Long-term evolution of Streptococcus mitis and Streptococcus pneumoniae leads to higher genetic diversity within rather than between human populations.

Evaluation of the apportionment of genetic diversity of human bacterial commensals within and between human populations is an important step in the characterization of their evolutionary potential. Recent studies showed a correlation between the genomic diversity of human commensal strains and that of their host, but the strength of this correlation and of the geographic structure among human populations is a matter of debate. Here, we studied the genomic diversity and evolution of the phylogenetically related oro-nasopharyngeal healthy-carriage Streptococcus mitis and Streptococcus pneumoniae, whose lifestyles range from stricter commensalism to high pathogenic potential. A total of 119 S. mitis genomes showed higher within- and among-host variation than 810 S. pneumoniae genomes in European, East Asian and African populations. Summary statistics of the site-frequency spectrum for synonymous and non-synonymous variation and ABC modelling showed this difference to be due to higher ancestral bacterial population effective size (Ne) in S. mitis, whose genomic variation has been maintained close to mutation-drift equilibrium across (at least many) generations, whereas S. pneumoniae has been expanding from a smaller ancestral bacterial population. Strikingly, both species show limited differentiation among human populations. As genetic differentiation is inversely proportional to the product of effective population size and migration rate (Nem), we argue that large Ne have led to similar differentiation patterns, even if m is very low for S. mitis. We conclude that more diversity within than among human populations and limited population differentiation must be common features of the human microbiome due to large Ne.

Diverse regulatory pathways modulate bet hedging of competence induction in epigenetically-differentiated phase variants of Streptococcus pneumoniae.

Despite enabling Streptococcus pneumoniae to acquire antibiotic resistance and evade vaccine-induced immunity, transformation occurs at variable rates across pneumococci. Phase variants of isolate RMV7, distinguished by altered methylation patterns driven by the translocating variable restriction-modification (tvr) locus, differed significantly in their transformation efficiencies and biofilm thicknesses. These differences were replicated when the corresponding tvr alleles were introduced into an RMV7 derivative lacking the locus. RNA-seq identified differential expression of the type 1 pilus, causing the variation in biofilm formation, and inhibition of competence induction in the less transformable variant, RMV7domi. This was partly attributable to RMV7domi's lower expression of ManLMN, which promoted competence induction through importing N-acetylglucosamine. This effect was potentiated by analogues of some proteobacterial competence regulatory machinery. Additionally, one of RMV7domi's phage-related chromosomal island was relatively active, which inhibited transformation by increasing expression of the stress response proteins ClpP and HrcA. However, HrcA increased competence induction in the other variant, with its effects depending on Ca2+ supplementation and heat shock. Hence the heterogeneity in transformation efficiency likely reflects the diverse signalling pathways by which it is affected. This regulatory complexity will modulate population-wide responses to synchronising quorum sensing signals to produce co-ordinated yet stochastic bet hedging behaviour.

An innate pathogen sensing strategy involving ubiquitination of bacterial surface proteins.

Sensing of pathogens by ubiquitination is a critical arm of cellular immunity. However, universal ubiquitination targets on microbes remain unidentified. Here, using in vitro, ex vivo, and in vivo studies, we identify the first protein-based ubiquitination substrates on phylogenetically diverse bacteria by unveiling a strategy that uses recognition of degron-like motifs. Such motifs form a new class of intra-cytosolic pathogen-associated molecular patterns (PAMPs). Their incorporation enabled recognition of nonubiquitin targets by host ubiquitin ligases. We find that SCFFBW7 E3 ligase, supported by the regulatory kinase, glycogen synthase kinase 3ß, is crucial for effective pathogen detection and clearance. This provides a mechanistic explanation for enhanced risk of infections in patients with chronic lymphocytic leukemia bearing mutations in F-box and WD repeat domain containing 7 protein. We conclude that exploitation of this generic pathogen sensing strategy allows conservation of host resources and boosts antimicrobial immunity.

Development of a Novel Ex Vivo Porcine Hepatic Segmental Perfusion Proof-of-Concept Model Towards More Ethical Translational Research.

