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Pharyngeal colonisation by the commensal bacterium Neisseria lactamica inhibits colonisation by Neisseria meningitidis and has an inverse epidemiological association with meningococcal disease. The mechanisms that underpin this relationship are unclear, but could involve the induction of cross-reactive immunity. In this study, we aimed to evaluate whether colonisation with N lactamica induces N lactamica-specific B-cell responses that are cross-reactive with N meningitidis.In this randomised, placebo-controlled, human infection trial at University Hospital Southampton Clinical Research Facility (Southampton, UK), healthy adults aged 18-45 years were randomly assigned (2:1) to receive intranasal inoculation with either 10Of 50 participants assessed for eligibility between Sept 5, 2018, and March 3, 2019, 31 were randomly assigned (n=20 N lactamica, n=11 PBS). Among the 17 participants who were colonised with N lactamica, the median baselines compared with peak post-colonisation N lactamica-specific plasma-cell frequencies (per 10Natural immunity to N meningitidis after colonisation with N lactamica might be due to cross-reactive adaptive responses. Exploitation of this microbial mechanism with a genetically modified live vector could protect against N meningitidis colonisation and disease.Wellcome Trust, Medical Research Council, and NIHR Southampton Biomedical Research Centre.

Background: Heterologous COVID vaccine priming schedules are immunogenic and effective. This report aims to understand the persistence of immune response to the viral vectored, mRNA and protein-based COVID-19 vaccine platforms used in homologous and heterologous priming combinations, which will inform the choice of vaccine platform in future vaccine development. Methods: Com-COV2 was a single-blinded trial in which adults ≥ 50 years, previously immunised with single dose ‘ChAd’ (ChAdOx1 nCoV-19, AZD1222, Vaxzevria, Astrazeneca) or ‘BNT’ (BNT162b2, tozinameran, Comirnaty, Pfizer/BioNTech), were randomised 1:1:1 to receive a second dose 8–12 weeks later with either the homologous vaccine, or ‘Mod’ (mRNA-1273, Spikevax, Moderna) or ‘NVX’ (NVX-CoV2373, Nuvaxovid, Novavax). Immunological follow-up and the secondary objective of safety monitoring were performed over nine months. Analyses of antibody and cellular assays were performed on an intention-to-treat population without evidence of COVID-19 infection at baseline or for the trial duration. Findings: In April/May 2021, 1072 participants were enrolled at a median of 9.4 weeks after receipt of a single dose of ChAd (N = 540, 45% female) or BNT (N = 532, 39% female) as part of the national vaccination programme. In ChAd-primed participants, ChAd/Mod had the highest anti-spike IgG from day 28 through to 6 months, although the heterologous vs homologous geometric mean ratio (GMR) dropped from 9.7 (95% CI (confidence interval): 8.2, 11.5) at D28 to 6.2 (95% CI: 5.0, 7.7) at D196. The heterologous/homologous GMR for ChAd/NVX similarly dropped from 3.0 (95% CI:2.5,3.5) to 2.4 (95% CI:1.9, 3.0). In BNT-primed participants, decay was similar between heterologous and homologous schedules with BNT/Mod inducing the highest anti-spike IgG for the duration of follow-up. The adjusted GMR (aGMR) for BNT/Mod compared with BNT/BNT increased from 1.36 (95% CI: 1.17, 1.58) at D28 to 1.52 (95% CI: 1.21, 1.90) at D196, whilst for BNT/NVX this aGMR was 0.55 (95% CI: 0.47, 0.64) at day 28 and 0.62 (95% CI: 0.49, 0.78) at day 196. Heterologous ChAd-primed schedules produced and maintained the largest T-cell responses until D196. Immunisation with BNT/NVX generated a qualitatively different antibody response to BNT/BNT, with the total IgG significantly lower than BNT/BNT during all follow-up time points, but similar levels of neutralising antibodies. Interpretation: Heterologous ChAd-primed schedules remain more immunogenic over time in comparison to ChAd/ChAd. BNT-primed schedules with a second dose of either mRNA vaccine also remain more immunogenic over time in comparison to BNT/NVX. The emerging data on mixed schedules using the novel vaccine platforms deployed in the COVID-19 pandemic, suggest that heterologous priming schedules might be considered as a viable option sooner in future pandemics. ISRCTN:27841311 EudraCT:2021-001275-16.

High quality health care research must involve patients and the public. This ensures research is important, relevant and acceptable to those it is designed to benefit. The world's first human challenge study with SARS-CoV-2 undertook detailed public involvement to inform study design despite the urgency to review and establish the study. The work was integral to the UK Research Ethics Committee review and approval of the study. Discussion with individuals from ethnic minorities within the UK population supported decision-making around the study exclusion criteria. Public review of study materials for consent processes led to the addition of new information, comparisons and visual aids to help volunteers consider the practicalities and risks involved in participating. A discussion exploring the acceptability of a human challenge study with SARS-CoV-2 taking place in the UK, given the current context of the pandemic, identified overall support for the study. Public concern for the wellbeing of trial participants, as a consequence of isolation, was identified. We outline our approach to public involvement and its impact on study design.

Objectives\udThis study aimed to assess whether the presence of bacteria or viruses in the upper airway of children presenting with uncomplicated lower respiratory tract infection (LRTI) predicts the benefit of antibiotics.\udMethods\udChildren between 6 months and 12 years presenting to UK general practices with an acute LRTI were randomized to receive amoxicillin 50 mg/kg/d for 7 days or placebo. Children not randomized (ineligible or clinician/parental choice) could participate in a parallel observational study. The primary outcome was the duration of symptoms rated moderately bad or worse. Throat swabs were taken and analyzed for the presence of bacteria and viruses by multiplex PCR.\udResults\udSwab results were available for most participants in the trial (306 of 432; 71%) and in the observational (182 of 326; 59%) studies. Bacterial pathogens potentially sensitive to amoxicillin (Haemophilus influenzae, Moraxella catarrhalis, Streptococcus pneumoniae) were detected among 51% of the trial placebo group and 49% of the trial antibiotic group. The median difference in the duration of symptoms rated moderately bad or worse between antibiotic and placebo was similar when potentially antibiotic-susceptible bacteria were present (median: –1 day; 99% CI, –12.3 to 10.3) or not present (median: –1 day; 99% CI, –4.5 to 2.5). Furthermore, bacterial genome copy number did not predict benefit. There were similar findings for all secondary outcomes and when including the data from the observational study.\udDiscussion\udThere was no clear evidence that antibiotics improved clinical outcomes conditional on the presence or concentration of bacteria or viruses in the upper airway. Before deploying microbiologic point-of-care tests for children with uncomplicated LRTI in primary care, rigorous validating trials are needed.

