Whole-body imaging of bacterial infection and antibiotic response

We describe imaging of green fluorescent protein (GFP)-expressing bacteria from outside intact infected animals. This simple, non-intrusive technique can show in great detail the spatial-temporal behavior of the infectious process. The bacteria, expressing the GFP, are sufficiently bright as to be clearly visible from outside the infected animal and recorded with simple equipment. Introduced bacteria can be whole-body imaged in most mouse organs, including the peritoneal cavity, stomach, small intestine, and colon. This imaging technology affords a powerful approach to visualizing the infection process, determining the tissue specificity of infection, the spatial migration of the infectious agents and the response to antimicrobial agents.     

Bacteriophage proteins associated with the bacterial membrane after bacteriophage T7 infection.

The majority of the phage-induced proteins made after T7 infection of Escherichia coli are tightly associated with the bacterial membrane. Many of these have been identified. Selective extraction of proteins from these membranes by the detergent Sarkosyl or the chaotropic agent guanidine-hydrochloride indicated that most of these proteins are an integral part of the cytoplasmic membrane and the cell wall. No major changes in the distribution of bacterial proteins in the membrane were observed as a consequence of phage T7 infection.     

Increased polyclonal immunoglobulin reactivity toward human and bacterial proteins is associated with clinical protection in human Plasmodium infection

Introduction

By 2030, more than 50% of the African population will live in urban areas. Controlling malaria reduces the disease burden and further improves economic development. As a complement to treated nets and prompt access to treatment, measures targeted against the larval stage of Anopheles sp. mosquitoes are a promising strategy for urban areas. However, a precise knowledge of the geographic location and potentially of ecological characteristics of breeding sites is of major importance for such interventions.

Methods

In total 151 km² of central Dar es Salaam, the biggest city of Tanzania, were systematically searched for open mosquito breeding sites. Ecologic parameters, mosquito larvae density and geographic location were recorded for each site. Logistic regression analysis was used to determine the key ecological factors explaining the different densities of mosquito larvae.

Results

A total of 405 potential open breeding sites were examined. Large drains, swamps and puddles were associated with no or low Anopheles sp. larvae density. The probability of Anopheles sp. larvae to be present was reduced when water was identified as "turbid". Small breeding sites were more commonly colonized by Anopheles sp. larvae. Further, Anopheles gambiae s.l. larvae were found in highly organically polluted habitats.

Conclusions

Clear ecological characteristics of the breeding requirements of Anopheles sp. larvae could not be identified in this setting. Hence, every stagnant open water body, including very polluted ones, have to be considered as potential malaria vector breeding sites.     

Prolonged antibiotic use induces intestinal injury in mice that is repaired after removing antibiotic pressure: implications for empiric antibiotic therapy

Metabolic profiling of urine and fecal extracts, histological investigation of intestinal ilea, and fecal metagenomics analyses were used to investigate effects of prolonged antibiotic use in mice. The study provides insight into the effects of extended empiric antibiotic therapy in humans. Mice were administered a broad-spectrum antibiotic for four consecutive days followed by oral gavage with Clostridium butyricum, an opportunistic gram-positive pathogenic bacteria commonly isolated in fecal and blood cultures of necrotizing enterocolitis patients. Metagenomics data indicated loss of bacterial diversity after 4 days on antibiotics that was restored after removing antibiotic pressure. Histological analyses indicated damage to ileal villi after antibiotic treatment that underwent repair after lifting antibiotic pressure. Metabolic profiling confirmed intestinal injury in antibiotic-treated mice indicated by increased urinary trans-4-hydroxy-l-proline, a breakdown product of collagen present in connective tissue of ileal villi that may serve as a biomarker for antibiotic-induced injury in at risk populations.     

Chromosomal loci associated with antibiotic hypersensitivity in pulmonary isolates of Pseudomonas aeruginosa

