Effect of colonisation with Neisseria lactamica on cross-reactive anti-meningococcal B-cell responses: a randomised, controlled, human infection trial.

BACKGROUND: 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. METHODS: 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 105 colony-forming units of N lactamica in 1 mL phosphate-buffered saline (PBS) or 1 mL PBS alone. Participants and researchers conducting participant sampling and immunological assays were masked to allocation. The primary endpoint was the frequency of circulating N lactamica-specific plasma cells and memory B cells after N lactamica inoculation (day 7-28) compared with baseline values (day 0), measured using enzyme-linked immunospot assays. The secondary endpoint was to measure the frequency of N meningitidis-specific B cells. In a second study, we measured the effect of duration of N lactamica colonisation on seroconversion by terminating carriage at either 4 days or 14 days with single-dose oral ciprofloxacin. The studies are now closed to participants. The trials are registered with ClinicalTrials.gov, NCT03633474 and NCT03549325. FINDINGS: Of 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 105 peripheral blood mononuclear cells) were 0·0 (IQR 0·0-0·0) versus 5·0 (1·5-10·5) for IgA-secreting plasma cells (p<0·0001), and 0·0 (0·0-0·0) versus 3·0 (1·5-9·5) for IgG-secreting plasma cells (p<0·0001). Median N lactamica-specific IgG memory-B-cell frequencies (percentage of total IgG memory B cells) increased from 0·0024% (0·0000-0·0097) at baseline to 0·0384% (0·0275-0·0649) at day 28 (p<0·0001). The frequency of N meningitidis-specific IgA-secreting and IgG-secreting plasma cells and memory B cells also increased signficantly in participants who were colonised with N lactamica. Upper respiratory tract symptoms were reported in ten (50%) of 20 participants who were inoculated with N lactamica and six (55%) of 11 participants who were inoculated with PBS (p>0·99). Three additional adverse events (two in the N lactamica group and one in the PBS group) and no serious adverse events were reported. In the second study, anti-N lactamica and anti-N meningitidis serum IgG titres increased only in participants who were colonised with N lactamica for 14 days. INTERPRETATION: Natural 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. FUNDING: Wellcome Trust, Medical Research Council, and NIHR Southampton Biomedical Research Centre.

Controlled human infection with Neisseria lactamica in late pregnancy to measure horizontal transmission and microbiome changes in mother-neonate pairs: a single-arm interventional pilot study protocol.

INTRODUCTION: Infant upper respiratory microbiota are derived partly from the maternal respiratory tract, and certain microbiota are associated with altered risk of infections and respiratory disease. Neisseria lactamica is a common pharyngeal commensal in young children and is associated with reduced carriage and invasive disease by Neisseria meningitidis. Nasal inoculation with N. lactamica safely and reproducibly reduces N. meningitidis colonisation in healthy adults. We propose nasal inoculation of pregnant women with N. lactamica, to establish if neonatal pharyngeal colonisation occurs after birth, and to characterise microbiome evolution in mother-infant pairs over 1 month post partum. METHODS AND ANALYSIS: 20 healthy pregnant women will receive nasal inoculation with N. lactamica (wild type strain Y92-1009) at 36-38 weeks gestation. Upper respiratory samples, as well as optional breastmilk, umbilical cord blood and infant venous blood samples, will be collected from mother-infant pairs over 1 month post partum. We will assess safety, N. lactamica colonisation (by targeted PCR) and longitudinal microevolution (by whole genome sequencing), and microbiome evolution (by 16S rRNA gene sequencing). ETHICS AND DISSEMINATION: This study has been approved by the London Central Research Ethics Committee (21/PR/0373). Findings will be published in peer-reviewed open-access journals as soon as possible. TRIAL REGISTRATION NUMBER: NCT04784845.

Neisseria lactamica Controlled Human Infection Model.