Purpose of review: there has been an exponential increase in research intoinfant microbiome evolution, and it appears that pharyngeal microbiota are associated with clinical phenotypes(e.g. infection and asthma). While broad consensus views are emerging, significant challenges and uncertainties remain.Recent findings: infant pharyngeal microbiome research is limited by low biomass, high temporal diversity, and lack of agreed standards for sampling, DNA sequencing, and taxonomic reporting. Analysis of amplicon sequence variants and improvedcost and availability of whole genome sequencing are promising options for improving taxonomic resolution of such studies. Infant respiratory microbiomes arise, at least in part, from maternal flora (e.g. the respiratory tract and breastmilk), and are associated with environmental and clinical factors (e.g.mode of feeding and delivery, siblings, daycare attendance, birth season, and antibiotic usage). Interventional research to modify the infant pharyngeal microbiota hasrecentlybeen reported, using dietary supplements. Summary: further work is needed to improve characterisation ofthe infant pharyngeal microbiomes, including routes of bacterial acquisition, role of environmental factors, and associations with disease phenotypes. Methodological standards are desirable to facilitate more reproducible, comparable research. Improved understanding may enable manipulation of infant pharyngeal microbiota to improve clinical outcomes.

Background Bordetella pertussis is among the leading causes of vaccine-preventable deaths and morbidity globally. Human asymptomatic carriage as a reservoir for community transmission of infections might be a target of future vaccine strategies, but has not been demonstrated. Our objective was to demonstrate that asymptomatic nasopharyngeal carriage of Bordetella pertussis is inducible in humans and to define the microbiological and immunological features of presymptomatic infection. Methods Healthy subjects aged 18–45 years with an antipertussis toxin immunoglobin G (IgG) concentration of Results There were 34 participants challenged, in groups of 4 or 5. The dose was gradually escalated from 103 colony-forming units (0% colonized) to 105 colony-forming units (80% colonized). Minor symptoms were reported in a minority of participants. Azithromycin eradicated colonization in 48 hours in 88% of colonized individuals. Antipertussis toxin IgG seroconversion occurred in 9 out of 19 colonized participants and in none of the participants who were not colonized. Nasal wash was a more sensitive method to detect colonization than pernasal swabs. No shedding of Bordetella pertussis was detected in systematically collected environmental samples. Conclusions Bordetella pertussis colonization can be deliberately induced and leads to a systemic immune response without causing pertussis symptoms. Clinical Trials Registration NCT03751514.

OBJECTIVES: Escherichia coli is the leading cause of bacteraemia. In an era of emerging multi-drug-resistant strains, development of effective preventative strategies will be informed by knowledge of strain diversity associated with specific infective syndromes/patient groups. We hypothesised that the number of virulence factor (VF) genes amongst bacteraemia isolates from neutropaenic patients would be lower than isolates from immunocompetent patients.METHODS: Immunocompetent and neutropaenic adults with E. coli bacteraemia were recruited prospectively and the source of bacteraemia determined. VF gene profiles were established in silico following whole genome sequencing.RESULTS: Isolates from individual patients were monoclonal. Strains from immunocompetent patients with urinary tract infective foci (UTIF) harboured more VF genes (median number of VF genes 16, range 8-24) than isolates from both immunocompetent patients with non-UTIF (10, 2-22, p = 0.0058) and neutropaenic patients with unknown focus of infection (NPUFI) (8, 3-13, p < 0.0001). Number of VF genes (OR 1.21, 95% CIs 1.01-1.46, p = 0.039) and urinary catheter/recurrent urinary tract infection (OR 12.82, 95% CIs 1.24-132.65, p = 0.032) were independent predictors of bacteraemia secondary to UTIF vs. non-UTIF in immunocompetent patients. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst UTIF- vs non-UTIF-originating isolates amongst immunocompetent patients, while papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTIF- vs NPUFI-associated isolates.CONCLUSIONS: Bacteraemia-associated E. coli strains originating from UTIF have distinct VF gene profiles from strains associated with non-UTIF- and NPUFI. This diversity must be addressed in the design of future vaccines to ensure adequate coverage of strains responsible for site-specific disease.

Meningococcal disease is a severe consequence of infection with Neisseria meningitidis, a pathobiont of the pharynx. This organism is panmitic so virulent clones transformed with new genetic material can emerge and cause severe outbreaks. The key to sustainable prevention is to restrict carriage of disease-causing strains and thus reduce the chances of transmission between human hosts.Meningococcal population biology has changed recently with emergence of virulent strains linked to a number of sublineages of clonal complex 11. These strains have variously expressed the capsular material of serogroups C and W and caused severe disease in various countries. Glycoconjugate vaccines including quadrivalent (ACWY) and now pentavalent (ACWYX) vaccines are highly immunogenic and prevent disease and carriage due to their respective serogroups. For NmB, new vaccines (4CMenB and MenB-FHbp) containing conserved outer membranes proteins have been deployed and are immunogenic and protective at population level, but clones exist which do not express cognate antigens. In contrast to glycoconjugate vaccines they may not have potent carriage-reducing activity. Mass chemoprophylaxis is gaining credence as an alternative strategy is effective, but has significant shortcomings in sustainability.Meningococcal disease is well defined genomically for epidemiological purposes. There is potential for unpredictable emergence of clones that may have reduced susceptibility even to modern vaccines, and continued surveillance and vigilance is necessary. However, tremendous strides have been made in recent years.

Purpose of review Nonpathogenic commensal Neisseria are rarely considered in the clinical setting despite evidence that they can cause invasive opportunistic infections. In contrast, they may offer protection against pathogenic Neisseria, and such relationships are being actively explored in experimental studies. Recent findings Recent case reports are presented of invasive infection caused by nonpathogenic Neisseria in patients on novel biologic therapies. On the other hand, Neisseria lactamica, a nonpathogenic commensal, has been shown in human challenge studies to inhibit colonization by Neisseria meningitidis. Experimental mouse models have also explored the inhibitory effects of nonpathogenic Neisseria on Neisseria gonnhoreae infection. Cutting-edge advances in metagenomics and microbiomics are being used to understand the mechanisms underpinning these effects. Summary Clinicians should have increased awareness of nonpathogenic Neisseria. First, as new immunomodulating therapies become licenced, the interactions that maintain balance between commensals and their human hosts may be altered. Second, these bacteria are showing promise in their capacity to exclude pathogenic Neisseria species from their anatomical niches.

IntroductionNeisseria lactamicais a commensal organism found in the human nasopharynx and is closely related to the pathogenN. meningitidis(meningococcus). Carriage ofN. lactamicais associated with reduced meningococcal carriage and disease. We summarise an ethically approved protocol for an experimental human challenge study using a genetically modified strain ofN. lactamicathat expresses the meningococcal antigen NadA. We aim to develop a model to study the role of specific bacterial antigens in nasopharyngeal carriage and immunity, to evaluate vaccines for their efficacy in preventing colonisation and to provide a proof of principle for the development of bacterial medicines.Methods and analysisHealthy adult volunteers aged 18–45 years will receive an intranasal inoculation of either the NadA containing strain ofN. lactamicaor a genetically modified, but wild-type equivalent control strain. These challenge volunteers will be admitted for 4.5 days observation following inoculation and will then be discharged with strict infection control rules. Bedroom contacts of the challenge volunteers will also be enrolled as contact volunteers. Safety, colonisation, shedding, transmission and immunogenicity will be assessed over 90 days after which carriage will be terminated with antibiotic eradication therapy.Ethics and disseminationThis study has been approved by the Department for Environment, Food and Rural Affairs and South Central Oxford A Research Ethics Committee (reference: 18/SC/0133). Findings will be published in peer-reviewed open-access journals as soon as possible.Trial registration numberNCT03630250; Pre-results.

