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
of primary human AM obtained from PLHIV and HIV-uninfected adults. We found that pneumococcus survived intracellularly in AMs at least 24 hours post
infection, and this was more frequent in PLHIV than HIV-uninfected adults. Corroborating these findings,
evidence showed that PLHIV had a higher propensity for harboring
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
intracellularly could contribute to the increased risk of pneumococcal pneumonia in PLHIV.
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.
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.
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.
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.
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.
Oxford University, Wolfson Foundation, MRC, NIH, NIHR, and MRC and BBSRC studentships supported the work.
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.
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.
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.
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.
University of Oxford and a Royal Society Wolfson grant funded biosafety facility.
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.
K1 capsule type Escherichia coli is the predominant cause of neonatal meningitis. In an intravenous (IV) infection model with a clinical K1 isolate IHE 3034 adult mice die of sepsis within 24 hours. ...However, by utilising cefazolin, a first-generation cephalosporin known for its inability to pass through the blood brain barrier, it is possible to prevent bacteraemia and study the early phases of E. coli meningitis in the brain. Mice were infected IV with E. coli IHE 3034 and treated with cefazolin at 12 and 24h intraperitoneal to ensure the survival of animals to the determined end points. The results of the experiment show IHE 3034 is present in the brain as early as 12h and it remains in the tissue 72h post infection even if blood is clear. Despite this, at the cellular level, clear foci formation can be observed in the meninges and the choroid plexus around the vascular endothelial cells at 48h, showing IHE 3034’s ability for intracellular replication inside the meningeal region The foci do not appear to be present at 24 or 72h, indicating for a secondary replication site around the major vascular endothelia sites of the brain. This further promotes the bacterial load increase through cell bursting in the tissue with or without a sustained bacterial influx from the blood. These findings show that K1 E. coli can form foci in the brain which may lead to escalated disease progression and severe morbidity following an initially successful clearance of bloodborne bacteria during sepsis.
During bacteremic pneumonia, the prevailing dogma is that bacteria seed from the lungs into the blood. Recently, we have shown that experimental murine sepsis is preceded by intracellular replication ...within splenic macrophages (Ercoli
Nat Microbiol
2018), which shed into the bloodstream initiating invasive disease. Here we aimed to investigate a role for the spleen in the pathogenesis of bacteraemia following pneumonia. We analysed by confocal microscopy the fate of pneumococci during
ex vivo
human spleen perfusions (REC reference: 18/EM/0057), in spleens during pneumonia in non-human primates (Reyes
PLOS one
2016) and mice. During ex vivohumanspleenperfusion, clusters of pneumococci were observed within macrophages and the size of bacterial clusters increased over time. To associate these infectious foci to invasive pneumococcal disease during pneumonia, we analysed spleens in a baboon pneumonia model, and detected pneumococcal clusters in splenic macrophages. To test the functional relevance of these data, we treated intranasally-challenged mice with a single, non-therapeutic sub-MIC dose of azithromycin, known to concentrate inside macrophages. Data showed that bacterial lung-counts were identical in treated and untreated mice. Untreated mice showed signs of disease, had high blood and spleen-counts, whereas mice treated with the non-therapeutic dose showed no signs of disease, had low spleen-counts and no bacteraemia. Thus, the number of pneumococci in the spleen, not the lung, correlates to blood-counts during bacterial pneumonia. We hypothesise that after initial control of invasive infection by the spleen, bacteraemia associated with pneumonia arises from a sub-set of splenic macrophages that are permissive for bacterial replication.
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