Separating Bacteria by Capsule Amount Using a Discontinuous Density Gradient.

Capsule is a key virulence factor in many bacterial species, mediating immune evasion and resistance to various physical stresses. While many methods are available to quantify and compare capsule production between different strains or mutants, there is no widely used method for sorting bacteria based on how much capsule they produce. We have developed a method to separate bacteria by capsule amount, using a discontinuous density gradient. This method is used to compare capsule amounts semi-quantitatively between cultures, to isolate mutants with altered capsule production, and to purify capsulated bacteria from complex samples. This method can also be coupled with transposon-insertion sequencing to identify genes involved in capsule regulation. Here, the method is demonstrated in detail, including how to optimize the gradient conditions for a new bacterial species or strain, and how to construct and run the density gradient

The Capsule Regulatory Network of Klebsiella pneumoniae Defined by density-TraDISort.

Klebsiella pneumoniae infections affect infants and the immunocompromised, and the recent emergence of hypervirulent and multidrug-resistant K. pneumoniae lineages is a critical health care concern. Hypervirulence in K. pneumoniae is mediated by several factors, including the overproduction of extracellular capsule. However, the full details of how K. pneumoniae capsule biosynthesis is achieved or regulated are not known. We have developed a robust and sensitive procedure to identify genes influencing capsule production, density-TraDISort, which combines density gradient centrifugation with transposon insertion sequencing. We have used this method to explore capsule regulation in two clinically relevant Klebsiella strains, K. pneumoniae NTUH-K2044 (capsule type K1) and K. pneumoniae ATCC 43816 (capsule type K2). We identified multiple genes required for full capsule production in K. pneumoniae, as well as putative suppressors of capsule in NTUH-K2044, and have validated the results of our screen with targeted knockout mutants. Further investigation of several of the K. pneumoniae capsule regulators identified-ArgR, MprA/KvrB, SlyA/KvrA, and the Sap ABC transporter-revealed effects on capsule amount and architecture, serum resistance, and virulence. We show that capsule production in K. pneumoniae is at the center of a complex regulatory network involving multiple global regulators and environmental cues and that the majority of capsule regulatory genes are located in the core genome. Overall, our findings expand our understanding of how capsule is regulated in this medically important pathogen and provide a technology that can be easily implemented to study capsule regulation in other bacterial species.IMPORTANCE Capsule production is essential for K. pneumoniae to cause infections, but its regulation and mechanism of synthesis are not fully understood in this organism. We have developed and applied a new method for genome-wide identification of capsule regulators. Using this method, many genes that positively or negatively affect capsule production in K. pneumoniae were identified, and we use these data to propose an integrated model for capsule regulation in this species. Several of the genes and biological processes identified have not previously been linked to capsule synthesis. We also show that the methods presented here can be applied to other species of capsulated bacteria, providing the opportunity to explore and compare capsule regulatory networks in other bacterial strains and species

Dominant Role of Nucleotide Substitution in the Diversification of Serotype 3 Pneumococci over Decades and during a Single Infection

Streptococcus pneumoniae of serotype 3 possess a mucoid capsule and cause disease associated with high mortality rates relative to other pneumococci. Phylogenetic analysis of a complete reference genome and 81 draft sequences from clonal complex 180, the predominant serotype 3 clone in much of the world, found most sampled isolates belonged to a clade affected by few diversifying recombinations. However, other isolates indicate significant genetic variation has accumulated over the clonal complex’s entire history. Two closely related genomes, one from the blood and another from the cerebrospinal fluid, were obtained from a patient with meningitis. The pair differed in their behaviour in a mouse model of disease and in their susceptibility to antimicrobials, with at least some of these changes attributable to a mutation that upregulated the patAB efflux pump. This indicates clinically important phenotypic variation can accumulate rapidly through small alterations to the genotype

Detection and replication of Moku virus in honey bees and social wasps

Transmission of honey bee viruses to other insects, and vice versa, has previously been reported and the true ecological importance of this phenomenon is still being realized. Members of the family Vespidae interact with honey bees via predation or through the robbing of brood or honey from colonies, and these activities could result in virus transfer. In this study we screened Vespa velutina and Vespa crabro collected from Europe and China and also honey bees and Vespula vulgaris from the UK for Moku virus (MV), an Iflavirus first discovered in the predatory social wasp Vespula pensylvanica in Hawaii. MV was found in 71% of Vespula vulgaris screened and was also detected in UK Vespa crabro. Only seven percent of Vespa velutina individuals screened were MV-positive and these were exclusively samples from Jersey. Of 69 honey bee colonies screened, 43% tested positive for MV. MV replication was confirmed in Apis mellifera and Vespidae species, being most frequently detected in Vespula vulgaris. MV sequences from the UK were most similar to MV from Vespula pensylvanica compared to MV from Vespa velutina in Belgium. The implications of the transfer of viruses between the Vespidae and honey bees are discussed

Female partners of patients after surgical prostate cancer treatment: interactions with physicians and support needs

