De novo mutations mediate phenotypic switching in an opportunistic human lung pathogen.

Bacteria evolving within human hosts encounter selective tradeoffs that render mutations adaptive in one context and deleterious in another. Here, we report that the cystic fibrosis-associated pathogen Burkholderia dolosa overcomes in-human selective tradeoffs by acquiring successive point mutations that alternate phenotypes. We sequenced the whole genomes of 931 respiratory isolates from two recently infected patients and an epidemiologically-linked, chronically-infected patient. These isolates are contextualized using 112 historical genomes from the same outbreak strain. Within both newly infected patients, diverse parallel mutations that disrupt O-antigen expression quickly arose, comprising 29% and 63% of their B. dolosa communities by 3 years. The selection for loss of O-antigen starkly contrasts with our previous observation of parallel O-antigen-restoring mutations after many years of chronic infection in the historical outbreak. Experimental characterization revealed that O-antigen loss increases uptake in immune cells while decreasing competitiveness in the mouse lung. We propose that the balance of these pressures, and thus whether O-antigen expression is advantageous, depends on tissue localization and infection duration. These results suggest that mutation-driven alternation during infection may be more frequent than appreciated and is underestimated without dense temporal sampling.

The Challenges of Vaccine Trial Participation among Underserved and Hard-to-Reach Communities: An Internal Expert Consultation of the VACCELERATE Consortium.

Underserved and hard-to-reach population groups are under-represented in vaccine trials. Thus, we aimed to identify the challenges of vaccine trial participation of these groups in member countries of the VACCELERATE network. Seventeen National Coordinators (NC), each representing their respective country (15 European countries, Israel, and Turkey), completed an online survey. From 15 eligible groups, those that were more frequently declared underserved/hard-to-reach in vaccine research were ethnic minorities (76.5%), persons experiencing homelessness (70.6%), illegal workers and refugees (64.7%, each). When prioritization for education on vaccine trials was considered, ethnic groups, migrants, and immigrants (5/17, 29.4%) were the groups most frequently identified by the NC as top targets. The most prominent barriers in vaccine trial participation affecting all groups were low levels of health literacy, reluctance to participate in trials due to engagement level, and low levels of trust in vaccines/vaccinations. This study highlighted population groups considered underserved/hard-to-reach in countries contained within the European region, and the respective barriers these groups face when participating in clinical studies. Our findings aid with the design of tailored interventions (within-and across-countries of the European region) and with the development of strategies to overcome major barriers in phase 2 and phase 3 vaccine trial participation.

A urokinase-associated outbreak of Ralstonia mannitolilytica bloodstream infections in haemodialysis patients in north-eastern Italy, January to April 2023.

An outbreak of Ralstonia mannitolilytica bloodstream infections occurred in four hospitals in north-eastern Italy, involving 20 haemodialysis patients with tunnelled central vascular catheter access. We identified as the outbreak source a batch of urokinase vials imported from India contaminated with R. mannitolilytica. Whole genome sequences of the clinical and urokinase strains were highly related, and only urokinase-treated patients were reported with R. mannitolilytica infections (attack rate = 34%; 95% confidence interval: 22.1-47.4). Discontinuation of the contaminated urokinase terminated the outbreak.

VACCELERATE Site Network: Real-time definition of clinical study capacity in Europe.