Neisseria lactamica is a nonpathogenic commensal of the human upper respiratory tract that has been associated with protection against N. meningitidis colonization and disease. We have previously utilized the N. lactamica controlled human infection model to investigate the protective effect of N. lactamica colonization on N. meningitidis colonization, the nature of cross-reactive immune responses mounted toward N. meningitidis following N. lactamica colonization, and the microevolution of N. lactamica over a 5-month colonization period. More recently, we have assessed the possibility of utilizing genetically modified strains of N. lactamica to enable use of the commensal as a vehicle for prolonged exposure of the nasopharynx of humans to antigens of interest, expressed in carried organisms. A controlled infection with N. lactamica expressing the meningococcal antigen NadA has been executed and the results demonstrate that this strategy is effective at generating immune responses to the target antigen. Throughout this chapter, we outline in a step-by-step manner the methodologies utilized when performing controlled human infection with N. lactamica including procedures relating to: (1) the dilution of N. lactamica stock vials to derive intranasal inocula, (2) the delivery of intranasal inocula to human volunteers, (3) the determination of N. lactamica colonization status following intranasal inoculation using oropharyngeal swabbing and nasal wash sampling, (4) the microbiological procedures utilized to identify N. lactamica colonization among study volunteers, and (5) the identification of N. lactamica colonies as strain Y92-1009 using polymerase chain reaction.

A recombinant commensal bacteria elicits heterologous antigen-specific immune responses during pharyngeal carriage.

The human nasopharynx contains a stable microbial ecosystem of commensal and potentially pathogenic bacteria, which can elicit protective primary and secondary immune responses. Experimental intranasal infection of human adults with the commensal Neisseria lactamica produced safe, sustained pharyngeal colonization. This has potential utility as a vehicle for sustained release of antigen to the human mucosa, but commensals in general are thought to be immunologically tolerated. Here, we show that engineered N. lactamica, chromosomally transformed to express a heterologous vaccine antigen, safely induces systemic, antigen-specific immune responses during carriage in humans. When the N. lactamica expressing the meningococcal antigen Neisseria Adhesin A (NadA) was inoculated intranasally into human volunteers, all colonized participants carried the bacteria asymptomatically for at least 28 days, with most (86%) still carrying the bacteria at 90 days. Compared to an otherwise isogenic but phenotypically wild-type strain, colonization with NadA-expressing N. lactamica generated NadA-specific immunoglobulin G (IgG)- and IgA-secreting plasma cells within 14 days of colonization and NadA-specific IgG memory B cells within 28 days of colonization. NadA-specific IgG memory B cells were detected in peripheral blood of colonized participants for at least 90 days. Over the same period, there was seroconversion against NadA and generation of serum bactericidal antibody activity against a NadA-expressing meningococcus. The controlled infection was safe, and there was no transmission to adult bedroom sharers during the 90-day period. Genetically modified N. lactamica could therefore be used to generate beneficial immune responses to heterologous antigens during sustained pharyngeal carriage.

Microevolution of Neisseria lactamica during nasopharyngeal colonisation induced by controlled human infection.

Neisseria lactamica is a harmless coloniser of the infant respiratory tract, and has a mutually-excluding relationship with the pathogen Neisseria meningitidis. Here we report controlled human infection with genomically-defined N. lactamica and subsequent bacterial microevolution during 26 weeks of colonisation. We find that most mutations that occur during nasopharyngeal carriage are transient indels within repetitive tracts of putative phase-variable loci associated with host-microbe interactions (pgl and lgt) and iron acquisition (fetA promotor and hpuA). Recurrent polymorphisms occurred in genes associated with energy metabolism (nuoN, rssA) and the CRISPR-associated cas1. A gene encoding a large hypothetical protein was often mutated in 27% of the subjects. In volunteers who were naturally co-colonised with meningococci, recombination altered allelic identity in N. lactamica to resemble meningococcal alleles, including loci associated with metabolism, outer membrane proteins and immune response activators. Our results suggest that phase variable genes are often mutated during carriage-associated microevolution.

