranous colitis, and death. In this study, we sampled C. difficile from clinical, environmental, and canine reservoirs in Flagstaff, Arizona, USA, to understand the distribution and transmission of the pathogen in a One Health framework; Flagstaff is a medium-sized, geographically isolated city with a single hospital system, making it an ideal site to characterize genomic overlap between sequenced C. difficile isolates across reservoirs. An analysis of 562 genomes from Flagstaff isolates identified 65 sequence types (STs), with eight STs being found across all three reservoirs and another nine found across two reservoirs. A screen of toxin genes in the pathogenicity locus identified nine STs where all isolates lost the toxin genes needed for CDI manifestation ( tcdB , tcdA ), demonstrating the widespread distribution of non-toxigenic C. difficile (NTCD) isolates in all three reservoirs; 15 NTCD genomes were sequenced from symptomatic, clinical samples, including two from mixed infections that contained both tcdB+ and tcdB - isolates. A comparative single nucleotide polymorphism (SNP) analysis of clinically derived isolates identified 78 genomes falling within clusters separated by ≤2 SNPs, indicating that ~19 % of clinical isolates are associated with potential healthcare-associated transmission clusters; only symptomatic cases were sampled in this study, and we did not sample asymptomatic transmission. Using this same SNP threshold, we identified genomic overlap between canine and soil isolates, as well as putative transmission between environmental and human reservoirs. The core genome of isolates sequenced in this study plus a representative set of public C. difficile genomes ( n =136), was 2690 coding region sequences, which constitutes ~70 % of an individual C. difficile genome; this number is significantly higher than has been published in some other studies, suggesting that genome data quality is important in understanding the minimal number of genes needed by C. difficile . This study demonstrates the close genomic overlap among isolates sampled across reservoirs, which was facilitated by maximizing the genomic search space used for comprehensive identification of potential transmission events. Understanding the distribution of toxigenic and non-toxigenic C. difficile across reservoirs has implications for surveillance sampling strategies, characterizing routes of infections, and implementing mitigation measures to limit human infection.

As the size of reference sequence databases and high-throughput sequencing datasets continue to grow, it is becoming computationally infeasible to use traditional alignment to large genome databases for taxonomic classification of metagenomic reads. Exact matching approaches can rapidly assign taxonomy and summarize the composition of microbial communities, but they sacrifice accuracy and can lead to false positives. Full alignment tools provide higher confidence assignments and can assign sequences from genomes that diverge from reference sequences; however, full alignment tools are computationally intensive. To address this, we designed MTSv specifically for alignment-based taxonomic assignment in metagenomic analysis. This tool implements an FM-index assisted q-gram filter and SIMD accelerated Smith-Waterman algorithm to find alignments. However, unlike traditional aligners, MTSv will not attempt to make additional alignments to a TaxID once an alignment of sufficient quality has been found. This improves efficiency when many reference sequences are available per taxon. MTSv was designed to be flexible and can be modified to run on either memory or processor constrained systems. Although MTSv cannot compete with the speeds of exact k-mer matching approaches, it is reasonably fast and has higher precision than popular exact matching approaches. Because MTSv performs a full alignment it can classify reads even when the genomes share low similarity with reference sequences and provides a tool for high confidence pathogen detection with low off-target assignments to near neighbor species., Competing Interests: The authors declare that they have no competing interests., (© 2022 Furstenau et al.)

