Synergistic activity of mobile genetic element defences in Streptococcus pneumoniae

A diverse set of mobile genetic elements (MGEs) transmit between Streptococcus pneumoniae cells, but many isolates remain uninfected. The best-characterised defences against horizontal transmission of MGEs are restriction-modification systems (RMSs), of which there are two phase-variable examples in S. pneumoniae. Additionally, the transformation machinery has been proposed to limit vertical transmission of chromosomally integrated MGEs. This work describes how these mechanisms can act in concert. Experimental data demonstrate RMS phase variation occurs at a sub-maximal rate. Simulations suggest this may be optimal if MGEs are sometimes vertically inherited, as it reduces the probability that an infected cell will switch between RMS variants while the MGE is invading the population, and thereby undermine the restriction barrier. Such vertically inherited MGEs can be deleted by transformation. The lack of between-strain transformation hotspots at known prophage att sites suggests transformation cannot remove an MGE from a strain in which it is fixed. However, simulations confirmed that transformation was nevertheless effective at preventing the spread of MGEs into a previously uninfected cell population, if a recombination barrier existed between co-colonising strains. Further simulations combining these effects of phase variable RMSs and transformation found they synergistically inhibited MGEs spreading, through limiting both vertical and horizontal transmission

Post-vaccine epidemiology of serotype 3 pneumococci identifies transformation inhibition through prophage-driven alteration of a non-coding RNA

Background: The respiratory pathogen Streptococcus pneumoniae (the pneumococcus) is a genetically diverse bacterium associated with over 101 immunologically distinct polysaccharide capsules (serotypes). Polysaccharide conjugate vaccines (PCVs) have successfully eliminated multiple targeted serotypes, yet the mucoid serotype 3 has persisted despite its inclusion in PCV13. This capsule type is predominantly associated with a single globally disseminated strain, GPSC12 (clonal complex 180).Methods: A genomic epidemiology study combined previous surveillance datasets of serotype 3 pneumococci to analyse the population structure, dynamics, and differences in rates of diversification within GPSC12 during the period of PCV introductions. Transcriptomic analyses, whole genome sequencing, mutagenesis, and electron microscopy were used to characterise the phenotypic impact of loci hypothesised to affect this strain's evolution.Results: GPSC12 was split into clades by a genomic analysis. Clade I, the most common, rarely underwent transformation, but was typically infected with the prophage phi OXC141. Prior to the introduction of PCV13, this Glade's composition shifted towards a phi OXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also phi OXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the similar to 100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage phi OXC141 was found to modify csRNA3, a non-coding RNA that inhibits the induction of transformation. This alteration was identified in -30% of all pneumococci and was particularly common in the unusually clonal serotype 1 GPSC2 strain. RNA-seq and quantitative reverse transcriptase PCR experiments using a genetically tractable pneumococcus demonstrated the altered csRNA3 was more effective at inhibiting production of the competence-stimulating peptide pheromone. This resulted in a reduction in the induction of competence for transformation.Conclusion: This interference with the quorum sensing needed to induce competence reduces the risk of the prophage being deleted by homologous recombination. Hence the selfish prophage-driven alteration of a regulatory RNA limits cell-cell communication and horizontal gene transfer, complicating the interpretation of post-vaccine population dynamics

Neutralization of schwann cell-secreted VEGF is protective to in vitro and in vivo experimental diabetic neuropathy

The pathogenetic role of vascular endothelial growth factor (VEGF) in long-term retinal and kidney complications of diabetes has been demonstrated. Conversely, little is known in diabetic neuropathy. We examined the modulation of VEGF pathway at mRNA and protein level on dorsal root ganglion (DRG) neurons and Schwann cells (SC) induced by hyperglycaemia. Moreover, we studied the effects of VEGF neutralization on hyperglycemic DRG neurons and streptozotocin-induced diabetic neuropathy. Our findings demonstrated that DRG neurons were not affected by the direct exposition to hyperglycaemia, whereas showed an impairment of neurite outgrowth ability when exposed to the medium of SC cultured in hyperglycaemia. This was mediated by an altered regulation of VEGF and FLT-1 receptors. Hyperglycaemia increased VEGF and FLT-1 mRNA without changing their intracellular protein levels in DRG neurons, decreased intracellular and secreted protein levels without changing mRNA level in SC, while reduced the expression of the soluble receptor sFLT-1 both in DRG neurons and SC. Bevacizumab, a molecule that inhibits VEGF activity preventing the interaction with its receptors, restored neurite outgrowth and normalized FLT-1 mRNA and protein levels in co-cultures. In diabetic rats, it both prevented and restored nerve conduction velocity and nociceptive thresholds. We demonstrated that hyperglycaemia early affected neurite outgrowth through the impairment of SC-derived VEGF/FLT-1 signaling and that the neutralization of SC-secreted VEGF was protective both in vitro and in vivo models of diabetic neuropathy

Whole-genome analysis uncovers loss of blaZ associated with carriage isolates belonging to methicillin-resistant Staphylococcus aureus (MRSA) clone ST5-VI in Cape Verde