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Bordetella pertussis is the causative agent of whooping cough, commonly referred to as pertussis. Although the incidence of pertussis was reduced through vaccination, during the last thirty years it has returned to high levels in a number of countries. This resurgence has been linked to the switch from the use of whole-cell to acellular vaccines. Protection afforded by acellular vaccines appears to be short-lived compared to that afforded by whole cell vaccines. In order to inform future vaccine improvement by identifying immune correlates of protection, a human challenge model of B. pertussis colonisation has been developed. Accurate measurement of colonisation status in this model has required development of a qPCR-based assay to enumerate B. pertussis in samples that distinguishes between viable and dead bacteria. Here we report the development of this assay and its performance in the quantification of B. pertussis from human challenge model samples. This assay has future utility in diagnostic labs and in research where a quantitative measure of both B. pertussis number and viability is required.

IntroductionWe summarise an ethically approved protocol for the development of an experimental human challenge colonisation model. GloballyBordetella pertussisis one of the leading causes of vaccine-preventable death. Many countries have replaced whole cell vaccines with acellular vaccines over the last 20 years during which pertussis appears to be resurgent in a number of countries in the developed world that boast high immunisation coverage. The acellular vaccine provides relatively short-lived immunity and, in contrast to whole cell vaccines, may be less effective against colonisation and subsequent transmission. To improve vaccine strategies, a greater understanding of humanB. pertussiscolonisation is required. This article summarises a protocol and does not contain any results.Methods and analysisA controlled human colonisation model will be developed over two phases. In phase A, a low dose of the inoculum will be given intranasally to healthy participants. This dose will be escalated or de-escalated until colonisation is achieved in approximately 70% (95% CI 47% to 93%) of the exposed volunteers without causing disease. The colonisation period, shedding and exploratory immunology will be assessed during a 17-day inpatient stay and follow-up over 1 year. The dose of inoculum that achieves 70% colonisation will then be confirmed in phase B, comparing healthy participants exposed toB. pertussiswith a control group receiving a sham inoculum.Ethics and disseminationThis study has been approved by the ethical committee reference: 17/SC/0006, 24 February 2017. Findings will be published in peer-reviewed open access journals as soon as possible.

Summary Objective Infected macrophages play a role in the dissemination of Salmonella and may serve as a reservoir of infection in asymptomatic carriers. However, relatively little is known about the early molecular interactions of the bacteria with these cells. We have recently shown that members of the tetraspanin family of membrane proteins are involved in the initial adhesion of a range of bacteria to host cells. This study investigated the role of tetraspanins in Salmonella enterica subsp. enterica serovar Typhimurium ( S. Typhimurium) infection of human monocyte-derived macrophages (MDM). Methods The role of tetraspanins was studied by the use of tetraspanins recombinant proteins as well as monoclonal antibodies targeted against different tetraspanins. Knockdown of the tetraspanin CD63 was carried out by siRNA to further study the role of CD63 in Salmonella uptake. Results Recombinant proteins representing the large extracellular domains of tetraspanins inhibited binding of S. Typhimurium to human MDM by ∼50%, whereas tetraspanin-specific antibodies showed varying effects, with some enhancing (anti-CD37) and some inhibiting (anti-CD81, anti-CD63) binding. Inhibition of the S. Typhimurium-MDM interaction by anti-CD63 mAb appeared to be mediated by antibody induced internalization, suggesting that surface expression of CD63 is required for S. Typhimurium binding. Knockdown of CD63 in human MDM using siRNA greatly reduced S. Typhimurium binding, confirming the importance of CD63. However, ectopic expression of CD63 in the non-phagocytic cell line HEK293 was insufficient to mediate bacterial binding. Conclusion Bacterial adhesion is the first step in infection by pathogens that invade and replicate within host cells. Taken together, the results here describe a role for tetraspanins in binding of S. Typhimurium to human macrophages and highlight the particular importance of CD63 in this process.

Mannose binding lectin (MBL2) is a soluble pattern recognition receptor that is key to generating innate immune responses to invasive infection, including against the cardinal Gram-negative bacterium Neisseria meningitidis. Individuals homozygous or heterozygous for any of three variant alleles of MBL2 (O/O or A/O genotypes) have deficient concentrations of MBL2 in circulating blood, but previous studies linking MBL deficiency to susceptibility to meningococcal disease have not revealed a consistent association. We genotyped 741 patients with microbiologically – proven meningococcal disease and correlated MBL2 genotype with plasma bacterial load of N. meningitidis with blood samples taken during hospital admission. We show that individuals with genotypes compatible with MBL2 deficiency have higher measurable levels of bacterial plasma genomic load with the greatest effect seen in children

Meningococcal disease is caused by a limited range of clonal complexes of Neisseria meningitidis. The disease occurs in people who lack bactericidal antibodies to this pathogen, and therefore the patients are reliant on innate immunity or components of acquired immunity other than bactericidal antibodies. Gene variants that influence the function of innate and acquired immune response components have been associated with altered host susceptibility to meningococcal disease, and some genetic factors have also been associated with more severe disease. Identification of genetic factors associated with meningococcal disease will enhance our understanding of this rare but dangerous condition which causes death and serious morbidity in young, previously fit individuals. Genetic variations in the gene cluster encoding IL-1 and in key genes including TNF, SP-A2 and CFH have been associated with susceptibility to meningococcal disease. Understanding the mechanisms underlying genetic susceptibility to meningococcal disease will permit the development of novel therapeutic measures for the treatment of Gram-negative sepsis. To enable the discovery of new mechanisms of the disease, future research will move away from small-scale association studies and instead include analysis of large patient cohorts with accurately linked clinical and demographic information.

Neisseria meningitidis is an exclusively human pathogen that has evolved primarily to colonize the nasopharynx rather than to cause systemic disease. Colonization is the most frequent outcome following meningococcal infection and a prerequisite for invasive disease. The mechanism of colonization involves attachment of the organism to epithelial cells via bacterial type IV pili (Tfp), but subsequent events during colonization remain largely unknown. We analyzed 576 N. meningitidis mutants for their capacity to colonize human nasopharyngeal tissue in an organ culture model to identify bacterial genes required for colonization. Eight colonization-defective mutants were isolated. Two mutants were unable to express Tfp and were defective for adhesion to epithelial cells, which is likely to be the basis of their attenuation in nasopharyngeal tissue. Three other mutants are predicted to have lost previously uncharacterized surface molecules, while the remaining mutants have transposon insertions in genes of unknown function. We have identified novel meningococcal colonization factors, and this should provide insights into the survival of this important pathogen in its natural habitat.

This document is an update of Guidelines published in 2005 and now includes scientific publications through to May 2010. It provides evidence-based recommendations for the most common management questions occurring in routine clinical practice in the management of adult patients with LRTI. Topics include management outside hospital, management inside hospital (including community-acquired pneumonia (CAP), acute exacerbations of COPD (AECOPD), acute exacerbations of bronchiectasis) and prevention. Background sections and graded evidence tables are also included. The target audience for the Guideline is thus all those whose routine practice includes the management of adult LRTI.