Introduction Ex vivo machine perfusion describes the technique where organs are continuously perfused and oxygenated extracorporeally (at physiological conditions) to maintain the organs' viability. To our knowledge, there are currently no reported studies describing ex vivo perfusion of a single hepatic segment. Here, we describe the development of a porcine ex vivo hepatic segmental perfusion model to demonstrate proof of concept and support further research into the ex vivo perfusion of the human liver using discarded tissue.  Methods Whole livers were retrieved from abattoir-derived pigs and connected to a normothermic extracorporeal perfusion circuit. Constant segmental perfusion via the common or segmental hepatic artery and portal vein with heparinised autologous blood was established. The viability of the perfused organ was assessed by monitoring perfusion pressures, flow rates and histology samples. Results Following perfusion and optimisation of the model for three hepatic segments, the third perfusion demonstrated viable hepatocytes centrally after 4 h of segmental perfusion. Conclusion Ex vivo hepatic segmental perfusion is technically challenging but its success in a porcine model and the principles learned should facilitate the development of an analogous human model using discarded tissue following formal liver resections. The model would use a healthy liver segment following a major formal resection such as a hemi-hepatectomy and ex vivo perfusion performed via a segmental hepatic artery and portal vein. If successful this model would represent a significant development and enable ethical translation research to assess the response of human livers to a variety of stressors, including toxicity and infection.

A Narrative Review of the Applications of Ex-vivo Human Liver Perfusion.

Ex-vivo perfusion describes the extra-corporeal delivery of fluid to an organ or tissue. Although it has been widely studied in the context of organ preservation and transplantation, it has also proven to be an invaluable tool in the development of novel models for translational pre-clinical research. Here, we review the literature reporting ex-vivo human liver perfusion experiments to further understand current perfusion techniques and protocols together with their applications. A computerised search was made of Ovid, MEDLINE, and Embase using the search words "ex-vivo liver or hepatic perfusion". All relevant studies in English describing experiments using ex-vivo perfusion of human livers between 2016 and 2021, inclusive, were included. Of 21 reviewed studies, 19 used ex-vivo human liver perfusion in the context of allogeneic liver transplantation. The quality and size of the studies varied considerably. Human liver perfusion was almost exclusively limited to whole organs and "split" livers, although one study did describe the successful perfusion of tissue sections following a partial hepatectomy. This review of recent literature involving ex-vivo human liver perfusion demonstrates that the technique is not limited to whole liver perfusion. Split-liver perfusion is extremely valuable allowing one lobe to act as a control and increasing the number available for research. This review also highlights the present lack of any reports of segmental liver perfusion. The discarded donor liver is a scarce resource, and the successful use of segmental perfusion has the potential to expand the available experimental models to facilitate pre-clinical experimentation.

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 ϕOXC141. Prior to the introduction of PCV13, this clade's composition shifted towards a ϕOXC141-negative subpopulation in a systematically sampled UK collection. In the post-PCV13 era, more rapidly recombining non-Clade I isolates, also ϕOXC141-negative, have risen in prevalence. The low in vitro transformation efficiency of a Clade I isolate could not be fully explained by the ~100-fold reduction attributable to the serotype 3 capsule. Accordingly, prophage ϕOXC141 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.

Intracellular survival of Streptococcus pneumoniae in human alveolar macrophages is augmented with HIV infection.

People Living with HIV (PLHIV) are at an increased risk of pneumococcal pneumonia than HIV-uninfected adults, but the reasons for this are still not well understood. We investigated whether alveolar macrophages (AM) mediated control of pneumococcal infection is impaired in PLHIV compared to HIV-uninfected adults. We assessed anti-bactericidal activity against Streptococcus pneumoniae of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post ex vivo infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings, in vivo evidence showed that PLHIV had a higher propensity for harboring S. pneumoniae within their AMs than HIV-uninfected adults. Moreover, bacterial intracellular survival in AMs was associated with extracellular propagation of pneumococcal infection. Our data suggest that failure of AMs to eliminate S. pneumoniae intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV.

Diurnal Differences in Intracellular Replication Within Splenic Macrophages Correlates With the Outcome of Pneumococcal Infection.