492a and 492c were two strains of Pseudomonas aeruginosa isolated from the sputum of a patient with cystic fibrosis. The strains were closely related but expressed different antibiograms. 492c was hypersensitive (10-100 times more sensitive than 492a) to the beta-lactam antibiotics carbenicillin, methicillin, flucloxacillin, mecillinam and cefuroxime and the non-beta-lactam, nalidixic acid. 492c also showed enhanced sensitivity (4-8 times more sensitive than 492a) to chloramphenicol, trimethoprim and novobiocin. 492a and PAO8 expressed similar levels of antibiotic resistance, except for trimethoprim, to which 492a was five times more sensitive than PAO8. Two genes associated with antibiotic hypersensitivity were mapped in the 30 min region of the chromosome, by means of R68.45-mediated plate matings between a Leu - mutant of 492c and PAO8, followed by transductional analysis using phage F116L. The first of these genes, blsA1, was closely linked to nalB, and in a PAO background, was associated with hypersensitivity to the beta-lactams and a moderate increase in sensitivity to chloramphenicol, trimethoprim, nalidixic acid and novobiocin. A further increase in sensitivity to the latter three antibiotics was associated with the second gene, tpsA1, which mapped between ser-3 and hisV. This gene could also be transferred to PAO from 492a, thus 492c could have arisen from 492a in vivo following a single chromosomal mutation at the blsA locus. Isolation of a blsA mutant of PAO969 provided further evidence for this theory.     

The interleukin-6 -174 promoter polymorphism is associated with extrapulmonary bacterial dissemination in Streptococcus pneumoniae infection

Interleukin-6 (IL-6) is required for the clearance of bacteria in pneumococcal pneumonia. The abundance of endogenous IL-6 production on infectious stimuli is associated with genotypic differences in the -174 promoter region of IL-6 (-174 G-->C), showing increased IL-6 levels in patients carrying the GG genotype. One hundred patients with culturally proven pneumococcal disease were analyzed for distribution of the G-/C-alleles in the IL-6 -174 promoter region in comparison to 50 age-matched controls. Extrapulmonary pneumococcal dissemination, including septic metastasis, endocardial and meningeal infection, was used as parameter for impaired clearance of the bacteria. No significant differences in the allele distribution were observed between patients and controls. Within the patient group, the interleukin-6 GG homozygous carriers were less likely to develop extrapulmonary pneumococcal infection (10.3% versus 30.9%; OR 0.26, 95% CI 0.07-0.94, p=0.04). The IL-6 GG genotype, encoding for enhanced IL-6 secretion on bacterial stimuli, reduces the risk of bacterial spread to extrapulmonary sites in pneumococcal infection, possibly due to a more effective clearance of the pathogen from the blood and the respiratory tract.     

Epidemic pulmonary infection associated with Mycobacterium xenopi indigenous in sewage-sludge

Mycobacterium xenopi was isolated from the sputum of 21 patients with clinical signs of pulmonary disease and of 52 asymptomatic subjects living in the environment of a sludge pool. M. xenopi was cultured in high numbers from sludge samples. The infections were assumed to occur partly via dry sludge particles scattered by the wind in summer, and partly by sludge used as fertilizer.     

Advanced paternal age is associated with lower facial attractiveness

In view of disease risk, Kong et al. (2012) demonstrated that most of the new mutations are explained by the age of the father at conception. Accordingly, paternal age effects have been found for a variety of offspring traits, from physical and mental health to intelligence. Here, we investigated whether facial attractiveness is significantly associated with paternal age. We used the Wisconsin Longitudinal Study (n = 4018 male and 4416 female high school graduates) to analyze the association between an individual's father's age at birth and that individual's facial attractiveness (estimated by rating the high school yearbook photographs from 1957), controlling for sex, age as well as mother's age. We find that subject's facial attractiveness decreased with advancing paternal but not maternal age, suggesting that facial attractiveness might be a cue of an individual's new mutation load.     

Dectin-1 and DC-SIGN polymorphisms associated with invasive pulmonary Aspergillosis infection

The recognition of pathogen-derived structures by C-type lectins and the chemotactic activity mediated by the CCL2/CCR2 axis are critical steps in determining the host immune response to fungi. The present study was designed to investigate whether the presence of single nucleotide polymorphisms (SNPs) within DC-SIGN, Dectin-1, Dectin-2, CCL2 and CCR2 genes influence the risk of developing Invasive Pulmonary Aspergillosis (IPA). Twenty-seven SNPs were selected using a hybrid functional/tagging approach and genotyped in 182 haematological patients, fifty-seven of them diagnosed with proven or probable IPA according to the 2008 EORTC/MSG criteria. Association analysis revealed that carriers of the Dectin-1(rs3901533 T/T) and Dectin-1(rs7309123 G/G) genotypes and DC-SIGN(rs4804800 G), DC-SIGN(rs11465384 T), DC-SIGN(7248637 A) and DC-SIGN(7252229 C) alleles had a significantly increased risk of IPA infection (OR = 5.59 95%CI 1.37-22.77; OR = 4.91 95%CI 1.52-15.89; OR = 2.75 95%CI 1.27-5.95; OR = 2.70 95%CI 1.24-5.90; OR = 2.39 95%CI 1.09-5.22 and OR = 2.05 95%CI 1.00-4.22, respectively). There was also a significantly increased frequency of galactomannan positivity among patients carrying the Dectin-1(rs3901533_T) allele and Dectin-1(rs7309123_G/G) genotype. In addition, healthy individuals with this latter genotype showed a significantly decreased level of Dectin-1 mRNA expression compared to C-allele carriers, suggesting a role of the Dectin-1(rs7309123) polymorphism in determining the levels of Dectin-1 and, consequently, the level of susceptibility to IPA infection. SNP-SNP interaction (epistasis) analysis revealed significant interactions models including SNPs in Dectin-1, Dectin-2, CCL2 and CCR2 genes, with synergistic genetic effects. Although these results need to be further validated in larger cohorts, they suggest that Dectin-1, DC-SIGN, Dectin-2, CCL2 and CCR2 genetic variants influence the risk of IPA infection and might be useful in developing a risk-adapted prophylaxis.     