Among 384 patients with confirmed meningococcal disease, the likelihood of detecting Neisseria meningitidis DNA in cerebrospinal fluid (CSF) increased with age, serogroup B infection, and prehospitalization antibiotic treatment. Plasma and CSF genomic bacterial loads of non-B N. meningitidis serogroups correlated significantly. Serogroup B-infected patients with genotype TNF2 (-308A) had significantly higher CSF bacterial loads.

The tetraspanins are a superfamily of transmembrane proteins with diverse functions and can form extended microdomains within the plasma membrane in conjunction with partner proteins, which probably includes receptors for bacterial adhesins. Neisseria meningitidis , the causative agent of meningococcal disease, attaches to host nasopharyngeal epithelial cells via type IV pili and opacity (Opa) proteins. We examined the role of tetraspanin function in Neisseria meningitidis adherence to epithelial cells. Tetraspanins CD9, CD63, and CD151 were expressed by HEC-1-B and DETROIT 562 cells. Coincubation of cells with antibodies against all three tetraspanin molecules used individually or in combination, with recombinant tetraspanin extracellular domains (EC2), or with small interfering RNAs (siRNAs) significantly reduced adherence of Neisseria meningitidis . In contrast, recombinant CD81, a different tetraspanin, had no effect on meningococcal adherence. Antitetraspanin antibodies reduced the adherence to epithelial cells of Neisseria meningitidis strain derivatives expressing Opa and pili significantly more than isogenic strains lacking these determinants. Adherence to epithelial cells of strains of Staphylococcus aureus , Neisseria lactamica , Escherichia coli , and Streptococcus pneumoniae was also reduced by pretreatment of cells with tetraspanin antibodies and recombinant proteins. These data suggest that tetraspanins are required for optimal function of epithelial adhesion platforms containing specific receptors for Neisseria meningitidis and potentially for multiple species of bacteria.

Background Escherichia coli is the leading cause of bacteremia with multi-drug-resistant strains proving increasingly problematic. Knowledge of the strain diversity associated with site-specific infections will inform the development of new preventative strategies, e.g. vaccines. We hypothesized that virulence factor (VF) scores of bacteremia strains from neutropenia patients with unknown infective foci (NPUIF—likely due to bowel translocation) would be lower than those from immunocompetent patients. Methods Immunocompetent (n = 49) and neutropaenic adults (n = 8) with E. coli bacteremia were prospectively enrolled and the focus of bacteremia determined. Neutropenia patients were enrolled only if there was no identifiable infective focus. Multi-locus sequence typing and VF score (31 known VFs included) data were derived in silico following whole-genome sequencing and the results compared between patient groups. Results Bacteremia strains from immunocompetent patients with urinary tract infective foci (UTI-foci) harbored significantly more VFs (median VF score 16, range 8–24) than strains from both immunocompetent patients with non-UTI-foci (10, 2–22, P = 0.006) and NPUIF (8, 3–13, P < 0.0001). VF scores of strains from non-UTI-foci were not significantly different to those from NPUIF (10, 2–22 vs. 8, 3–13, respectively. P = 0.28). Logistic regression analysis demonstrated that VF score (OR 1.21, 95% CIs 1.01–1.46, P = 0.039) and recurrent urinary tract infection/urinary tract infection (OR 12.82, 95% CIs 1.24–132.65, P = 0.032) were independent predictors of bacteremia secondary to UTI-foci vs. non-UTI-foci in immunocompetent patients. Hence, for every unit increase in VF score, the odds of a bacteremia strain originating from UTI-foci increased by 1.21. papA, papC, papE/F, papG, agn43, tia, iut, fyuA, kpsM and sat were significantly more prevalent amongst strains associated with UTI-foci vs. non-UTI-foci amongst immunocompetent patients. papC, papE/F, papG, agn43, tia, fyuA, hlyA, usp and clb were significantly more prevalent amongst UTI-foci- vs. NPUIF-associated strains. Conclusion UTI-associated E. coli bacteremia strains have distinct VF profiles from those originating from non-UTI-foci and NPUIF. Future vaccines must consider this diversity to ensure adequate coverage of strains associated with site-specific disease. Disclosures All authors: No reported disclosures.

Background Bordetella pertussis is one of the leading causes of vaccine preventable death and morbidity globally. Over the last 20 years, pertussis has resurged worldwide, even in territories with high immunization coverage. To improve vaccine strategies, a greater understanding of human B. pertussis infection and immunity is required. This study aims to develop a safe controlled human B. pertussis infection model and to define natural immune responses against wild-type B. pertussis in order to facilitate the development of bioassays and next-generation pertussis vaccines. Methods In this first-in-human controlled infection model, healthy volunteers aged 18–45 years with an anti-pertussis toxin (PT) IgG level of Results 24 volunteers were challenged in groups of 4–5. The dose was gradually escalated from 103 colony forming units (cfu) to 105 cfu. Colonization rate ranged from 0% (dose 103 cfu) to 80% (105 cfu). Amongst this initial cohort, no significant safety concerns or symptoms attributed to B. pertussis disease were reported. Eradication was achieved by 48 hours in 100% of colonized volunteers. At least 4-fold rise in anti-PT IgG by day 28 in comparison to baseline was observed in 5 out of 8 volunteers who had >1,000 cfu/mL viable B. pertussis in the nasal wash and in one volunteer without detectable colonization. Nasal wash cultures were more sensitive in detecting colonization than nasopharyngeal swab cultures. No shedding of B. pertussis was detected in systematically collected environmental samples. Conclusion This is the first study to demonstrate safe deliberate induction of B. pertussis colonization. It shows that asymptomatic B. pertussis colonization occurs and causes a systemic immune response. The model that we have developed will be a valuable tool to further investigate B. pertussis colonization and vaccine development. Disclosures K. Kester, Sanofi: Employee, Salary. S. Faust, Pfizer, Merck, Sanofi, AstraZeneca/Medimmune: Scientific Advisor, all honoraria paid to institution with no personal payments of any kind.

Summary Various genes regulate the intensity of the inflammatory and coagulation response to infection and therefore might determine the severity and outcome of meningococcal disease. We systematically reviewed the published work for case–control studies on the influence of host genetics on severity and outcome in meningococcal disease and identified 27 studies including 7245 patients, with an overall mortality of 10% (range 1–19%). Despite flaws in the methods of the studies there was a clear association of host genetics with mortality and severity in meningococcal disease. Polymorphisms in SERPINE1 (odds ratio [OR] 2·23, 95% CI 1·48–3·35), IL1RN (OR 1·85, 95% CI 1·25–2·76) and IL1B (OR 1·81, 95% CI 1·09–2·97) were associated with mortality in our meta-analyses. In conclusion, gene variation influences severity and mortality in meningococcal disease. Polymorphisms might have potential as prognostic markers or to determine tailor-made adjunctive therapy. Carefully designed, prospective, whole-genome association studies and randomised clinical trials of treatments in specific genetic subgroups are needed.