Background: The coronavirus disease 2019 pandemic has impacted the health of the population. Technological advances become alternative tools for the promotion of lifestyle and prevention of non-communicable diseases in university personnel. Objective: This study analyzed the effect of an intervention based on a telehealth program and investigated its efficacy in improving lifestyle, body mass index, and glucose concentration in university staff during the coronavirus disease 2019 pandemic. Methods: A pre-experimental study was conducted between August and December 2021 on 100 workers of a private university located in Tarapoto, Peru. Information was collected on lifestyle practices and beliefs, body mass index, and glucose contraction before and after the intervention. After the intervention, 78 participants were analyzed; 22 workers did not respond to the invitation. Results: Comparison between pretest and follow-up showed an increase in lifestyle practices and beliefs (from 93.33 ± 9.44 to 96.23 ± 9.32, p  < 0.01), although this value is considered an unhealthy lifestyle. Moreover, a decrease in body mass index (from 25.23 ± 3.08 to 24.98 ± 2.98, p  < 0.05) and glucose concentration (from 98.35 ± 6.70 to 97.45 ± 5.61, p  < 0.01) was observed. Conclusion: Telehealth-based educational interventions could be effective in university staff during the coronavirus disease 2019 pandemic, which, in turn, would support improved lifestyles and adequate body mass index and glucose levels., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2022.)

Burkholderia pseudomallei is a soil-dwelling bacterium endemic to Southeast Asia and northern Australia that causes the disease, melioidosis. Although the global genomic diversity of clinical B. pseudomallei isolates has been investigated, there is limited understanding of its genomic diversity across small geographic scales, especially in soil. In this study, we obtained 288 B. pseudomallei isolates from a single soil sample (~100g; intensive site 2, INT2) collected at a depth of 30cm from a site in Ubon Ratchathani Province, Thailand. We sequenced the genomes of 169 of these isolates that represent 7 distinct sequence types (STs), including a new ST (ST1820), based on multi-locus sequence typing (MLST) analysis. A core genome SNP phylogeny demonstrated that all identified STs share a recent common ancestor that diverged an estimated 796-1260 years ago. A pan-genomics analysis demonstrated recombination between clades and intra-MLST phylogenetic and gene differences. To identify potential differential virulence between STs, groups of BALB/c mice (5 mice/isolate) were challenged via subcutaneous injection (500 CFUs) with 30 INT2 isolates representing 5 different STs; over the 21-day experiment, eight isolates killed all mice, 2 isolates killed an intermediate number of mice (1-2), and 20 isolates killed no mice. Although the virulence results were largely stratified by ST, one virulent isolate and six attenuated isolates were from the same ST (ST1005), suggesting that variably conserved genomic regions may contribute to virulence. Genomes from the animal-challenged isolates were subjected to a bacterial genome-wide association study to identify genomic regions associated with differential virulence. One associated region is a unique variant of Hcp1, a component of the type VI secretion system, which may result in attenuation. The results of this study have implications for comprehensive sampling strategies, environmental exposure risk assessment, and understanding recombination and differential virulence in B. pseudomallei., Competing Interests: The authors have declared that no competing interests exist.

Clostridioides difficile is a pathogen often associated with hospital-acquired infection or antimicrobial-induced disease; however, increasing evidence indicates infections can result from community or environmental sources. Most genomic sequencing of C. difficile has focused on clinical strains, although evidence is growing that C. difficile spores are widespread in soil and water in the environment. In this study, we sequenced 38 genomes collected from soil and water isolates in Flagstaff (AZ, USA) and Slovenia in an effort targeted towards environmental surveillance of C. difficile . At the average nucleotide identity (ANI) level, the genomes were divergent to C. difficile at a threshold consistent with different species. A phylogenetic analysis of these divergent genomes together with Clostridioides genomes available in public repositories confirmed the presence of three previously described, cryptic Clostridioide s species and added two additional clades. One of the cryptic species (C-III) was almost entirely composed of Arizona and Slovenia genomes, and contained distinct sub-groups from each region (evidenced by SNP and gene-content differences). A comparative genomics analysis identified multiple unique coding sequences per clade, which can serve as markers for subsequent environmental surveys of these cryptic species. Homologues to the C. difficile toxin genes, tcdA and tcdB , were found in cryptic species genomes, although they were not part of the typical pathogenicity locus observed in C. difficile , and in silico PCR suggested that some would not amplify with widely used PCR diagnostic tests. We also identified gene homologues in the binary toxin cluster, including some present on phage and, for what is believed to be the first time, on a plasmid. All isolates were obtained from environmental samples, so the function and disease potential of these toxin homologues is currently unknown. Enzymatic profiles of a subset of cryptic isolates ( n =5) demonstrated differences, suggesting that these isolates contain substantial metabolic diversity. Antimicrobial resistance (AMR) was observed across a subset of isolates ( n =4), suggesting that AMR mechanisms are intrinsic to the genus, perhaps originating from a shared environmental origin. This study greatly expands our understanding of the genomic diversity of Clostridioides . These results have implications for C. difficile One Health research, for more sensitive C. difficile diagnostics, as well as for understanding the evolutionary history of C. difficile and the development of pathogenesis.