Genomes of Escherichia coli bacteraemia isolates originating from urinary tract foci contain more virulence-associated genes than those from non-urinary foci and neutropaenic hosts.

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.

Extra-intestinal pathogenic Escherichia coli (ExPEC): Disease, carriage and clones.

Extra-intestinal pathogenic Escherichia coli (ExPEC) have a complex phylogeny, broad virulence factor (VF) armament and significant genomic plasticity, and are associated with a spectrum of host infective syndromes ranging from simple urinary tract infection to life-threatening bacteraemia. Their importance as pathogens has come to the fore in recent years, particularly in the context of the global emergence of hyper-virulent and antibiotic resistant strains. Despite this, the mechanisms underlying ExPEC transmission dynamics and clonal selection remain poorly understood. Large-scale epidemiological and clinical studies are urgently required to ascertain the mechanisms underlying these processes to enable the development of novel evidence-based preventative and therapeutic strategies. In the current review, we provide a concise summary of the methods utilised for ExPEC phylogenetic delineation before exploring in detail the associations between ExPEC VFs and site-specific disease. We then consider the role of ExPEC as an intestinal colonist and outline known associations between ExPEC clonal variation, specific disease syndromes and antibiotic resistance.

Hepatic Tract Plug-Embolisation After Biliary Stenting. Is It Worthwhile?

PURPOSE: PTC and stenting procedures are associated with significant risks including life-threatening haemorrhage, sepsis, renal failure and high mortality rates. PTC tract closure methods are utilised to reduce haemorrhagic complications despite little evidence to support their use. The current study assesses the incidence of haemorrhagic complications following PTC and stenting procedures, both prior to and following the introduction of a dedicated expanding gelatin foam-targeted embolisation liver tract closure technique. MATERIALS AND METHODS: Haemorrhagic complications were retrospectively identified in patients undergoing PTC procedures both prior to (subgroup 1) and following (subgroup 2) the introduction of a dedicated targeted liver tract closure method between 9/11/2010 and 10/08/2012 in a single tertiary referral centre. Mean blood Hb decrease following PTC was established in subgroups 1 and 2. Kaplan-Meier life-table analysis was performed to compare survival outcomes between subgroups using the log-rank test. RESULTS: Haemorrhagic complications were significantly reduced following the introduction of the targeted PTC tract closure method [(12 vs. 3 % of subgroups 1 (n = 101) and 2 (n = 92), respectively (p = 0.027)]. Mean blood Hb decrease following PTC was 1.40 versus 0.68 g/dL in subgroups 1 and 2, respectively (p = 0.069). 30-day mortality was 14 and 12 % in subgroups 1 and 2, respectively. 50 % of the entire cohort had died by 174 days post-PTC. CONCLUSION: Introduction of liver tract embolisation significantly reduced haemorrhagic complications in our patient cohort. Utilisation of this method has the potential to reduce the morbidity and mortality burden associated with post-PTC haemorrhage by preventing bleeding from the liver access tract.

Nasal Inoculation of the Commensal Neisseria lactamica Inhibits Carriage of Neisseria meningitidis by Young Adults: A Controlled Human Infection Study.

BACKGROUND: Herd protection by meningococcal vaccines is conferred by population-level reduction of meningococcal nasopharyngeal colonization. Given the inverse epidemiological association between colonization by commensal Neisseria lactamica and meningococcal disease, we investigated whether controlled infection of human volunteers with N. lactamica prevents colonization by Neisseria meningitidis. METHODS: In a block-randomized human challenge study, 310 university students were inoculated with 10(4) colony-forming units of N. lactamica or were sham-inoculated, and carriage was monitored for 26 weeks, after which all participants were reinoculated with N. lactamica and resampled 2 weeks later. RESULTS: At baseline, natural N. meningitidis carriage in the control group was 22.4% (36/161), which increased to 33.6% (48/143) by week 26. Two weeks after inoculation of N. lactamica, 33.6% (48/143) of the challenge group became colonized with N. 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 of N. 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 with N. lactamica replicated the result. Genome sequencing showed that inhibition affected multiple meningococcal sequence types. CONCLUSIONS: The inhibition of meningococcal carriage by N. lactamica is even more potent than after glycoconjugate meningococcal vaccination. Neisseria lactamica or its components could be a novel bacterial medicine to suppress meningococcal outbreaks. This observation explains the epidemiological observation of natural immunity conferred by carriage of N. lactamica. CLINICAL TRIALS REGISTRATION: NCT02249598.