BACKGROUND: Diagnostic polymerase chain reaction (PCR) detection of Neisseria meningitidis has enabled accurate quantification of the bacterial load in patients with meningococcal disease. METHODS: Quantification of the N. meningitidis DNA level by real time-PCR was conducted on whole-blood samples obtained from patients presenting with meningococcal disease to hospitals throughout England and Wales over a 3-year period. Levels were correlated with clinical outcome, infecting serogroup, and host factors including, interleukin-1 genotype (IL-1). RESULTS: Bacterial loads were available for 1045 patients and were not associated with the age of the patient, delay in sample submission, or administration of antibiotics prior to admission. The median log bacterial load was higher in 95 patients who died (5.29 log(10)copies/mL; interquartile range, 4.41-6.30 log(10)copies/mL) than in 950 patients who survived (3.79 log(10)copies/mL; interquartile range, 2.87-4.71 log(10)copies/mL). Logistic regression revealed that age (odds ratio, 1.04 per 1-year increase in age) and bacterial load (odds ratio, 2.04 per log(10)-copies/mL increase) had a statistically significant effect on the risk of death. Infection with N. meningitidis serogroup C was associated with increased risk of death and an increased bacterial load. Also associated with a higher bacterial load were prolonged hospitalization (duration, >10 days); digit, limb, or soft-tissue loss; and requirement of hemodialysis. Carriage of IL-1RN(+2018) was associated with increased mortality (odds ratio, 2.14; P=.07) but not with a higher bacterial load. CONCLUSIONS: In meningococcal disease, bacterial load is associated with likelihood of death, development of permanent disease sequelae, and prolonged hospitalization. The bacterial load was relatively higher in patients infected with N. meningitidis serogroup C than in those infected with other serogroups. The effects of age and IL-1 genotype on mortality are independent of a high genomic bacterial load.

The aim of this study was to show that sequential intravenous and oral moxifloxacin monotherapy (400 mg once per day) is as efficacious and safe as a combination regimen (intravenous ceftriaxone, 2 g once per day, plus sequential intravenous and oral levofloxacin, 500 mg twice per day) in patients hospitalized with community-acquired pneumonia.We conducted a prospective, multicenter, randomized, double-blind noninferiority trial. Patients with a Pneumonia Severity Index (PSI) of III-V were stratified on the basis of PSI risk class before randomization. The primary efficacy end point was clinical response at test of cure (4-14 days after the completion of treatment). Secondary efficacy end points were clinical and bacteriological response at end of treatment (days 7-14) and at follow-up assessment (21-28 days after the end of treatment), overall mortality, and mortality attributable to pneumonia.Seven hundred thirty-three patients were enrolled in the study (368 in the moxifloxacin arm and 365 in the comparator arm); 49% had a PSI of IV, and 10% had a PSI of V. Of 569 patients (291 in the moxifloxacin arm and 278 in the comparator arm) valid for per-protocol analysis, the overall clinical cure rates at test of cure were 86.9% for moxifloxacin and 89.9% for the comparator regimen (95% confidence interval, -8.1% to 2.2%). Bacteriological success at test of cure was 83.3% for moxifloxacin and 85.1% for the comparator regimen (95% confidence interval, -15.4% to 11.8%). There were no significant differences between moxifloxacin and comparator treatments in the incidence of treatment-emergent adverse events or in mortality.Monotherapy with sequential intravenous/oral moxifloxacin was noninferior to treatment with ceftriaxone plus levofloxacin combination therapy in patients with community-acquired pneumonia who required hospitalization.

Primary antibody deficiencies are characterized by decreased serum levels of immunoglobulin isotypes and increased susceptibility to infection by various microorganisms including encapsulated bacteria. This study was performed in order to evaluate bactericidal antibody response of these patients to polysaccharide meningococcal vaccine. Twenty-four antibody deficient children of mean age 11.2+/-3.5 years, and 15 sex and age-matched healthy volunteers were enrolled. All subjects received meningococcal polysaccharide vaccine A+C; blood samples were collected before vaccination and 3 weeks after vaccination. Following vaccination, the serum bactericidal antibody (SBA) geometric mean titre was significantly increased compared to the prevaccination level in the patient group (8.98 versus 1.63, P0.001) and the control group (12.13 versus 1.26, P0.001). All controls had a protective SBA response (SBA titre ofor=8 post-vaccination or rise ofor=4-fold from pre- to post-vaccination), whereas only 16 of 24 patients (66.6%) had a protective response (P=0.014). The non-responder patients included 5 cases with common variable immunodeficiency, two cases with hyper IgM syndrome, and one case with IgG subclass deficiency. This study indicates that some patients with primary antibody deficiencies can produce protective post-vaccination titres similar to the normal population, despite the common perception that patients with primary antibody deficiencies respond poorly to vaccination. This supports the use of polysaccharide-containing vaccines in these patients.

Summary Objectives Invasive pneumococcal disease (IPD) is associated with a high mortality despite antimicrobial therapy, but may be preventable by pneumococcal vaccination. The extent of previous exposure to pneumococcal capsular polysaccharide vaccination prior to an episode of IPD in hospitalised adults in the United Kingdom is unclear. Methods We conducted a retrospective cohort study in adults with IPD admitted to either of two teaching hospitals in Sheffield, United Kingdom during 1992–2000. Receipt of pneumococcal vaccination, risk factors for IPD, death and disability were determined. Results The number of cases of IPD was 552 and 187/230 patient records from one site were reviewed. According to UK pneumococcal vaccination guidelines 59% of patients should have received the vaccine and 76% of patients if updated guidelines, which include age>65 years as an indication, are applied. In patients with known risk factors, excluding age, only 8% had been vaccinated. The mortality from IPD was 21% and an additional 6% suffered major complications. Conclusions In patients hospitalised with IPD there is a high rate of pre-existing risk factors and a low rate of administration of pneumococcal vaccination. IPD incurs significant mortality, morbidity and economic cost and there is potential for reducing this by improved uptake of pneumococcal vaccination.

WOS: 000378628600001, PubMed ID: 27062097, European Clinical Microbiology and Infectious Disease Society (ESCMID), Funded by a grant of the European Clinical Microbiology and Infectious Disease Society (ESCMID). All authors report no conflicts of interest relevant to this article.

Background Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensalNeisseria lactamicaand meningococcal disease, we investigated whether controlled infection of human volunteers withN. lactamicaprevents colonization byNeisseria meningitidis. Methods In a block-randomized human challenge study, 310 university students were inoculated with 10^4 colony-forming units ofN. lactamicaor were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated withN. lactamicaand resampled 2 weeks later. Results At baseline, naturalN. meningitidiscarriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation ofN. lactamica, 33.6% (48/143) of the challenge group became colonized withN. lactamica. In this group, meningococcal carriage reduced from 24.2% (36/149) at inoculation to 14.7% (21/143) 2 weeks after inoculation (−9.5%;P= .006). The inhibition of meningococcal carriage was only observed in carriers ofN. lactamica, was due both to displacement of existing meningococci and to inhibition of new acquisition, and persisted over at least 16 weeks. Crossover inoculation of controls withN. lactamicareplicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types. Results The inhibition of meningococcal carriage byN. lactamicais even more potent than after glycoconjugate meningococcal vaccination.Neisseria lactamicaor its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage ofN. lactamica.