Circadian rhythms affect the progression and severity of bacterial infections including those caused by Streptococcus pneumoniae, but the mechanisms responsible for this phenomenon remain largely elusive. Following advances in our understanding of the role of replication of S. pneumoniae within splenic macrophages, we sought to investigate whether events within the spleen correlate with differential outcomes of invasive pneumococcal infection. Utilising murine invasive pneumococcal disease (IPD) models, here we report that infection during the murine active phase (zeitgeber time 15; 15h after start of light cycle, 3h after start of dark cycle) resulted in significantly faster onset of septicaemia compared to rest phase (zeitgeber time 3; 3h after start of light cycle) infection. This correlated with significantly higher pneumococcal burden within the spleen of active phase-infected mice at early time points compared to rest phase-infected mice. Whole-section confocal microscopy analysis of these spleens revealed that the number of pneumococci is significantly higher exclusively within marginal zone metallophilic macrophages (MMMs) known to allow intracellular pneumococcal replication as a prerequisite step to the onset of septicaemia. Pneumococcal clusters within MMMs were more abundant and increased in size over time in active phase-infected mice compared to those in rest phase-infected mice which decreased in size and were present in a lower percentage of MMMs. This phenomenon preceded significantly higher levels of bacteraemia alongside serum IL-6 and TNF-α concentrations in active phase-infected mice following re-seeding of pneumococci into the blood. These data greatly advance our fundamental knowledge of pneumococcal infection by linking susceptibility to invasive pneumococcal infection to variation in the propensity of MMMs to allow persistence and replication of phagocytosed bacteria. These findings also outline a somewhat rare scenario whereby the active phase of an organism's circadian cycle plays a seemingly counterproductive role in the control of invasive infection.

Analyzing Macrophage Infection at the Organ Level.

Classical in vivo infection models are oftentimes associated with speculation due to the many physiological factors that are unseen or not accounted for when analyzing experimental outputs, especially when solely utilizing the classic approach of tissue-derived colony-forming unit (CFU) enumeration. To better understand the steps and natural progression of bacterial infection, the pathophysiology of individual organs with which the bacteria interact in their natural course of infection must be considered. In this case, it is not only important to isolate organs as much as possible from additional physiological processes, but to also consider the dynamics of the bacteria at the cellular level within these organs of interest. Here, we describe in detail two models, ex vivo porcine liver and spleen coperfusion and a murine infection model, and the numerous associated experimental outputs produced by these models that can be taken and used together to investigate the pathogen-host interactions within tissues in depth.

Streptococcus pneumoniae: 'captain of the men of death' and financial burden.

Streptococcus pneumoniae may inhabit the upper respiratory tract of humans without causing harm but it also causes diseases with high morbidity and mortality. It has excellent adaptive capabilities thanks to its ability to shuffle its genetic content by acquiring and incorporating DNA from other bacteria and is highly competent for genetic transformation. Sugar sensing, cleavage and transport ensure its fitness and survival in the host, and intracellular survival in macrophages has been linked to virulence. The polysaccharide capsule and toxin pneumolysin are the most important virulence determinants. Polysaccharide-based vaccines provide protection against the serotypes represented in vaccine formulations.

Interaction of Klebsiella pneumoniae with tissue macrophages in a mouse infection model and ex-vivo pig organ perfusions: an exploratory investigation.

BACKGROUND: Hypervirulent Klebsiella pneumoniae (hvKp) strains of capsule type K1 and K2 cause invasive infections associated with hepatic abscesses, which can be difficult to treat and are frequently associated with relapsing infections. Other K pneumoniae strains (non-hvKp), including lineages that have acquired carbapenem resistance, do not manifest this pathology. In this work we aimed to test the hypothesis that within-macrophage replication is a key mechanism underpinning abscess formation in hvKp infections. METHODS: In this exploratory investigation, to study the pathophysiology of abscess formation, mice were intravenously infected with 106 colony forming units (CFU) of either hvKp isolates (six strains) or non-hvKp isolates (seven strains). Intracellular bacterial replication and neutrophil influx in liver and spleen was quantified by fluorescence microscopy of sliced cryopreserved organs of mice collected 30 min, 6 h, and 24 h after infection with the aim to provide data of bacterial association to Kupffer cells in the liver and to the different tissue macrophages in the spleen. Microbiological and microscopy analysis of an ex-vivo model of pig liver and spleen infection were used to confirm within-macrophage replication. Pig organs were perfused with heparinised, autologous pig's blood and injected with 6·5 × 107 CFU of hvKp K2 sequence type 25 strain GMR151. Blood and tissue biopsies collected before infection and 30 min, 1 h, 2 h, 3 h, 4 h, and 5 h after infection were used to measure bacterial counts and to identify the subcellular localisation of bacteria by immunohistochemistry analysis. FINDINGS: We show that hvKp resisted phagocyte-mediated clearance and replicated in mouse liver macrophages to form clusters 6 h after infection, with a mean of 7·0 bacteria per Kupffer cell (SD 6·2); however, non-hvKp were efficiently cleared (mean 1·5 bacteria per cell [SD 1·1]). HvKp infection promoted neutrophil recruitment to sites of infection, which in the liver resulted in histopathological signs of abscess formation as early as 24 h post-infection. Experiments in pig organs which share a high functional and anatomical resemblance to human organs, provided strong evidence for the propensity of hvKp to replicate within the hepatic macrophages. INTERPRETATION: These findings show subversion of innate immune processes in the liver by K pneumoniae and resistance to Kupffer cell mediated clearance as an explanation for the propensity of hvKp strains to cause hepatic abscesses. FUNDING: University of Oxford and a Royal Society Wolfson grant funded biosafety facility.