By late 2020, the coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), had caused tens of millions of infections and over 1 million deaths worldwide. A protective vaccine and more effective therapeutics are urgently needed. We evaluated a new poly(ADP-ribose) polymerase (PARP) inhibitor, stenoparib, that recently advanced to phase II clinical trials for treatment of ovarian cancer, for activity against human respiratory coronaviruses, including SARS-CoV-2, in vitro Stenoparib exhibits dose-dependent suppression of SARS-CoV-2 multiplication and spread in Vero E6 monkey kidney and Calu-3 human lung adenocarcinoma cells. Stenoparib was also strongly inhibitory to the human seasonal respiratory coronavirus HCoV-NL63. Compared to remdesivir, which inhibits viral replication downstream of cell entry, stenoparib impedes entry and postentry processes, as determined by time-of-addition (TOA) experiments. Moreover, a 10 μM dosage of stenoparib-below the approximated 25.5 μM half-maximally effective concentration (EC 50 )-combined with 0.5 μM remdesivir suppressed coronavirus growth by more than 90%, indicating a potentially synergistic effect for this drug combination. Stenoparib as a stand-alone or as part of combinatorial therapy with remdesivir should be a valuable addition to the arsenal against COVID-19. IMPORTANCE New therapeutics are urgently needed in the fight against COVID-19. Repurposing drugs that are either already approved for human use or are in advanced stages of the approval process can facilitate more rapid advances toward this goal. The PARP inhibitor stenoparib may be such a drug, as it is currently in phase II clinical trials for the treatment of ovarian cancer and its safety and dosage in humans have already been established. Our results indicate that stenoparib possesses strong antiviral activity against SARS-CoV-2 and other coronaviruses in vitro. This activity appears to be based on multiple modes of action, where both pre-entry and postentry viral replication processes are impeded. This may provide a therapeutic advantage over many current options that have a narrower target range. Moreover, our results suggest that stenoparib and remdesivir in combination may be especially potent against coronavirus infection., (Copyright © 2021 Stone et al.)

Background: University teachers, as agents of socialization, are one of the most influential groups in improving public health, and their nutritional knowledge can influence both the eating behavior of students and the family., Objective: This study was carried out to evaluate the effect of a telehealth program on the level of nutritional knowledge and body mass index (BMI) of university teachers., Methods: A pre-experimental study was carried out from August to December 2020. A pretest and a posttest were applied. The number of teachers in the initial test was 88 and those included in the final evaluation, 83; and they were selected by nonprobability sampling for convenience. The telehealth-based program lasted 18 weeks and included basic information on nutrition and motivational messages. Sociodemographic and anthropometric data were collected through a predesigned questionnaire before and after the educational program., Results: Posttest knowledge scores increased significantly from 6.40 ± 1.70 to 10.59 ± 1.18 ( P  < .001). In relation to time, significant variations are observed in weight measurements ( P  < .001), BMI ( P  < .001), and basic nutritional knowledge ( P  < .001) before and after the intervention program., Conclusion: The telehealth-based intervention improved basic nutritional knowledge levels and BMI. Likewise, the current findings provide evidence on the importance and need to develop future nutritional education programs that comprehensively encompass all study plans for the benefit of the university community.