BACKGROUND: The inconsistent European vaccine trial landscape rendered the continent of limited interest for vaccine developers. The VACCELERATE consortium created a network of capable clinical trial sites throughout Europe. VACCELERATE identifies and provides access to state-of-the-art vaccine trial sites to accelerate clinical development of vaccines. METHODS: Login details for the VACCELERATE Site Network (vaccelerate.eu/site-network/) questionnaire can be obtained after sending an email to. Interested sites provide basic information, such as contact details, affiliation with infectious disease networks, main area of expertise, previous vaccine trial experience, site infrastructure and preferred vaccine trial settings. In addition, sites can recommend other clinical researchers for registration in the network. If directly requested by a sponsor or sponsor representative, the VACCELERATE Site Network pre-selects vaccine trial sites and shares basic study characteristics provided by the sponsor. Interested sites provide feedback with short surveys and feasibility questionnaires developed by VACCELERATE and are connected with the sponsor to initiate the site selection process. RESULTS: As of April 2023, 481 sites from 39 European countries have registered in the VACCELERATE Site Network. Of these, 137 (28.5 %) sites have previous experience conducting phase I trials, 259 (53.8 %) with phase II, 340 (70.7 %) with phase III, and 205 (42.6 %) with phase IV trials, respectively. Infectious diseases were reported as main area of expertise by 274 sites (57.0 %), followed by any kind of immunosuppression by 141 (29.3 %) sites. Numbers are super additive as sites may report clinical trial experience in several indications. Two hundred and thirty-one (47.0 %) sites have the expertise and capacity to enrol paediatric populations and 391 (79.6 %) adult populations. Since its launch in October 2020, the VACCELERATE Site Network has been used 21 times for academic and industry trials, mostly interventional studies, focusing on different pathogens such as fungi, monkeypox virus, Orthomyxoviridae/influenza viruses, SARS-CoV-2, or Streptococcus pneumoniae/pneumococcus. CONCLUSIONS: The VACCELERATE Site Network enables a constantly updated Europe-wide mapping of experienced clinical sites interested in executing vaccine trials. The network is already in use as a rapid-turnaround single contact point for the identification of vaccine trials sites in Europe.

Diagnosis of Imported Dengue and Zika Virus Infections in Italy from November 2015 to November 2022: Laboratory Surveillance Data from a National Reference Laboratory.

Dengue (DENV) and Zika (ZIKV) viruses are mosquito-borne human pathogens. In Italy, the presence of the competent vector Aedes albopictus increases the risk of autochthonous transmission, and a national plan for arboviruses prevention, surveillance, and response (PNA 2020-2025) is in place. The results of laboratory diagnosis of both viruses by the National Reference Laboratory for arboviruses (NRLA) from November 2015 to November 2022 are presented. Samples from 655 suspected cases were tested by both molecular and serological assays. Virus and antibody kinetics, cross-reactivity, and diagnostic performance of IgM ELISA systems were analysed. Of 524 cases tested for DENV, 146 were classified as confirmed, 7 as probable, while 371 were excluded. Of 619 cases tested for ZIKV, 44 were classified as confirmed, while 492 were excluded. All cases were imported. Overall, 75.3% (110/146) of DENV and 50% (22/44) of ZIKV cases were confirmed through direct virus detection methods. High percentages of cross reactivity were observed between the two viruses. The median lag time from symptoms onset to sample collection was 7 days for both DENV molecular (range 0-20) and NS1 ELISA (range 0-48) tests, with high percentages of positivity also after 7 days (39% and 67%, respectively). For ZIKV, the median lag time was 5 days (range 0-22), with 16% positivity after 7 days. Diagnostic performance was assessed with negative predictive values ranging from 92% to 95% for the anti-DENV systems, and of 97% for the ZIKV one. Lower positive predictive values were seen in the tested population (DENV: 55% to 91%, ZIKV: 50%). DENV and ZIKV diagnosis by molecular test is the gold standard, but sample collection time is a limitation. Serological tests, including Plaque Reduction Neutralization Test, are thus necessary. Co-circulation and cross-reactivity between the two viruses increase diagnostic difficulty. Continuous evaluation of diagnostic strategies is essential to improve laboratory testing.

Multicomponent Pseudomonas aeruginosa Vaccines Eliciting Th17 Cells and Functional Antibody Responses Confer Enhanced Protection against Experimental Acute Pneumonia in Mice.