Splenic macrophages as the source of bacteraemia during pneumococcal pneumonia.

BACKGROUND: Severe community-acquired pneumococcal pneumonia is commonly associated with bacteraemia. Although it is assumed that the bacteraemia solely derives from pneumococci entering the blood from the lungs it is unknown if other organs are important in the pathogenesis of bacteraemia. Using three models, we tested the relevance of the spleen in pneumonia-associated bacteraemia. METHODS: We used human spleens perfused ex vivo to explore permissiveness to bacterial replication, a non-human primate model to check for splenic involvement during pneumonia and a mouse pneumonia-bacteraemia model to demonstrate that splenic involvement correlates with invasive disease. FINDINGS: Here we present evidence that the spleen is the reservoir of bacteraemia during pneumonia. We found that in the human spleen infected with pneumococci, clusters with increasing number of bacteria were detectable within macrophages. These clusters also were detected in non-human primates. When intranasally infected mice were treated with a non-therapeutic dose of azithromycin, which had no effect on pneumonia but concentrated inside splenic macrophages, bacteria were absent from the spleen and blood and importantly mice had no signs of disease. INTERPRETATION: We conclude that the bacterial load in the spleen, and not lung, correlates with the occurrence of bacteraemia. This supports the hypothesis that the spleen, and not the lungs, is the major source of bacteria during systemic infection associated with pneumococcal pneumonia; a finding that provides a mechanistic basis for using combination therapies including macrolides in the treatment of severe community-acquired pneumococcal pneumonia. FUNDING: Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.

Genetic Background and Antibiotic Resistance Profiles of K. pneumoniae NDM-1 Strains Isolated from UTI, ABU, and the GI Tract, from One Hospital in Poland, in Relation to Strains Nationally and Worldwide.

In recent years, there has been an observed increase in infections caused by carbapenem-resistant Klebsiella pneumonia (Kp) strains. The aim of this study was the phenotypic and genotypic analysis of eight K. pneumoniae NDM (Kp NDM) isolates, recovered in Poland during the years 2016 and 2018 from seven patients with urinary tract infections (UTIs), asymptomatic bacteriuria (ABU), or colonization of the gut. PCR melting profile genotyping indicated a close relationship between the strains derived from 2018, which were not related to the strain isolated in 2016. WGS results were analyzed in relation to international Kp isolates. Clonal and phylogenetic analyses were performed based on multilocus sequence typing (MLST) and single nucleotide polymorphisms (SNPs) of the core genome. The metallo-ß-lactamase was assigned to the NDM-1 type and the sequence was identified as ST11. Eleven antimicrobial resistance genes were detected, mostly from plasmid contigs. Unprecedented profiles of plasmid replicons were described with the IncFII/pKPX-1 dominant replicon. In terms of the KL24 and O2v1 capsular antigen profiles, these isolates corresponded to Greek strains. Strains isolated from UTI, ABU, and colonization GI tract patients were not carrying environment-specific virulence genes. Based on the assessment of strain relationships at the genome level and their direction of evolution, the international character of the sublines was demonstrated, with a documented epidemic potential in Poland and Greece. In conclusion, some groups of patients, e.g., renal transplant recipients or those with complicated UTIs, who are frequently hospitalized and undergoing antibiotic therapy, should be monitored not only for the risk of UTI, but also for colonization by Kp NDM strains.

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.

A Virulence Associated Siderophore Importer Reduces Antimicrobial Susceptibility of Klebsiella pneumoniae.