Antimicrobial resistance (AMR) in the nosocomial pathogen, Acinetobacter baumannii , is becoming a serious public health threat. While some mechanisms of AMR have been reported, understanding novel mechanisms of resistance is critical for identifying emerging resistance. One of the first steps in identifying novel AMR mechanisms is performing genotype/phenotype association studies; however, performing these studies is complicated by the plastic nature of the A. baumannii pan-genome. In this study, we compared the antibiograms of 12 antimicrobials associated with multiple drug families for 84 A. baumannii isolates, many isolated in Arizona, USA. in silico screening of these genomes for known AMR mechanisms failed to identify clear correlations for most drugs. We then performed a bacterial genome wide association study (bGWAS) looking for associations between all possible 21-mers; this approach generally failed to identify mechanisms that explained the resistance phenotype. In order to decrease the genomic noise associated with population stratification, we compared four phylogenetically-related pairs of isolates with differing susceptibility profiles. RNA-Sequencing (RNA-Seq) was performed on paired isolates and differentially-expressed genes were identified. In these isolate pairs, five different potential mechanisms were identified, highlighting the difficulty of broad AMR surveillance in this species. To verify and validate differential expression, amplicon sequencing was performed. These results suggest that a diagnostic platform based on gene expression rather than genomics alone may be beneficial in certain surveillance efforts. The implementation of such advanced diagnostics coupled with increased AMR surveillance will potentially improve A. baumannii infection treatment and patient outcomes., (Copyright © 2020 Roe, Williamson, Vazquez, Kyger, Valentine, Bowers, Phillips, Harrison, Driebe, Engelthaler and Sahl.)

Although acute melioidosis is the most common outcome of Burkholderia pseudomallei infection, we have documented a case, P314, where disease severity lessened with time, and the pathogen evolved towards a commensal relationship with the host. In the current study, we used whole-genome sequencing to monitor this long-term symbiotic relationship to better understand B. pseudomallei persistence in P314's sputum despite intensive initial therapeutic regimens. We collected and sequenced 118 B. pseudomallei isolates from P314's airways over a >16-year period, and also sampled the patient's home environment, recovering six closely related B. pseudomallei isolates from the household water system. Using comparative genomics, we identified 126 SNPs in the core genome of the 124 isolates or 162 SNPs/indels when the accessory genome was included. The core SNPs were used to construct a phylogenetic tree, which demonstrated a close relationship between environmental and clinical isolates and detailed within-host evolutionary patterns. The phylogeny had little homoplasy, consistent with a strictly clonal mode of genetic inheritance. Repeated sampling revealed evidence of genetic diversification, but frequent extinctions left only one successful lineage through the first four years and two lineages after that. Overall, the evolution of this population is nonadaptive and best explained by genetic drift. However, some genetic and phenotypic changes are consistent with in situ adaptation. Using a mouse model, P314 isolates caused greatly reduced morbidity and mortality compared to the environmental isolates. Additionally, potentially adaptive phenotypes emerged and included differences in the O-antigen, capsular polysaccharide, motility, and colony morphology. The >13-year co-existence of two long-lived lineages presents interesting hypotheses that can be tested in future studies to provide additional insights into selective pressures, niche differentiation, and microbial adaptation. This unusual melioidosis case presents a rare example of the evolutionary progression towards commensalism by a highly virulent pathogen within a single human host., Competing Interests: The authors have declared that no competing interests exist.