Immune unresponsiveness to secondary heterologous bacterial infection after sepsis induction is TRAIL dependent

Sepsis is the leading cause of death in most intensive care units, and patients who survive the hyperinflammation that develops early during sepsis later display severely compromised immunity. Not only is there apoptosis of lymphoid and myeloid cells during sepsis that depletes these critical cellular components of the immune system, but also the remaining immune cells show decreased function. Using a cecal-ligation and puncture (CLP) model to induce intra-abdominal polymicrobial peritonitis, we recently established a link between the apoptotic cells generated during sepsis and induction of sepsis-induced suppression of delayed-type hypersensitivity. The present study extends this earlier work to include a secondary heterologous bacterial infection (OVA(257)-expressing Listeria monocytogenes [LM-OVA]) subsequent to sepsis initiation to investigate sepsis-induced alterations in the control of this secondary infection and the associated naive Ag-specific CD8 T cell response. We found that CLP-treated wild-type (WT) mice had a reduced ability to control the LM-OVA infection, which was paralleled by suppressed T cell responses, versus sham-treated WT mice. In contrast, CLP-treated Trail(-/-) and Dr5(-/-) mice were better able to control the secondary bacterial infection, and the Ag-specific CD8 T cell response was similar to that seen in sham-treated mice. Importantly, administration of a blocking anti-TRAIL mAb to CLP-treated WT mice was able to restore the ability to control the LM-OVA infection and generate Ag-specific CD8 T cell responses like those seen in sham-treated mice. These data further implicate TRAIL-dependent immune suppression during sepsis and suggest TRAIL neutralization may be a potential therapeutic target to restore cellular immunity in septic patients.     

Genetic variation in inflammasome genes is associated with outcome in bacterial meningitis

Bacterial meningitis is a severe and deadly disease, most commonly caused by Streptococcus pneumoniae. Disease outcome has been related to severity of the inflammatory response in the subarachnoid space. Inflammasomes are intracellular signaling complexes contributing to this inflammatory response. The role of genetic variation in inflammasome genes in bacterial meningitis is largely unknown. In a prospective nationwide cohort of patients with pneumococcal meningitis, we performed a genetic association study and found that single-nucleotide polymorphisms in the inflammasome genes CARD8 (rs2043211) and NLRP1 (rs11621270) are associated with poor disease outcome. Levels of the inflammasome associated cytokines interleukin (IL)-1β and IL-18 in cerebrospinal fluid also correlated with clinical outcome, but were not associated with the CARD8 and NLRP1 polymorphisms. Our results implicate an important role of genetic variation in inflammasome genes in the regulation of inflammatory response and clinical outcome in patients with bacterial meningitis.     

Treating cancer with infection: a review on bacterial cancer therapy

There is an increasing need for new cancer therapies. The antitumour effect of bacterial infection has been well observed and practiced throughout history. Bacteria are well‐suited to serve as anticancer agents due to their intrinsic mobility, cell toxicity, immunogenicity, and preferential accumulation within the anoxic tumour environment. Furthermore, advances in biotechnology and molecular techniques have made it easier than ever to engineer bacteria as both therapeutic agents themselves and as therapeutic vectors. Here, we review bacteriolytic therapy and immunotherapy strategies, and examine the development of bacteria as vehicles for cell‐ and tissue‐targeted delivery of genetic cancer therapeutics.     