Novel negative pressure wound therapy with instillation and the management of diabetic foot infections.

PURPOSE OF REVIEW: The use of negative pressure wound therapy with instillation (NPWTi) in complex or difficult-to-treat acute and chronic wounds has expanded rapidly since the introduction of commercially available NPWTi systems. We summarize the evidence related to NPWTi and particularly focus on the application of this technology in diabetic foot ulcers, diabetic foot infections and postoperative diabetic wounds. RECENT FINDINGS: The benefits of negative pressure wound therapy (NPWT) are well documented in the treatment of complex acute and chronic wounds, including noninfected postoperative diabetic wounds and diabetic foot ulcers. Combining intermittent wound irrigation with NPWT may offer additional benefits compared to NPWT alone, including further reduction of wound bed bioburden, increased granulation tissue formation and provision of wound irrigation in a sealed environment, thus preventing potential cross-contamination events. Recently, available evidence suggests that adjunctive NPWTi may be superior to standard NPWT in the management of diabetic infections following surgical debridement and may promote granulation tissue formation in slow-to-heal wounds. SUMMARY: Available evidence relating to the utilization of NPWTi in diabetic foot infections is promising but limited in quality, being derived mostly from case series or small retrospective or prospective studies. In order to confirm or refute the potential benefits of NPWTi in this patient cohort, well designed randomized controlled studies are required that compare NPWTi to NPWT or standard wound care methodologies.

Percutaneous breast implant herniation: a rare complication of miliary TB.

We describe the case of a 46-year-old female patient treated for disseminated tuberculosis (TB) infection involving the lungs, urinary tract and skin. Following initiation of antituberculous therapy, the patient's right breast implant eroded through the overlying skin and was seen to be herniating through the resulting defect. The breast implant was removed under local anaesthetic and histological analysis of the resected tissue demonstrated granuloma formation consistent with periprosthetic TB. Wound healing following implant removal was poor and future breast augmentation surgery was only considered following completion of 12 months anti-TB treatment. This case constitutes the first report in the literature of percutaneous breast implant herniation resulting from periprosthetic infection with TB. A high index of suspicion is required to ensure early detection and timely management of TB and, in cases where periprosthetic pus aspirate is sterile, mycobacterial infection must be actively excluded.

Genetic susceptibility to meningococcal infection.

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.

Renal failure and leukocytosis are predictors of a complicated course of Clostridium difficile infection if measured on day of diagnosis.

Nonsevere Clostridium difficile infection (CDI) and severe CDI, which carries a higher risk than nonsevere CDI for treatment failure and CDI recurrence, are difficult to distinguish at the time of diagnosis. To investigate the prognostic value of 3 markers of severe CDI suggested by recent guidelines (fever, leukocytosis, and renal failure), we used the database of 2 randomized controlled trials, which contained information for 1105 patients with CDI. Leukocytosis (risk ratio [RR], 2.29; 95% confidence interval [CI], 1.63-3.21) and renal failure (RR, 2.52; 95% CI, 1.82-3.50) were associated with treatment failure. Fever, although associated with treatment failure (RR, 2.45; 95% CI, 1.07-5.61), was rare. Renal failure was the only significant predictor of recurrence (RR, 1.45; 95% CI, 1.05-2.02). Different timing of measurements of leukocyte count and serum creatinine level around the CDI diagnosis led to a different severity classification in many cases. In conclusion, both leukocytosis and renal failure are useful predictors, although timing of measurement is important.