Clostridioides difficile is a ubiquitous, diarrhoeagenic pathogen often associated with healthcare-acquired infections that can cause a range of symptoms from mild, self-limiting disease to toxic megacolon and death. Since the early 2000s, a large proportion of C. difficile cases have been attributed to the ribotype 027 (RT027) lineage, which is associated with sequence type 1 (ST1) in the C. difficile multilocus sequence typing scheme. The spread of ST1 has been attributed, in part, to resistance to fluoroquinolones used to treat unrelated infections, which creates conditions ideal for C. difficile colonization and proliferation. In this study, we analysed 27 isolates from a healthcare network in northern Arizona, USA, and 1352 publicly available ST1 genomes to place locally sampled isolates into a global context. Whole genome, single nucleotide polymorphism analysis demonstrated that at least six separate introductions of ST1 were observed in healthcare facilities in northern Arizona over an 18-month sampling period. A reconstruction of transmission networks identified potential nosocomial transmission of isolates, which were only identified via whole genome sequence analysis. Antibiotic resistance heterogeneity was observed among ST1 genomes, including variability in resistance profiles among locally sampled ST1 isolates. To investigate why ST1 genomes are so common globally and in northern Arizona, we compared all high-quality C. difficile genomes and identified that ST1 genomes have gained and lost a number of genomic regions compared to all other C. difficile genomes; analyses of other toxigenic C. difficile sequence types demonstrate that this loss may be anomalous and could be related to niche specialization. These results suggest that a combination of antimicrobial resistance and gain and loss of specific genes may explain the prominent association of this sequence type with C. difficile infection cases worldwide. The degree of genetic variability in ST1 suggests that classifying all ST1 genomes into a quinolone-resistant hypervirulent clone category may not be appropriate. Whole genome sequencing of clinical C. difficile isolates provides a high-resolution surveillance strategy for monitoring persistence and transmission of C. difficile and for assessing the performance of infection prevention and control strategies.

The global dissemination of Klebsiella pneumoniae and Klebsiella pneumoniae carbapenemase (KPC) has been largely attributed to a few high-risk sequence types (STs) (ST258, ST11, ST512) associated with human disease. ST101 is an emerging clone that has been identified in different parts of the world with the potential to become a global, persistent public health threat. Recent research suggests the ST101 lineage is associated with an 11% increase in mortality rate in comparison to non-ST101 infections. In this study, we generated a high-quality, near-finished genome assembly of a multidrug-resistant (MDR) isolate from Italy (isolate 4743) that is a single locus variant of ST101 (ST1685). We demonstrate that the 4743 genome contains virulence features such as an integrative conjugative element carrying the yersiniabactin siderophore (ICEKp3), the mannose-resistant Klebsiella -like (type III) fimbriae cluster (mrkABCDFHIJ), the ferric uptake system (kfuABC), the yersiniabactin receptor gene fyuA , a capsular K type K17, and an O antigen type of O1. K. pneumoniae 4743 carries the bla KPC-2 carbapenemase gene along with genes conferring resistance to aminoglycosides, beta-lactams, fluoroquinolones, fosfomycin, macrolides, lincosamides, and streptogramin B. A comparative genomics analysis of 44 ST101 genomes as well as newly sequenced isolate 4743 identified variable antimicrobial resistance (AMR) resistance profiles and incompatibility plasmid types, but similar virulence factor profiles. Using Bayesian methodologies, we estimate the common ancestor for the ST101 lineage emerged in 1990 (95% HPD: 1965 to 2007) and isolates within the lineage acquired bla KPC after the divergence from its parental clonal group and dissemination. The identification of virulence factors and antibiotic resistance genes acquired by this newly emerging clone provides insight into the reported increased mortality rates and highlights its potential success as a persistent nosocomial pathogen. With a combination of both colistin resistance, carbapenem resistance, and several known virulence factors, the ST101 genetic repertoire may be a "perfect storm" allowing for a newly emerging, high-risk, extensively antibiotic resistant clone. This high-risk clone appears adept at acquiring resistance and may perpetuate the dissemination of extensive antimicrobial resistance. Greater focus on the acquisition of virulence factors and antibiotic resistance genes is crucial for understanding the spread of antibiotic resistance.

