A Newly Discovered Bordetella Species Carries a Transcriptionally Active CRISPR-Cas with a Small Cas9 Endonuclease

Background Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated genes (cas) are widely distributed among bacteria. These systems provide adaptive immunity against mobile genetic elements specified by the spacer sequences stored within the CRISPR. Methods The CRISPR-Cas system has been identified using Basic Local Alignment Search Tool (BLAST) against other sequenced and annotated genomes and confirmed via CRISPRfinder program. Using Polymerase Chain Reactions (PCR) and Sanger DNA sequencing, we discovered CRISPRs in additional bacterial isolates of the same species of Bordetella. Transcriptional activity and processing of the CRISPR have been assessed via RT-PCR. Results Here we describe a novel Type II-C CRISPR and its associated genes—cas1, cas2, and cas9—in several isolates of a newly discovered Bordetella species. The CRISPR-cas locus, which is absent in all other Bordetella species, has a significantly lower GC-content than the genome-wide average, suggesting acquisition of this locus via horizontal gene transfer from a currently unknown source. The CRISPR array is transcribed and processed into mature CRISPR RNAs (crRNA), some of which have homology to prophages found in closely related species B. hinzii. Conclusions Expression of the CRISPR-Cas system and processing of crRNAs with perfect homology to prophages present in closely related species, but absent in that containing this CRISPR-Cas system, suggest it provides protection against phage predation. The 3,117-bp cas9 endonuclease gene from this novel CRISPR-Cas system is 990 bp smaller than that of Streptococcus pyogenes, the 4,017-bp allele currently used for genome editing, and which may make it a useful tool in various CRISPR-Cas technologies

Comparing plasma and faecal measures of steroid hormones in Adelie penguins Pygoscelis adeliae

Physiological measurements of both stress and sex hormones are often used to estimate the consequences of natural or human-induced change in ecological studies of various animals. Different methods of hormone measurement exist, potentially explaining variation in results across studies; methods should be cross-validated to ensure that they correlate. We directly compared faecal and plasma hormone measurements for the first time in a wild free-living species, the Adelie penguin (Pygoscelis adeliae). Blood and faecal samples were simultaneously collected from individual penguins for comparison and assayed for testosterone and corticosterone (or their metabolites). Sex differences and variability within each measure, and correlation of values across measures were compared. For both hormones, plasma samples showed greater variation than faecal samples. Males had higher mean corticosterone concentrations than females, but the difference was only statistically significant in faecal samples. Plasma testosterone, but not faecal testosterone, was significantly higher in males than females. Correlation between sample types was poor overall, and weaker in females than in males, perhaps because measures from plasma represent hormones that are both free and bound to globulins, whereas measures from faeces represent only the free portion. Faecal samples also represent a cumulative measure of hormones over time, as opposed to a plasma ‘snapshot’ concentration. Our data indicate that faecal sampling appears more suitable for assessing baseline hormone concentrations, whilst plasma sampling may best define immediate responses to environmental events. Consequently, future studies should ensure that they select the most appropriate matrix and method of hormone measurement to answer their research questions

Comparative analysis of subtyping methods against a whole-genome-sequencing standard for Salmonella enterica serotype Enteritidis.

A retrospective investigation was performed to evaluate whole-genome sequencing as a benchmark for comparing molecular subtyping methods for Salmonella enterica serotype Enteritidis and survey the population structure of commonly encountered S. enterica serotype Enteritidis outbreak isolates in the United States. A total of 52 S. enterica serotype Enteritidis isolates representing 16 major outbreaks and three sporadic cases collected between 2001 and 2012 were sequenced and subjected to subtyping by four different methods: (i) whole-genome single-nucleotide-polymorphism typing (WGST), (ii) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA), (iii) clustered regularly interspaced short palindromic repeats combined with multi-virulence-locus sequence typing (CRISPR-MVLST), and (iv) pulsed-field gel electrophoresis (PFGE). WGST resolved all outbreak clusters and provided useful robust phylogenetic inference results with high epidemiological correlation. While both MLVA and CRISPR-MVLST yielded higher discriminatory power than PFGE, MLVA outperformed the other methods in delineating outbreak clusters whereas CRISPR-MVLST showed the potential to trace major lineages and ecological origins of S. enterica serotype Enteritidis. Our results suggested that whole-genome sequencing makes a viable platform for the evaluation and benchmarking of molecular subtyping methods