Ecosystem productivity is associated with bacterial phylogenetic distance in surface marine waters

Understanding the link between community diversity and ecosystem function is a fundamental aspect of ecology. Systematic losses in biodiversity are widely acknowledged but the impact this may exert on ecosystem functioning remains ambiguous. There is growing evidence of a positive relationship between species richness and ecosystem productivity for terrestrial macro‐organisms, but similar links for marine micro‐organisms, which help drive global climate, are unclear. Community manipulation experiments show both positive and negative relationships for microbes. These previous studies rely, however, on artificial communities and any links between the full diversity of active bacterial communities in the environment, their phylogenetic relatedness and ecosystem function remain hitherto unexplored. Here, we test the hypothesis that productivity is associated with diversity in the metabolically active fraction of microbial communities. We show in natural assemblages of active bacteria that communities containing more distantly related members were associated with higher bacterial production. The positive phylogenetic diversity–productivity relationship was independent of community diversity calculated as the Shannon index. From our long‐term (7‐year) survey of surface marine bacterial communities, we also found that similarly, productive communities had greater phylogenetic similarity to each other, further suggesting that the traits of active bacteria are an important predictor of ecosystem productivity. Our findings demonstrate that the evolutionary history of the active fraction of a microbial community is critical for understanding their role in ecosystem functioning.     

Anti-inflammatory response is associated with mortality and severity of infection in sepsis

Using a murine model of sepsis, we found that the balance of tissue pro- to anti-inflammatory cytokines directly correlated with severity of infection and mortality. Sepsis was induced in C57BL/6 mice by cecal ligation and puncture (CLP). Liver tissue was analyzed for levels of IL-1beta, IL-1 receptor antagonist (IL-1ra), tumor necrosis factor (TNF)-alpha, and soluble TNF receptor 1 by ELISA. Bacterial DNA was measured using quantitative real-time PCR. After CLP, early predominance of proinflammatory cytokines (6 h) transitioned to anti-inflammatory predominance at 24 h. The elevated anti-inflammatory cytokines were mirrored by increased tissue bacterial levels. The degree of anti-inflammatory response compared with proinflammatory response correlated with the bacterial concentration. To modulate the timing of the anti-inflammatory response, mice were treated with IL-1ra before CLP. This resulted in decreased proinflammatory cytokines, earlier bacterial load, and increased mortality. These studies show that the initial tissue proinflammatory response to sepsis is followed by an anti-inflammatory response. The anti-inflammatory phase is associated with increased bacterial load and mortality. These data suggest that it is the timing and magnitude of the anti-inflammatory response that predicts severity of infection in a murine model of sepsis.     

Methicillin-resistant Staphylococcus aureus infection is associated with increased mortality

Methicillin resistance is a widespread and major source of treatment complication in Staphylococcus aureus infections. Whether infections with methicillin-resistant S. aureus are associated with a worse clinical outcome, such as higher mortality, has remained controversial. Analyzing data from a large, global multicenter study, Hanberger et al. demonstrate that methicillin-resistant S. aureus infections are associated with approximately 50% higher mortality in the intensive care unit and significantly more frequent among critically ill patients than infections with methicillin-susceptible S. aureus. These findings call for the implementation or continuation of active methicillin-resistant S. aureus surveillance measures.     

Complex and unexpected outcomes of antibiotic therapy against a polymicrobial infection

Antibiotics are our primary approach to treating complex infections, yet we have a poor understanding of how these drugs affect microbial communities. To better understand antimicrobial effects on host-associated microbial communities we treated cultured sputum microbiomes from people with cystic fibrosis (pwCF, n = 24) with 11 different antibiotics, supported by theoretical and mathematical modeling-based predictions in a mucus-plugged bronchiole microcosm. Treatment outcomes we identified in vitro that were predicted in silico were: 1) community death, 2) community resistance, 3) pathogen killing, and 4) fermenter killing. However, two outcomes that were not predicted when antibiotics were applied were 5) community profile shifts with little change in total bacterial load (TBL), and 6) increases in TBL. The latter outcome was observed in 17.8% of samples with a TBL increase of greater than 20% and 6.8% of samples with an increase greater than 40%, demonstrating significant increases in community carrying capacity in the presence of an antibiotic. An iteration of the mathematical model showed that TBL increase was due to antibiotic-mediated release of pH-dependent inhibition of pathogens by anaerobe fermentation. These dynamics were verified in vitro when killing of fermenters resulted in a higher community carrying capacity compared to a no antibiotic control. Metagenomic sequencing of sputum samples during antibiotic therapy revealed similar dynamics in clinical samples. This study shows that the complex microbial ecology dictates the outcomes of antibiotic therapy against a polymicrobial infection.Subject terms: Microbiome, Microbial ecology