Diverse members of the genus Clostridium produce botulinum neurotoxins (BoNTs), which cause a flaccid paralysis known as botulism. While multiple species of clostridia produce BoNTs, the majority of human botulism cases have been attributed to Clostridium botulinum groups I and II. Recent comparative genomic studies have demonstrated the genomic diversity within these BoNT-producing species. This report introduces a multiplex PCR assay for differentiating members of C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Coding region sequences unique to each of the four species/subgroups were identified by in silico analyses of thousands of genome assemblies, and PCR primers were designed to amplify each marker. The resulting multiplex PCR assay correctly assigned 41 tested isolates to the appropriate species or subgroup. A separate PCR assay to determine the presence of the ntnh gene (a gene associated with the botulinum neurotoxin gene cluster) was developed and validated. The ntnh gene PCR assay provides information about the presence or absence of the botulinum neurotoxin gene cluster and the type of gene cluster present ( ha positive [ ha + ] or orfX + ). The increased availability of whole-genome sequence data and comparative genomic tools enabled the design of these assays, which provide valuable information for characterizing BoNT-producing clostridia. The PCR assays are rapid, inexpensive tests that can be applied to a variety of sample types to assign isolates to species/subgroups and to detect clostridia with botulinum neurotoxin gene ( bont ) clusters. IMPORTANCE Diverse clostridia produce the botulinum neurotoxin, one of the most potent known neurotoxins. In this study, a multiplex PCR assay was developed to differentiate clostridia that are most commonly isolated in connection with human botulism cases: C. botulinum group I, C. sporogenes , and two major subgroups within C. botulinum group II. Since BoNT-producing and nontoxigenic isolates can be found in each species, a PCR assay to determine the presence of the ntnh gene, which is a universally present component of bont gene clusters, and to provide information about the type ( ha + or orfX + ) of bont gene cluster present in a sample was also developed. The PCR assays provide simple, rapid, and inexpensive tools for screening uncharacterized isolates from clinical or environmental samples. The information provided by these assays can inform epidemiological studies, aid with identifying mixtures of isolates and unknown isolates in culture collections, and confirm the presence of bacteria of interest., (Copyright © 2017 Williamson et al.)

Nosocomial acquisition of Clostridium difficile is well documented, yet recent studies have highlighted the importance of community acquired infections and identified community associated reservoirs for this pathogen. Multiple studies have implicated companion pets and farm animals as possible sources of community acquired C. difficile infections in humans. To explore the potential role of pet dogs in human C. difficile infections we systematically collected canine fecal samples (n = 197) in Flagstaff, AZ. Additionally, nineteen fecal samples were collected at a local veterinary clinic from diarrheic dogs. We used these combined samples to investigate important questions regarding C. difficile colonization in pet canines: 1) What is the prevalence and diversity of C. difficile in this companion pet population, and 2) Do C. difficile isolates collected from canines genetically overlap with isolates that cause disease in humans? We used a two-step sequence typing approach, including multilocus sequence typing to determine the overall genetic diversity of C. difficile present in Flagstaff canines, and whole-genome sequencing to assess the fine-scale diversity patterns within identical multilocus sequence types from isolates obtained within and among multiple canine hosts. We detected C. difficile in 17% of the canine fecal samples with 10% containing toxigenic strains that are known to cause human disease. Sequencing analyses revealed similar genotypes in dogs and humans. These findings suggest that companion pets are a potential source of community acquired C. difficile infections in humans., Competing Interests: The authors have declared that no competing interests exist.

Unlabelled: Whole-genome sequence (WGS) data are commonly used to design diagnostic targets for the identification of bacterial pathogens. To do this effectively, genomics databases must be comprehensive to identify the strict core genome that is specific to the target pathogen. As additional genomes are analyzed, the core genome size is reduced and there is erosion of the target-specific regions due to commonality with related species, potentially resulting in the identification of false positives and/or false negatives., Importance: A comparative analysis of 1,130 Burkholderia genomes identified unique markers for many named species, including the human pathogens B. pseudomallei and B. mallei Due to core genome reduction and signature erosion, only 38 targets specific to B. pseudomallei/mallei were identified. By using only public genomes, a larger number of markers were identified, due to undersampling, and this larger number represents the potential for false positives. This analysis has implications for the design of diagnostics for other species where the genomic space of the target and/or closely related species is not well defined., (Copyright © 2016 Sahl et al.)