Complete Genome Sequence and Comparative Metabolic Profiling of the Prototypical Enteroaggregative Escherichia coli Strain 042

Background \ud Escherichia coli can experience a multifaceted life, in some cases acting as a commensal while in other cases causing intestinal and/or extraintestinal disease. Several studies suggest enteroaggregative E. coli are the predominant cause of E. coli-mediated diarrhea in the developed world and are second only to Campylobacter sp. as a cause of bacterial-mediated diarrhea. Furthermore, enteroaggregative E. coli are a predominant cause of persistent diarrhea in the developing world where infection has been associated with malnourishment and growth retardation. \ud \ud Methods \ud In this study we determined the complete genomic sequence of E. coli 042, the prototypical member of the enteroaggregative E. coli, which has been shown to cause disease in volunteer studies. We performed genomic and phylogenetic comparisons with other E. coli strains revealing previously uncharacterised virulence factors including a variety of secreted proteins and a capsular polysaccharide biosynthetic locus. In addition, by using Biolog™ Phenotype Microarrays we have provided a full metabolic profiling of E. coli 042 and the non-pathogenic lab strain E. coli K-12. We have highlighted the genetic basis for many of the metabolic differences between E. coli 042 and E. coli K-12. \ud \ud Conclusion \ud This study provides a genetic context for the vast amount of experimental and epidemiological data published thus far and provides a template for future diagnostic and intervention strategies

The intriguing interaction of Escherichia coli with the host environment and innovative strategies to interfere with colonization: A summary of the 2019 E. coli and the Mucosal Immune System meeting

The 3rd E. coli and the Mucosal Immune System (ECMIS) meeting was held at Ghent University in Belgium from June 2-5, 2019. It brought together an international group of scientists interested in mechanisms of colonization, host response, and vaccine development. ECMIS distinguishes itself from related meetings on these enteropathogens by providing a greater emphasis on animal health and disease, and covering a broad range of pathotypes including enterohemorrhagic, enteropathogenic, enterotoxigenic, enteroaggregative, and extraintestinal pathogenic E. coli. As it is well-established that the genus Shigella represents a subspecies of E. coli, these organisms along with related enteroinvasive E. coli are also included. In addition, Tannerella forsythia, a periodontal pathogen, was presented as an example of a pathogen which uses its surface glycans for mucosal interaction. This review summarizes several highlights from the 2019 meeting and major advances to our understanding of the biology of these pathogens and their impact on the host

Global Distribution of O Serotypes and Antibiotic Resistance in Extraintestinal Pathogenic Escherichia coli Collected From the Blood of Patients With Bacteremia Across Multiple Surveillance Studies

Background: Extraintestinal pathogenic Escherichia coli (ExPEC) is the leading cause of bacteremia worldwide, with older populations having increased risk of invasive bacterial disease. Increasing resistance to first-line antibiotics and emergence of multidrug-resistant (MDR) strains represent major treatment challenges. ExPEC O serotypes are key targets for potential multivalent conjugate vaccine development. Therefore, we evaluated the O serotype distribution and antibiotic resistance profiles of ExPEC strains causing bloodstream infections across 4 regions. Methods: Blood culture isolates from patients aged ≥60 years collected during 5 retrospective E. coli surveillance studies in Europe, North America, Asia-Pacific, and South America (2011-2017) were analyzed. Isolates were O serotyped by agglutination; O genotyping was performed for nontypeable isolates. Antimicrobial susceptibility testing was also conducted. Results: Among 3217 ExPEC blood culture isolates, the most ubiquitous O serotype was O25 (n = 737 [22.9%]), followed by O2, O6, O1, O75, O15, O8, O16, O4, O18, O77 group, O153, O9, O101/O162, O86, and O13 (prevalence of ≥1%). The prevalence of these O serotypes was generally consistent across regions, apart from South America; together, these 16 O serotypes represented 77.6% of all ExPEC bacteremia isolates analyzed. The overall MDR frequency was 10.7%, with limited variation between regions. Within the MDR subset (n = 345), O25 showed a dominant prevalence of 63.2% (n = 218). Conclusions: Predominant O serotypes among ExPEC bacteremia isolates are widespread across different regions. O25 was the most prevalent O serotype overall and particularly dominant among MDR isolates. These findings may inform the design of multivalent conjugate vaccines that can target the predominant O serotypes associated with invasive ExPEC disease in older adults