SUMMARYData were extracted from the case records of UK patients admitted with laboratory-confirmed influenza A(H1N1)pdm09. White and non-White patients were characterized by age, sex, socioeconomic status, pandemic wave and indicators of pre-morbid health status. Logistic regression examined differences by ethnicity in patient characteristics, care pathway and clinical outcomes; multivariable models controlled for potential confounders. Whites (n = 630) and non-Whites (n = 510) differed by age, socioeconomic status, pandemic wave of admission, pregnancy, recorded obesity, previous and current smoking, and presence of chronic obstructive pulmonary disease. After adjustment fora prioriconfounders non-Whites were less likely to have received pre-admission antibiotics [adjusted odds ratio (aOR) 0·43, 95% confidence interval (CI) 0·28–0·68,P P = 0·018). However, there were no significant differences by ethnicity in delayed admission, severity at presentation for admission, or likelihood of severe outcome.

Background. Meningococcal disease occurs after colonization of the nasopharynx with Neisseria meningitidis. Surfactant protein (SP)-A and SP-D are pattern-recognition molecules of the respiratory tract that activate inflammatory and phagocytic defences after binding to microbial sugars. Variation in the genes of the surfactant proteins affects the expression and function of these molecules. Methods. Allele frequencies of SP-Al, SP-A2, and SP-D were determined by polymerase chain reaction in 303 patients with microbiologically proven meningococcal disease, including 18 patients who died, and 222 healthy control subjects. Results. Homozygosity of allele 1A 1 of SP-A2 increased the risk of meningococcal disease (odds ratio [OR], 7.4; 95% confidence interval [CI], 1.3-42.4); carriage of 1A 5 reduced the risk (OR, 0.3; 95% CI, 0.1-0.97). An analysis of the multiple single-nucleotide polymorphisms in SP-A demonstrated that homozygosity for alleles encoding lysine (in 1A 1 ) rather than glutamine (in 1A 5 ) at amino acid 223 in the carbohydrate recognition domain was associated with an increased risk of meningococcal disease (OR, 6.7; 95% CI, 1.4-31.5). Carriage of alleles encoding lysine at residue 223 was found in 61% of patients who died, compared with 35% of those who survived (OR adjusted for age, 2.9; 95% CI, 1.1-7.7). Genetic variation of SP-Al and SP-D was not associated with meningococcal disease. Conclusions. Gene polymorphism resulting in the substitution of glutamine with lysine at residue 223 in the carbohydrate recognition domain of SP-A2 increases susceptibility to meningococcal disease, as well as the risk of death.

Objectives: Disease caused by penicillin-resistant Streptococcus pneumoniae (PRSP) is associated with more suppurative complications than disease caused by penicillin-susceptible S. pneumoniae (PSSP). Exposure of S. pneumoniae to b-lactam antibiotics enhances the proinflammatory activation of human cells by pneumococci via Toll-like receptor-2 (TLR2). To test the hypothesis that penicillin resistance influences cellular TLR2 activation by b-lactam-exposed pneumococci, we compared TLR2 induction by PSSP (MIC 0.06 mg/L) and a high-level PRSP clinical isolate (159; MIC 16 mg/L) following exposure to penicillin and cefotaxime. Methods: Both organisms were treated with penicillin or cefotaxime at and around the MIC. TLR2 signalling was measured as relative IL-8 promoter activation in transfected HeLa cells. Results: On exposure to penicillin, log-phase PSSP and PRSP induced TLR2-proinflammatory activation at levels significantly higher than unexposed bacteria, and maximal in each case at the MIC. Transformants containing low-affinity penicillin-binding proteins (PBP) 2x, 1a and 2b exhibited stepwise resistance to cefotaxime and penicillin. TLR2 activation following penicillin treatment was dependent on an abnormal cell wall (PBP1a and 2x) and autolysis (PBP2b). High affinity PBP2x was required for this effect to be observed in log-phase pneumococci exposed to cefotaxime at the MIC. Cefotaximemediated TLR2 activation was not observed in lag-phase transformants exposed to sub-lethal concentrations. Conclusions: These data show that PRSP have similar TLR2-proinflammatory effects to PSSP when exposed to b-lactam antibiotics but the antibiotic concentration relative to the MIC is critical. This has implications for treatment of pneumococcal disease when tissue concentrations of antibiotic are close to the MIC.

Neurocysticercosis, infection with larval Taenia solium, is a common, serious neuroparasitic infection. Larval degeneration results in inflammatory cell influx and granuloma formation which leads to clinical symptomatology. The role of chemokines in such cell influx is unknown. We demonstrate that monocyte stimulation by T. solium larval antigen (TsAg) results in a differential profile of CXCL8/IL-8 (146.5+/-8.5ng/ml after 24h), CCL2/MCP-1 (267+/-4 ng/ml after 48 h) and CCL3/MIP-1alpha (1.72+/-0.43 ng/ml after 8 h) secretion. There was coordinate mRNA accumulation reaching maximum at 1h for CCL3 and 2 h for CXCL8 and CCL2. TsAg induced maximal nuclear binding of p65, p50 and c-rel subunits of the transcriptional regulator NF-kappaB by 2 h. IkappaBalpha but not IkappaBbeta was degraded within 10 min before resynthesis by 2 h. Pre-treatment with the broad-spectrum NF-kappaB inhibitor pyrrolidine dithiocarbamate caused complete abrogation of TsAg-induced CCL2 secretion (p=0.005) and 91% reduction of CXCL8 secretion (p=0.0003). TsAg was unable to induce CXCL8 promoter activity in Toll-like receptor (TLR)-2 or TLR-4/MD-2 transfected HeLa cells in the absence of lectins or other adaptor molecules. In summary, our data demonstrate that TsAg induces chemokine secretion via specific pathways dependent on NF-kappaB but not TLR-4/TLR-2, and indicate a potential mechanism whereby larval degeneration results in brain inflammation.

We obtained bronchoalveolar lavage (BAL) fluid from 45 Malawian adults, to measure the concentrations of innate pulmonary immune factors that are important in lung defense against infection. Increased concentrations of the beta -chemokine RANTES were found in BAL fluid from the human immunodeficiency virus (HIV)-1-infected subjects, compared with those in BAL fluid from the HIV-1-uninfected control subjects (mean, 86 pg/mL vs. 0 pg/mL; P.001). Lysozyme concentrations were also elevated in the HIV-1-infected subjects, compared with those in the HIV-1-uninfected control subjects (1.9 mu g/mL vs. 1.1 mu g/mL; P=.03), but were not elevated in the HIV-1-infected subjects who had recently recovered from invasive pneumococcal disease. Concentrations of lactoferrin and secretory leukocyte protease inhibitor (SLPI) were not different when the subjects were compared by HIV-1 serostatus. Concentrations of RANTES (R2=0.68 and P.0001) and SLPI (R2=0.29 and P=.001) correlated with BAL fluid HIV-1 load but not with plasma HIV-1 load.