Thiamine is one of several essential cofactors for ATP generation. Its deficiency, like in beriberi and in the Wernicke-Korsakoff syndrome, has been studied for many decades. However, its mechanism of action is still not completely understood at the cellular and molecular levels. Since it acts as a coenzyme for dehydrogenases of pyruvate, branched-chain keto acids, and ketoglutarate, its nutritional privation is partly a phenocopy of inborn errors of metabolism, among them maple syrup urine disease. In the present paper, we report metabolic and genomic findings in mice deprived of thiamine. They are similar to the ones we have previously found in biotin deficiency, another ATP generation cofactor. Here we show that thiamine deficiency substantially reduced the energy state in the liver and activated the energy sensor AMP-activated kinase. With this vitamin deficiency, several metabolic parameters changed: blood glucose was diminished and serum lactate was increased, but insulin, triglycerides, and cholesterol, as well as liver glycogen, were reduced. These results indicate a severe change in the energy status of the whole organism. Our findings were associated with modified hepatic levels of the mRNAs of several carbon metabolism genes: a reduction of transcripts for liver glucokinase and fatty acid synthase and augmentation of those for carnitine palmitoyl transferase 1 and phosphoenolpyruvate carboxykinase as markers for glycolysis, fatty acid synthesis, beta-oxidation, and gluconeogenesis, respectively. Glucose tolerance was initially increased, suggesting augmented insulin sensitivity, as we had found in biotin deficiency; however, in the case of thiamine, it was diminished from the 3rd week on, when the deficient animals became undernourished, and paralleled the changes in AKT and mTOR, 2 main proteins in the insulin signaling pathway. Since many of the metabolic and gene expression effects on mice deprived of thiamine are similar to those in biotin deficiency, it may be that they result from a more general impairment of oxidative phosphorylation due to a shortage of ATP generation cofactors. These findings may be relevant to energy-related disorders, among them several inborn errors of metabolism, as well as common energy disorders like obesity, diabetes, and neurodegenerative illnesses., (© 2017 S. Karger AG, Basel.)

Aim: To identify the clinical-epidemiological features and the factors related to the presence of disability in patients with migraine., Subjects and Methods: A cross-sectional study was conducted in a rural population at the Policlinica Mabay in the province of Granma, Cuba. The second International Headache Society classification was used. Sample size, which was calculated using the program EpiInfo 2002, was 360 adult workers over 18 years of age, who were selected by simple random sampling., Results: Sixty-one respondents were diagnosed with migraine (prevalence: 16.9%; 95% CI = 13.3-21.31). Prevalence among females was 24.3% (CI 95% = 18.7-30.82) and 7.1% (CI 95% = 3.8-12.73) among males. Chronic migraine was the most frequent, with 25 cases (prevalence: 6.9%; CI 95% = 4.63-10.21), followed by migraine without aura, with 18 diagnoses (prevalence: 5%; CI 95% = 3.08-7.93). Frequency of headaches was greater in females (30.17 Y 12 days versus 23.45 Y 10 days; p = 0.041), as was their duration (19.87 Y 6.95 h versus 15.73 Y 5.58 h in males; p = 0.027). Minimal disability affected 24.6% of patients with migraine and 39.3% suffered moderate disability. Higher degrees of disability were associated to being female, the time elapsed since onset of the disease (21.04 Y 10.1 years versus 15.33 Y 7.5 years; p = 0.017), the frequency of headaches in the last three months (30.28 Y 10.1 days with headache versus 25.25 Y 8.5; p = 0.042), the duration of the headache (23.3 Y 15.24 h versus 12.38 Y 10.9 h; p = 0.002) and chronic migraine (p = 0.016)., Conclusions: There is a great social and occupational burden of disability in patients with migraine in the geographical area under study, and females are affected by this condition to a greater extent.

Lipoblastomas are most commonly superficially-located tumors. Our experience with three examples of deep-seated lipoblastomas is reported. The CT scans of all cases showed low-attenuation densities (less than 0 Hansfield units), strongly suggesting fatty composition. Fine-needle aspiration biopsy (FNAB) smears contained uni- and multivacuolated lipoblasts, myxoid areas, and a plexiform capillary network. The combination of CT scan and FNAB findings seems to be enough for definitive preoperative diagnosis of lipoblastoma, allowing differential diagnosis with other deep-seated tumors.