Objectives: Surveillance studies for Staphylococcus aureus carriage are a primary tool to survey the prevalence of methicillin-resistant S. aureus (MRSA) in the general population, patients and healthcare workers. We have previously reported S. aureus carriage in various African countries, including Cape Verde. Methods: Whole-genome sequences of 106 S. aureus isolates from Cape Verde were determined. Results: Staphylococcus aureus carriage isolates in Cape Verde show high genetic variability, with the detection of 27 sequence types (STs) and three primary genetic clusters associated with ST152, ST15 and ST5. One transmission event with less than eight core-genome single nucleotide polymorphisms (cgSNP) differences was detected among the ST5-VI MRSA lineage. Genetic analysis confirmed the phenotypic resistance and allowed the identification of six independent events of plasmid or transposon loss associated with the deletion of blaZ in nine isolates. In the four ST5 MRSA isolates, loss of the blaZ plasmid coincided with the acquisition of SCCmec type VI and an unusual penicillin phenotype with a minimum inhibitory concentration (MIC) at the breakpoint, indicating an adaptation trend in this endemic lineage. Similar events of blaZ plasmid loss, with concomitant acquisition SCCmec elements, were detected among ST5 isolates from different geographical origins. Conclusion: Overall, the genome data allowed to place isolates in a phylogenetic context and to identify different blaZ gene deletions associated with plasmid or transposon loss. Genomic analysis unveiled adaptation and evolution trends, namely among emerging MRSA lineages in the country, which deserve additional consideration in the design of future infection control protocols

Rapid diagnosis of experimental meningitis by bacterial heat production in cerebrospinal fluid

BACKGROUND: Calorimetry is a nonspecific technique which allows direct measurement of heat generated by biological processes in the living cell. We evaluated the potential of calorimetry for rapid detection of bacterial growth in cerebrospinal fluid (CSF) in a rat model of bacterial meningitis. METHODS: Infant rats were infected on postnatal day 11 by direct intracisternal injection with either Streptococcus pneumoniae, Neisseria meningitidis or Listeria monocytogenes. Control animals were injected with sterile saline or heat-inactivated S. pneumoniae. CSF was obtained at 18 hours after infection for quantitative cultures and heat flow measurement. For calorimetry, 10 microl and 1 microl CSF were inoculated in calorimetry ampoules containing 3 ml trypticase soy broth (TSB). RESULTS: The mean bacterial titer (+/- SD) in CSF was 1.5 +/- 0.6 x 108 for S. pneumoniae, 1.3 +/- 0.3 x 106 for N. meningitidis and 3.5 +/- 2.2 x 104 for L. monocytogenes. Calorimetric detection time was defined as the time until heat flow signal exceeded 10 microW. Heat signal was detected in 10-microl CSF samples from all infected animals with a mean (+/- SD) detection time of 1.5 +/- 0.2 hours for S. pneumoniae, 3.9 +/- 0.7 hours for N. meningitidis and 9.1 +/- 0.5 hours for L. monocytogenes. CSF samples from non-infected animals generated no increasing heat flow (<10 microW). The total heat was the highest in S. pneumoniae ranging from 6.7 to 7.5 Joules, followed by L. monocytogenes (5.6 to 6.1 Joules) and N. meningitidis (3.5 to 4.4 Joules). The lowest detectable bacterial titer by calorimetry was 2 cfu for S. pneumoniae, 4 cfu for N. meningitidis and 7 cfu for L. monocytogenes. CONCLUSION: By means of calorimetry, detection times of <4 hours for S. pneumoniae and N. meningitidis and <10 hours for Listeria monocytogenes using as little as 10 microl CSF were achieved. Calorimetry is a new diagnostic method allowing rapid and accurate diagnosis of bacterial meningitis from a small volume of CSF

Islet transplantation and insulin administration relieve long-term complications and rescue the residual endogenous pancreatic cells

Islet transplantation is a poorly investigated Long-term strategy for insulin replacement and for treatment of complications in patients with diabetes. We investigated whether islet transplantation and insulin treatment can relieve diabetic neuropathy and rescue the residual endogenous pancreatic beta cells. We used a multimodal approach, with five groups of Sprague-Dawley rats studied for 8 months: control rats, diabetic rats, insulin-treated diabetic rats with moderate or mild hyperglycemia, and diabetic rats transplanted with microencapsulated islets. Islet transplantation normalized glycemia and increased body and muscle weight; it was also effective in reducing proteinuria and altered liver function. Transplantation significantly improved tail nerve conduction velocity, Na+-K+-ATPase activity, and morphological alterations in the sciatic nerve as evidenced by decrease in g-ratio; it also restored thermal and ameliorated mechanical nociceptive thresholds. Morphometric analysis of pancreas indicated a significant beta-cell volume increase in transplanted rats, compared with mildly and moderately hyperglycemic rats. Thus, allogeneic islet transplantation had a positive systemic effect in diabetic rats and induced regression of the established neuropathy and restitution of the typical characteristics of the islets. These findings strongly reinforce the need for improving glycemic control, not only to reverse established diabetic complications but also to improve beta-cell status in diabetic pancreas