The accessory genomes of many pathogenic bacteria include ABC transporters that scavenge metal by siderophore uptake and ABC transporters that contribute to antimicrobial resistance by multidrug efflux. There are mechanistic and recently recognized structural similarities between siderophore importer proteins and efflux pumps. Here we investigated the influence of siderophore importer YbtPQ on antimicrobial resistance of Klebsiella pneumoniae. YbtPQ is encoded in the yersiniabactin cluster in a prevalent mobile genetic element ICEKp, and is also common in pathogenicity islands of Escherichia coli and Yersinia species, where yersiniabactin enhances virulence. Deletion of ICEKp increased the susceptibility of K. pneumoniae to all antimicrobials tested. The mechanism was dependent on the yersiniabactin importer YbtPQ and may involve antimicrobial efflux, since it was affected by the inhibitor reserpine. The element ICEKp is naturally highly mobile, indeed the accessory genome of K. pneumoniae is recognized as a reservoir of genes for the emergence of hospital outbreak strains and for transfer to other Gram-negative pathogens. Introduction of ICEKp, or a plasmid encoding YbtPQ, to E. coli decreased its susceptibility to a broad range of antimicrobials. Thus a confirmed siderophore importer, on a rapidly evolving and highly mobile element capable of interspecies transfer, may have a secondary function exporting antimicrobials.

Pathogenic Differences of Type 1 Restriction-Modification Allele Variants in Experimental Listeria monocytogenes Meningitis.

Background:L. monocytogenes meningoencephalitis has a mortality rate of up to 50% and neurofunctional sequelae are common. Type I restriction-modification systems (RMS) are capable of adding methyl groups to the host genome. Some contain multiple sequence recognition (hsdS) genes that recombine, resulting in distinct DNA methylation patterns and patterns of gene expression. These phenotypic switches have been linked to virulence and have recently been discovered in multiple clonal complexes of L. monocytogenes. In the present study, we investigated the significant of RMS on L. monocytogenes virulence during the acute phase of experimental meningitis. Methods:L. monocytogenes strains containing RMS systems were identified, and purified clones enriched for single hsdS alleles were isolated. In vivo, 11-day old Wistar rats were infected with an inoculum containing (a) one of 4 single RMS allele variants (A, B, C, D) treated with amoxicillin (AMX 50 mg/kg/dosis, q8h), (b) a mixture of all 4 variants with or without AMX treatment, or (c) different mixtures of 2 RMS allele variants. At selected time points after infection, clinical and inflammatory parameters, bacterial titers and brain damage were determined. Changes in the relative frequency of the occurring RMS alleles in the inoculum and in CSF or cerebellum of infected animals were analyzed by capillary electrophoresis. Results: We have identified a phase variable RMS locus within L. monocytogenes CC4 and generated stocks that stably expressed each of the possible hsdS genes within that loci. Generation of these allele variants (A, B, C, D) allowed us to determine the methylation pattern associated with each hsdS through SMRT sequencing. In vivo infections with these single allele variants revealed differences in disease severity in that C induced the worst clinical outcome and more pronounced hippocampal apoptosis; D showed the most pronounced weight loss and the highest bacterial titer in the cerebellum. A caused the least severe disease. Conclusion: We identified that L. monocytogenes expressing hsdS (A) causes less damage than when other hsdS genes are expressed. While expression of hsdSC and D worsened the outcome in L. monocytogenes meningitis. We also demonstrate a competitive advantage of variants C and B over variant A in this model. Phenotypical switching may therefore represent a mechanism of virulence regulation during the acute phase of CNS infections with L. monocytogenes.

The New Klebsiellapneumoniae ST152 Variants with Hypermucoviscous Phenotype Isolated from Renal Transplant Recipients with Asymptomatic Bacteriuria-Genetic Characteristics by WGS.