<p>Abstract</p> <p>Background</p> <p>Few studies have explored the women's experiences as a result of a partners' diagnosis of prostate cancer. This study begins to explore women's interactions with physicians (primary care and urologist) and the support needs associated with the diagnosis and treatment of their partners' prostate cancer.</p> <p>Methods</p> <p>Two focus groups (n = 14) of women whose partners were diagnosed with prostate cancer (diagnoses' 1 - 18 months). A trained facilitator used open-ended questions to explore ideas. The framework approach was used to analyze the transcripts.</p> <p>Results</p> <p>Three main themes emerged: 1. <b>More support</b>. Validation and information is needed for women including emotional support and opportunities to share experiences. 2. <b>Role of the physician</b>. The transfer of care once specialized treatment is no longer needed remained poorly defined, which increased confusion and feelings of abandonment related to the role of the primary physician. 3. <b>Partners' relationship changes</b>. Men became more dependent on their partners for support and to act as the primary communicator and caregiver.</p> <p>Conclusions</p> <p>Additional research is needed in this field to confirm the importance of training primary care physicians to consider holistic treatment approaches that recognize the partner and family needs as important in the complete physical and emotional healing of their patients.</p

What's in a Name? Species-Wide Whole-Genome Sequencing Resolves Invasive and Noninvasive Lineages of Salmonella enterica Serotype Paratyphi B

For 100 years, it has been obvious that Salmonella enterica strains sharing the serotype with the formula 1,4,[ 5], 12: b:1,2-now known as ParatyphiB-can cause diseases ranging from serious systemic infections to self-limiting gastroenteritis. Despite considerable predicted diversity between strains carrying the common Paratyphi B serotype, there remain few methods that subdivide the group into groups that are congruent with their disease phenotypes. Paratyphi B therefore represents one of the canonical examples in Salmonella where serotyping combined with classical microbiological tests fails to provide clinically informative information. Here, we use genomics to provide the first high-resolution view of this serotype, placing it into a wider genomic context of the Salmonella enterica species. These analyses reveal why it has been impossible to subdivide this serotype based upon phenotypic and limited molecular approaches. By examining the genomic data in detail, we are able to identify common features that correlate with strains of clinical importance. The results presented here provide new diagnostic targets, as well as posing important new questions about the basis for the invasive disease phenotype observed in a subset of strains. IMPORTANCE Salmonella enterica strains carrying the serotype Paratyphi B have long been known to possess Jekyll and Hyde characteristics; some cause gastroenteritis, while others cause serious invasive disease. Understanding what makes up the population of strains carrying this serotype, as well as the source of their invasive disease, is a 100-year-old puzzle that we address here using genomics. Our analysis provides the first high-resolution view of this serotype, placing strains carrying serotype Paratyphi B into the wider genomic context of the Salmonella enterica species. This work reveals a history of disease dating back to the middle ages, caused by a group of distinct lineages with various abilities to cause invasive disease. By quantifying the key genomic differences between the invasive and noninvasive populations, we are able to identify key virulence-related targets that can form the basis of simple, rapid, point-of-care tests.Peer reviewe

Transition of plasmodium sporozoites into liver stage-like forms is regulated by the RNA binding protein pumilio

Many eukaryotic developmental and cell fate decisions that are effected post-transcriptionally involve RNA binding proteins as regulators of translation of key mRNAs. In malaria parasites (Plasmodium spp.), the development of round, non-motile and replicating exo-erythrocytic liver stage forms from slender, motile and cell-cycle arrested sporozoites is believed to depend on environmental changes experienced during the transmission of the parasite from the mosquito vector to the vertebrate host. Here we identify a Plasmodium member of the RNA binding protein family PUF as a key regulator of this transformation. In the absence of Pumilio-2 (Puf2) sporozoites initiate EEF development inside mosquito salivary glands independently of the normal transmission-associated environmental cues. Puf2- sporozoites exhibit genome-wide transcriptional changes that result in loss of gliding motility, cell traversal ability and reduction in infectivity, and, moreover, trigger metamorphosis typical of early Plasmodium intra-hepatic development. These data demonstrate that Puf2 is a key player in regulating sporozoite developmental control, and imply that transformation of salivary gland-resident sporozoites into liver stage-like parasites is regulated by a post-transcriptional mechanism

Superspreaders drive the largest outbreaks of hospital onset COVID-19 infections.

SARS-CoV-2 is notable both for its rapid spread, and for the heterogeneity of its patterns of transmission, with multiple published incidences of superspreading behaviour. Here, we applied a novel network reconstruction algorithm to infer patterns of viral transmission occurring between patients and health care workers (HCWs) in the largest clusters of COVID-19 infection identified during the first wave of the epidemic at Cambridge University Hospitals NHS Foundation Trust, UK. Based upon dates of individuals reporting symptoms, recorded individual locations, and viral genome sequence data, we show an uneven pattern of transmission between individuals, with patients being much more likely to be infected by other patients than by HCWs. Further, the data were consistent with a pattern of superspreading, whereby 21% of individuals caused 80% of transmission events. Our study provides a detailed retrospective analysis of nosocomial SARS-CoV-2 transmission, and sheds light on the need for intensive and pervasive infection control procedures