Gamma interferon (IFN-γ) is a critical cytokine in host defense against salmonella infections, but its role in phagocytic killing of intracellularSalmonellaspp. has been investigated mainly in animal rather than human cells. We measured the effect of recombinant IFN-γ (rIFN-γ) priming on bacterial internalization, intracellular killing, oxidative burst, and cytokine release during phagocytosis ofSalmonella entericaserovar Typhimurium by human monocyte-derived macrophages (MDM). Eleven-day-old MDM, primed for 72 h with rIFN-γ (100 ng/ml) exhibited an increased proportion of cells with associated bacteria (31% versus 26%,P= 0.036) and a 67% increase in internalized bacteria per cell compared to unprimed cells (P= 0.025). Retrieval of viable bacteria following internalization was reduced 3.6-fold in 72-h primed versus unprimed MDM (interquartile range, 3.1 to 6.4) at 0.5 h due to enhanced early intracellular killing, and this difference was maintained up to 24 h. In contrast, cells primed for only 24 h exhibited no increase in early killing. MDM were competent to produce an early oxidative burst when stimulated with phorbol myristate acetate, which was fully abrogated by the respiratory burst inhibitor diphenyleneiodonium chloride (DPI), but infection of MDM withS. entericaserovar Typhimurium did not cause an increase in the early respiratory burst under unprimed or primed conditions, and DPI had no effect on the early killing of bacteria by primed or unprimed MDM. During 24 h following infection, rIFN-γ-primed MDM released more interleukin-12 (IL-12) and less IL-10 relative to unprimed cells. We conclude that 72-h priming with rIFN-γ increases the efficiency of internalization and nonoxidative early intracellular killing ofS. entericaserovar Typhimurium by human macrophages and modifies subsequent cytokine release.

Nitric oxide (NO) contributes to mammalian host defense by direct microbicidal activity and as a signaling molecule of innate immune responses. Macrophages produce NO via the inducible NO synthase (iNOS). The genome of Neisseria meningitidis includes two genes, norB (encoding nitric oxide reductase) and cycP (encoding cytochrome c′ ), both of which detoxify NO in pure cultures of N. meningitidis . We show here that norB , and to a lesser extent cycP , enhance survival of N. meningitidis within primary human macrophages. Furthermore, accumulation of lysosome-associated membrane protein 1 (LAMP-1) is modified in phagosomes containing an isogenic norB mutant of N. meningitidis compared to the wild type. The survival enhancement conferred by norB and cycP is ablated by pretreatment of macrophages with the nitric oxide synthase inhibitor N (G)-monomethyl- l -arginine (L-NMMA). Despite this evidence that NO detoxification confers advantage, we find, using a highly sensitive chemiluminescence technique, that human macrophage-associated [NO] is low even after activation by lipopolysaccharide and interferon alpha. Furthermore, wild-type N. meningitidis further depletes cell-associated NO during phagocytosis by an active mechanism and survives relatively poorly in the presence of L-NMMA, suggesting that the wild-type organism may utilize NO for optimal survival during intracellular life. The natural habitat of N. meningitidis is the human nasopharynx. Using a nasopharyngeal mucosa organ culture system, we show that mutants lacking norB and cycP also survive poorly in nasopharyngeal tissue compared to wild-type N. meningitidis . These findings indicate that the meningococcus requires active NO detoxification systems for optimal survival during experimental nasopharyngeal colonization and processing by human phagocytic cells.

Severe Acute Respiratory Syndrome (SARS) is a potentially severe and highly infectious disease to which healthcare workers involved in the management of cases are particularly vulnerable. These guidelines briefly summarise optimal and safe practice for clinicians involved in the emergency care of patients with probable or confirmed SARS.

The Streptococcus pneumoniae cell-wall components peptidoglycan and lipoteichoic acid activate Toll-like receptor 2 (TLR2), which transduces an inflammatory response. After exposure to penicillin, type 2 S. pneumoniae strain D39, but not the isogenic autolysin-deficient mutant AL2, induced significantly enhanced interleukin-8 promoter activity in TLR2-transfected HeLa cells. Lag-phase D39 exhibited enhanced TLR2 activation after exposure to penicillin at levels below the minimum inhibitory concentration (MIC); in contrast, early log-phase S. pneumoniae were most active when exposed to the MIC. This enhancement was not ablated by heat treatment but was attenuated by autolysin inhibitors. The antimicrobial activity of moxifloxacin and erythromycin was not associated with TLR2 activation by S. pneumoniae. These data show that penicillin treatment of S. pneumoniae releases proinflammatory cell-wall components that activate TLR2 and that this activity is dependent on autolysin, the growth phase of the organism, and the antibiotic concentration.

We tested the hypothesis that human immunodeficiency virus (HIV)-infected adults have a specific defect in anti-pneumococcal capsular polysaccharide (Pn-specific) immunoglobulin (Ig) in fluid obtained from the lower respiratory tract. Higher levels of total IgG and IgM were present in bronchoalveolar lavage samples from HIV-infected subjects than in those from HIV-uninfected subjects. Pn-specific IgG and IgM in bronchoalveolar lavage samples were not significantly different between HIV-infected and -uninfected subjects. After pneumococcal infection, HIV-infected patients had higher bronchoalveolar lavage levels of Pn-specific IgG than HIV-infected patients without recent infection (geometric means, 387 vs. 30 ng/mL, P=.001).

Lipooligosaccharide (LOS) structure and capsular polysaccharide of Neisseria meningitidis each greatly influence the virulence of the organism and the quality of host innate immune responses. In this study, we found that production of the proinflammatory cytokine tumor necrosis factor (TNF) by a human monocyte-derived cell line (THP-1) exposed to strains of N . meningitidis lacking capsule and/or with truncated LOS was similar to that elicited by the isogenic wild-type strain. These mutants also exhibited no difference in induction of the interleukin-8 (IL-8) promoter in a transfected HeLa cell system of Toll-like receptor 2 (TLR2) and TLR4/MD2 signaling. However, purified LOS from diverse strains of Neisseria (both N . meningitidis and N . gonorrhoeae ) caused widely variant levels of IL-8 promoter induction in cells expressing MD2 that correlated with the production of TNF from THP-1 cells. These data suggest that although modification of the oligosaccharide chain of LOS and/or absence of capsule do not affect cell signaling mediated by TLR4/MD2, fine-structural differences in the LOS do influence signaling through TLR4/MD2 and, through this pathway, influence some of the proinflammatory responses elicited by Neisseria .

Survival of macrophage microbicidal activity is a prerequisite for invasive disease caused by the enteric pathogenSalmonella entericaserovar Typhimurium. Flavohemoglobins, such as those ofEscherichia coli,Salmonella, and yeast, play vital roles in protection of these microorganisms in vitro from nitric oxide (NO) and nitrosative stress. ASalmonella hmpmutant defective in flavohemoglobin (Hmp) synthesis exhibits growth that is hypersensitive to nitrosating agents. We found that respiration of this mutant exhibited increased inhibition by NO, whereas wild-type cells pregrown with sodium nitroprusside orS-nitrosoglutathione showed enhanced tolerance of NO. Most significantly,hmpmutants internalized by primary human peripheral monocyte-derived macrophages survived phagocytosis relatively poorly compared with similarly bound and internalized wild-type cells. That the enhanced sensitivity to macrophage microbicidal activity is due primarily to the failure ofSalmonellato detoxify NO was suggested by the ability ofl-NG-monomethyl arginine—an inhibitor of NO synthase—to eliminate the difference in killing between wild-type andhmpmutantSalmonellacells. These observations suggest thatSalmonellaHmp contributes to protection from NO-mediated inhibition by human macrophages.