We report three cases in which medical students who participated in clinical studies as normal volunteers were discovered, during the course of the research, to have potentially serious abnormalities. Their previously unsuspected conditions included chronic persistent hepatitis, a lesion in the cerebral cortex, and seropositivity for hepatitis B and HIV antibodies. These findings were surprising and distressing to both subject and investigator, and raised questions of the need for institutional guidelines, and the potential legal liability of the investigator. We discuss how improvements in Informed Consent Forms might aid in reducing these problems, by alerting the researcher and the subject to the possibility that unsuspected disease might be discovered during the course of a clinical research study.

"Dale's Principle" states that each neuron releases one and only one synaptic transmitter. Mental disorders and behavioral drug effects are attributed to activation or blockade of one or more of these specific transmitters. A series of biochemical, electrophysiological, and behavioral studies suggests the alternative view that at each monoaminergic synapse the action of the transmitter is modulated by several metabolically related substances: amine analogs (2-phenylethylamine [PEA], p-tyramine, etc.), deaminated products (aldehydes, acids, and alcohols), and possibly also amino acid precursors. In support of this view, the authors present evidence for the presence, synthesis, metabolism, and biological activity (at the cellular level, using microelectrode techniques) of amino acid, amines, and deaminated compounds metabolically related to catecholamines and sorotonin. That neuroamino acids exert direct effects (not mediated via their amine metabolites) is illustrated by the rapid effects of microiontophoretic dopa upon cortical unit activity, and by the observation that neither the lethargic effect of 5-hydroxytryptophan (considered to support Jouvet's serotonergic theory of sleep) nor the behavioral stimulant effects of dopa (considered to support the catecholamine theory of affective behavior) are significantly prevented by L-aromatic amino acid decarboxylase inhibitors. The biological activity of the deaminated metabolites of catecholamines and serotonin is illustrated by the effects of their microiontophoretic administration upon cortical units. Further, probenecid (an inhibitor of acid transport across the blood-brain barrier) is shown to qualitatively alter the effects of intraventricularly administered PEA and of its metabolite phenylacetic acid upon visual evoked potentials. Rabbit brain is shown to synthesize a series of pharmacologically active noncatecholic phenylethylamines as by-products of catecholamine metabolism. Amine modulators such as PEA differ from typical transmitters by their ability to cross biological barriers; inhibition of decarboxylase in peripheral tissues only (using alpha-methyldopa hydrazine) markedly depletes brain PEA (but not catecholamines). Because of the homeostatic control of the rate of transmitter synthesis and disposition, physiological, pharmacological, and pathological changes may be expected to affect more the tissue levels of related modulators. This modulator theory of drug action is illustrated by the effect of several psychotropic drugs upon the brain levels of PEA and of norepinephrine. For instance, amphetamine initially decreases and then increases brain PEA levels, without altering brain norepinephrine levels. The authors propose an expanded "Dale's Principle": each neuron is specific in that it releases at all its endings the same pool of chemical messengers, composed of one transmitter and metabolically related modulators, the relative proportion of which is determined by the physiological state of the cell (biochemical plasticity)...

The electrophysiological and behavioral effects of phenylethanolamine (OHPEA) and of its precursor 2-phenylethylamine (PEA) were studied in mice and rabbits. In animals pretreated with MAOI, PEA was found to exert strong amphetamine-like effects, EEG alerting, reduction of visual evoked responses, increased locomotor activity, and blockade of tonic seizures induced by electroshock. OHPEA exerted weaker amphetamine-like effects. Inhibition of dopamine-beta-hydroxylase increased most of the effects of PEA. In non-pretreated animals, OHPEA was found to shorten electroshock latency and to prolong the duration of visual evoked responses. PEA (but not OHPEA) potentiated the excitement induced by delta9-tetrahydrocannabinol in MAOI-pretreated mice. Reserpine pretreatment reduced but did not abolish the CNS effects of OHPEA and PEA. One may speculate that endogenous PEA is more likely to serve as a modulator for ergotropic functions than is endogenous OHPEA.