Distinct Genetic Architectures for Male and Female Inflorescence Traits of Maize

We compared the genetic architecture of thirteen maize morphological traits in a large population of recombinant inbred lines. Four traits from the male inflorescence (tassel) and three traits from the female inflorescence (ear) were measured and studied using linkage and genome-wide association analyses and compared to three flowering and three leaf traits previously studied in the same population. Inflorescence loci have larger effects than flowering and leaf loci, and ear effects are larger than tassel effects. Ear trait models also have lower predictive ability than tassel, flowering, or leaf trait models. Pleiotropic loci were identified that control elongation of ear and tassel, consistent with their common developmental origin. For these pleiotropic loci, the ear effects are larger than tassel effects even though the same causal polymorphisms are likely involved. This implies that the observed differences in genetic architecture are not due to distinct features of the underlying polymorphisms. Our results support the hypothesis that genetic architecture is a function of trait stability over evolutionary time, since the traits that changed most during the relatively recent domestication of maize have the largest effects

Perceptions about data-informed decisions: an assessment of information-use in high HIV-prevalence settings in South Africa

BACKGROUND: Information-use is an integral component of a routine health information system and essential to influence policy-making, program actions and research. Despite an increased amount of routine data collected, planning and resource-allocation decisions made by health managers for managing HIV programs are often not based on data. This study investigated the use of information, and barriers to using routine data for monitoring the prevention of mother-to-child transmission of HIV (PMTCT) programs in two high HIV-prevalence districts in South Africa. METHODS: We undertook an observational study using a multi-method approach, including an inventory of facility records and reports. The performance of routine information systems management (PRISM) diagnostic ‘Use of Information’ tool was used to assess the PMTCT information system for evidence of data use in 57 health facilities in two districts. Twenty-two in-depth interviews were conducted with key informants to investigate barriers to information use in decision-making. Participants were purposively selected based on their positions and experience with either producing PMTCT data and/or using data for management purposes. We computed descriptive statistics and used a general inductive approach to analyze the qualitative data. RESULTS: Despite the availability of mechanisms and processes to facilitate information-use in about two-thirds of the facilities, evidence of information-use (i.e., indication of some form of information-use in available RHIS reports) was demonstrated in 53% of the facilities. Information was inadequately used at district and facility levels to inform decisions and planning, but was selectively used for reporting and monitoring program outputs at the provincial level. The inadequate use of information stemmed from organizational issues such as the lack of a culture of information-use, lack of trust in the data, and the inability of program and facility managers to analyze, interpret and use information. CONCLCUSIONS: Managers’ inability to use information implied that decisions for program implementation and improving service delivery were not always based on data. This lack of data use could influence the delivery of health care services negatively. Facility and program managers should be provided with opportunities for capacity development as well as practice-based, in-service training, and be supported to use information for planning, management and decision-making

Negatively-Marked MCQ Assessments That Reward Partial Knowledge Do Not Introduce Gender Bias Yet Increase Student Performance and Satisfaction and Reduce Anxiety

Multiple-choice question (MCQ) examinations are increasingly used as the assessment method of theoretical knowledge in large class-size modules in many life science degrees. MCQ-tests can be used to objectively measure factual knowledge, ability and high-level learning outcomes, but may also introduce gender bias in performance dependent on topic, instruction, scoring and difficulty. The ‘Single Answer’ (SA) test is often used in which students choose one correct answer, in which they are unable to demonstrate partial knowledge. Negatively marking eliminates the chance element of guessing but may be considered unfair. Elimination testing (ET) is an alternative form of MCQ, which discriminates between all levels of knowledge, while rewarding demonstration of partial knowledge. Comparisons of performance and gender bias in negatively marked SA and ET tests have not yet been performed in the life sciences. Our results show that life science students were significantly advantaged by answering the MCQ test in elimination format compared to single answer format under negative marking conditions by rewarding partial knowledge of topics. Importantly, we found no significant difference in performance between genders in either cohort for either MCQ test under negative marking conditions. Surveys showed that students generally preferred ET-style MCQ testing over SA-style testing. Students reported feeling more relaxed taking ET MCQ and more stressed when sitting SA tests, while disagreeing with being distracted by thinking about best tactics for scoring high. Students agreed ET testing improved their critical thinking skills. We conclude that appropriately-designed MCQ tests do not systematically discriminate between genders. We recommend careful consideration in choosing the type of MCQ test, and propose to apply negative scoring conditions to each test type to avoid the introduction of gender bias. The student experience could be improved through the incorporation of the elimination answering methods in MCQ tests via rewarding partial and full knowledge