Streptococcus pneumoniae is a major cause of pneumonia, bacteremia, and meningitis, especially among adults infected with the human immunodeficiency virus (HIV). Alveolar macrophages (AMs) are critical components of cellular defense against bacterial infection and are both infected and affected by HIV. In this study, AMs obtained at bronchoscopy from 44 Malawian adults (24 HIV positive and 20 HIV negative) were exposed in vitro to opsonized S. pneumoniae and coagulase-negative staphylococci. AMs from HIV-positive and -negative volunteers showed no significant difference in binding to or internalization of either S. pneumoniae or coagulase-negative staphylococci. In HIV-positive subjects, the presence of detectable HIV in lung fluid was not associated with AM impairment. AMs from HIV-infected adults did not exhibit impaired pneumococcal phagocytosis in the assay used. This suggests that an alternative mechanism of susceptibility is operating in these individuals.

The influence of the innate immune protein mannose-binding lectin (MBL) on the response of human phagocytes to Neisseria meningitidis was investigated. MBL increased the association of killed meningococci with neutrophils, monocytes, and macrophages by increasing the proportion of cells that recognized bacteria. MBL down-regulated the normal change in expression of the leukocyte adhesion molecules CD11b and CD62L. In an ex vivo model, the addition of MBL to the blood of MBL-deficient donors influenced the production of monocyte-derived inflammatory cytokines. The addition of high concentrations of MBL (>6 microg/mL) profoundly decreased the production of interleukin (IL)-6, IL-1beta, and tumor necrosis factor-alpha by monocytes in response to meningococci, whereas lower concentrations enhanced the production of IL-6 and IL-1beta. These results suggest that MBL not only is involved in complement activation but also is a potent regulator of inflammatory pathways and, as such, may affect the severity of meningococcal disease.

Meningococcal disease severity correlates with circulating concentrations of lipopolysaccharide (LPS) and proinflammatory cytokines. Disruption of the lpxA gene of Neisseria meningitidis generated a viable strain that was deficient of detectable LPS. The potency of wild-type N. meningitidis to elicit tumor necrosis factor (TNF)-alpha production by human monocyte-derived macrophages was approximately 10-fold greater than that of the lpxA mutant. Killed wild-type N. meningitidis and its soluble products induced interleukin (IL)-8 and TNF-alpha secretion by transfected HeLa cells expressing Toll-like receptor (TLR) 4/MD2, but the lpxA mutant was inactive via this pathway. In contrast, both strains induced IL-8 promoter activity in TLR2-transfected HeLa cells. These data provide evidence that N. meningitidis contains components other than LPS that can elicit biological responses via pathways that are independent of the TLR4/MD2 receptor system, and TLR2 is one of these alternate pathways. These findings have implications for future therapeutic strategies against meningococcal disease on the basis of the blockade of TLRs and the modulation of LPS activity.

The aim of this study was to compare outcomes for patients with community-acquired pneumonia (CAP) caused by Legionella spp. following treatment with moxifloxacin or a range of comparator antimicrobial agents. Data were pooled from four sequential I.V./P.O. trials of moxifloxacin in the treatment of CAP. Comparators were ceftriaxone +/- erythromycin, amoxicillin/clavulanate +/- clarithromycin, trovafloxacin, levofloxacin, or ceftriaxone + levofloxacin. Legionella infection was diagnosed by culture, urine antigen testing and/or serology. Clinical success rates for the efficacy-valid (per protocol) populations were recorded at the test-of-cure visit (5-30 days post-therapy). Severity of CAP was determined using the modified American Thoracic Society criteria.Of 1786 efficacy-valid patients, 33 (1.8%) had documented infection with Legionella spp. (moxifloxacin: n=13; comparator: n=20). Of these, 30 cases were identified by serology and/or urine antigen detection and 3 by respiratory culture. The success rate of moxifloxacin vs. comparator therapy was 92.3% vs. 80.0% for the I.V./P.O. trials.Sequential (I.V./P.O.) moxifloxacin demonstrated clinical efficacy that was at least as good as that of comparator treatments for the treatment of CAP due to Legionella.

Human alveolar macrophages (HAM) are the major resident phagocytic cells of the gas-exchanging areas of the lung. Following contact with macrophages, bacteria enter phagosomes, which gradually acquire the characteristics of terminal phagolysosomes, with incorporation of lysosome-associated membrane protein (LAMP). We measured the binding of type 1Streptococcus pneumoniaeto the surface of HAM and then measured subsequent internalization and phagosomal incorporation of LAMP-1 under various opsonic conditions. Opsonization with serum containing immunoglobulin resulted in significantly greater binding of pneumococci to HAM compared with opsonization with immunoglobulin G (IgG)-depleted serum containing complement, which in turn resulted in marginally increased binding over that observed in the absence of opsonization. Internalization of opsonizedS. pneumoniaegradually increased to a maximum of 20% of bound bacteria by 120 min of warm incubation, with 20% of internalized pneumococci being localized within LAMP-containing compartments by 80 min. Internalization of opsonizedS. pneumoniaeby HAM correlated with a reduction of bacterial viability. When inocula were adjusted so that pneumococcal binding under different conditions was equalized, subsequent internalization, trafficking to LAMP-containing compartments, and reduction of bacterial viability were less efficient in the absence of opsonization than that observed following opsonization with adsorbed or IgG-replete adsorbed serum. Once bound to the surface of HAM, pneumococci opsonized with adsorbed serum with or without IgG were internalized, processed, and killed equally well. In conclusion, binding, intracellular trafficking, and killing ofS. pneumoniaeby HAM are each significantly increased by opsonization with serum containing immunogloblin and/or complement.

A number of national society guidelines exist for empiric management of community-acquired pneumonia but these are, to a large extent, not evidence-based, but based on clinical experience, in vitro data, pragmatism and common sense. Many randomized controlled trials of antibiotic therapy in community-acquired pneumonia have been conducted, but most of these have been powered to demonstrate equivalent efficacy of new treatments in comparison with conventional antimicrobial therapy. Development of new antibiotics has been driven by the emergence of penicillin-resistant Streptococcus pneumoniae, but so far there is no hard evidence that beta-lactam therapy fails in community-acquired pneumonia, at least with the higher doses of penicillins that are commonly used in hospital practice. Nonetheless, newer antibiotics have been deployed including macrolides and quinolones, and have demonstrated equivalent (and in some cases, marginally improved) efficacy to older antibiotic treatments in randomized control trials. A number of studies have shown that it is possible to stratify patients according to severity of illness, to in-patient or out-patient management protocols. These have been validated and refined.

Pneumonia caused by Streptococcus pneumoniae is characterized by neutrophil infiltration and variable epithelial injury. Neutrophil adhesion to alveolar epithelial pneumocytes (A549) was measured and demonstrated to be dose-dependent following preincubation of these (A549) pneumocytes with type 1 S. pneumoniae. Adhesion peaked at a bacteria-to-epithelial cell ratio of 5:1 after a 4-h incubation but was absent after 2 h and without FMLP. Filtered conditioned media (CM) from pneumococci cultured with (CM+) or without (CM-) epithelial cells were tested. CM+ induced significant adhesion in the absence of FMLP (P < .001); CM- had no effect. In the presence of FMLP, adhesion induced by both media was significantly greater than by FMLP alone (P < .001) and was significantly blocked (P < .01) by antibodies to CD11b and CD18. CM+ upregulated epithelial intercellular adhesion molecule 1 but CM- did not. These data provide new information concerning the interactions of S. pneumoniae, alveolar epithelial cells, and neutrophils.