Klebsiellapneumoniae (Kp) is one of the most important etiological factors of urinary tract infections in renal transplant (RTx) recipients. We described the antimicrobial susceptibility phenotypes and genomic features of two hypermucoviscous (HM) Kp isolates recovered from RTx recipients with asymptomatic bacteriuria (ABU). Using whole genome sequencing (WGS) data, we showed that the strains belong to the ST152 lineage with the KL149 capsular serotype, but without rmpA/magA genes, which is typical for HM+ hypervirulent Kp. These new strains carried virulence-associated genes that predispose for urinary tract infections (UTIs). Likewise, both strains carried the ecp gene encoding pilus common for extended-spectrum ß-lactamase (ESBL) Escherichiacoli. Although the two ST152 isolates were closely related and differed by only nine single nucleotide polymorphisms (SNPs) in their chromosomes, they had different plasmid compositions and chromosomal elements, with isolate KP28872 carrying an ESBL plasmid and an integrative conjugative element. These two isolates are an example of the high plasticity of the K. pneumoniae accessory genome. The identification of patients with ABU matched with the correct epidemiological profiling of isolates could facilitate interventions to prevent or rapidly treat K. pneumoniae infections.

Prevalence of phase variable epigenetic invertons among host-associated bacteria.

Type I restriction-modification (R-M) systems consist of a DNA endonuclease (HsdR, HsdM and HsdS subunits) and methyltransferase (HsdM and HsdS subunits). The hsdS sequences flanked by inverted repeats (referred to as epigenetic invertons) in certain Type I R-M systems undergo invertase-catalyzed inversions. Previous studies in Streptococcus pneumoniae have shown that hsdS inversions within clonal populations produce subpopulations with profound differences in the methylome, cellular physiology and virulence. In this study, we bioinformatically identified six major clades of the tyrosine and serine family invertases homologs from 16 bacterial phyla, which potentially catalyze hsdS inversions in the epigenetic invertons. In particular, the epigenetic invertons are highly enriched in host-associated bacteria. We further verified hsdS inversions in the Type I R-M systems of four representative host-associated bacteria and found that each of the resultant hsdS allelic variants specifies methylation of a unique DNA sequence. In addition, transcriptome analysis revealed that hsdS allelic variations in Enterococcus faecalis exert significant impact on gene expression. These findings indicate that epigenetic switches driven by invertases in the epigenetic invertons broadly operate in the host-associated bacteria, which may broadly contribute to bacterial host adaptation and virulence beyond the role of the Type I R-M systems against phage infection.

Combined therapy with ceftriaxone and doxycycline does not improve the outcome of meningococcal meningitis in mice compared to ceftriaxone monotherapy.

BACKGROUND: Meningococcal meningitis (MM) is a life-threatening disease associated with approximately 10% case fatality rates and neurological sequelae in 10-20% of the cases. Recently, we have shown that the matrix metalloproteinase (MMP) inhibitor BB-94 reduced brain injury in a mouse model of MM. The present study aimed to assess whether doxycycline (DOX), a tetracycline that showed a neuroprotective effect as adjuvant therapy in experimental pneumococcal meningitis (PM), would also exert a beneficial effect when given as adjunctive therapy to ceftriaxone (CRO) in experimental MM. METHODS: BALB/c mice were infected by the intracisternal route with a group C Neisseria meningitidis strain. Eighteen h post infection (hpi), animals were randomised for treatment with CRO [100 mg/kg subcutaneously (s.c.)], CRO plus DOX (30 mg/kg s.c.) or saline (control s.c.). Antibiotic treatment was repeated 24 and 40 hpi. Mouse survival and clinical signs, bacterial counts in cerebella, brain damage, MMP-9 and cyto/chemokine levels were assessed 48 hpi. RESULTS: Analysis of bacterial load in cerebella indicated that CRO and CRO + DOX were equally effective at controlling meningococcal replication. No differences in survival were observed between mice treated with CRO (94.4%) or CRO + DOX (95.5%), (p > 0.05). Treatment with CRO + DOX significantly diminished both the number of cerebral hemorrhages (p = 0.029) and the amount of MMP-9 in the brain (p = 0.046) compared to untreated controls, but not to CRO-treated animals (p > 0.05). Levels of inflammatory markers in the brain of mice that received CRO or CRO + DOX were not significantly different (p > 0.05). Overall, there were no significant differences in the parameters assessed between the groups treated with CRO alone or CRO + DOX. CONCLUSIONS: Treatment with CRO + DOX showed similar bactericidal activity to CRO in vivo, suggesting no antagonist effect of DOX on CRO. Combined therapy significantly improved mouse survival and disease severity compared to untreated animals, but addition of DOX to CRO did not offer significant benefits over CRO monotherapy. In contrast to experimental PM, DOX has no adjunctive activity in experimental MM.