The Gram-negative pathogen Pseudomonas aeruginosa is a common cause of pneumonia in hospitalized patients. Its increasing antibiotic resistance and widespread occurrence present a pressing need for vaccines. We previously showed that a P. aeruginosa type III secretion system protein, PopB, elicits a strong Th17 response in mice after intranasal (IN) immunization and confers antibody-independent protection against pneumonia in mice. In the current study, we evaluated the immunogenicity and protective efficacy in mice of the combination of PopB (purified with its chaperone protein PcrH) and OprF/I, an outer membrane hybrid fusion protein, compared with immunization with the proteins individually either by the intranasal (IN) or subcutaneous (SC) routes. Our results show that after vaccination, a Th17 recall response from splenocytes was detected only in mice vaccinated with PopB/PcrH, either alone or in combination with OprF/I. Mice immunized with the combination of PopB/PcrH and OprF/I had enhanced protection in an acute lethal P. aeruginosa pneumonia model, regardless of vaccine route, compared with mice vaccinated with either alone or adjuvant control. Immunization generated IgG titers against the vaccine proteins and whole P. aeruginosa cells. Interestingly, none of these antisera had opsonophagocytic killing activity, but antisera from mice immunized with vaccines containing OprF/I, had the ability to block IFN-γ binding to OprF/I, a known virulence mechanism. Hence, vaccines combining PopB/PcrH with OprF/I that elicit functional antibodies lead to a broadly and potently protective vaccine against P. aeruginosa pulmonary infections.

VACCELERATE Volunteer Registry: A European study participant database to facilitate clinical trial enrolment.

INTRODUCTION: The coronavirus disease 2019 (COVID-19) pandemic has evidenced the key role of vaccine design, obtention, production and administration to successfully fight against infectious diseases and to provide efficient remedies for the citizens. Although clinical trials were rapidly established during this pandemic, identifying suitable study subjects can be challenging. For this reason, the University Hospital Cologne established a volunteer registry for participation in clinical trials first in Germany, which has now been incorporated into the European VACCELERATE clinical trials network and grew to a European Volunteer Registry. As such, VACCELERATE's Volunteer Registry aims to become a common entry point for potential volunteers in future clinical trials in Europe. METHODS: Interested volunteers who would like to register for clinical trials in the VACCELERATE Volunteer Registry can access the registration questionnaire via http://www.vaccelerate.eu/volunteer-registry. Potential volunteers are requested to provide their current country and area of residence, contact information, including first and last name and e-mail address, age, gender, comorbidities, previous SARS-CoV-2 infection and vaccination status, and maximum distance willing to travel to a clinical trial site. The registry is open to both adults and children, complying with national legal consent requirements. RESULTS: As of May 2022, the questionnaire is available in 12 countries and 14 languages. Up to date, more than 36,000 volunteers have registered, mainly from Germany. Within the first year since its establishment, the VACCELERATE Volunteer Registry has matched more than 15,000 volunteers to clinical trials. The VACCELERATE Volunteer Registry will be launched in further European countries in the coming months. CONCLUSIONS: The VACCELERATE Volunteer Registry is an active single-entry point for European residents interested in COVID-19 clinical trials participation in 12 countries (i.e., Austria, Cyprus, Germany, Greece, Ireland, Lithuania, Norway, Portugal, Spain, Sweden and Turkey). To date, more than 15,000 registered individuals have been connected to clinical trials in Germany alone. The registry is currently in the implementation phase in 5 additional countries (i.e., Belgium, Czech Republic, Hungary, Israel and the Netherlands).

Rapid expansion and extinction of antibiotic resistance mutations during treatment of acute bacterial respiratory infections.

Acute bacterial infections are often treated empirically, with the choice of antibiotic therapy updated during treatment. The effects of such rapid antibiotic switching on the evolution of antibiotic resistance in individual patients are poorly understood. Here we find that low-frequency antibiotic resistance mutations emerge, contract, and even go to extinction within days of changes in therapy. We analyzed Pseudomonas aeruginosa populations in sputum samples collected serially from 7 mechanically ventilated patients at the onset of respiratory infection. Combining short- and long-read sequencing and resistance phenotyping of 420 isolates revealed that while new infections are near-clonal, reflecting a recent colonization bottleneck, resistance mutations could emerge at low frequencies within days of therapy. We then measured the in vivo frequencies of select resistance mutations in intact sputum samples with resistance-targeted deep amplicon sequencing (RETRA-Seq), which revealed that rare resistance mutations not detected by clinically used culture-based methods can increase by nearly 40-fold over 5-12 days in response to antibiotic changes. Conversely, mutations conferring resistance to antibiotics not administered diminish and even go to extinction. Our results underscore how therapy choice shapes the dynamics of low-frequency resistance mutations at short time scales, and the findings provide a possibility for driving resistance mutations to extinction during early stages of infection by designing patient-specific antibiotic cycling strategies informed by deep genomic surveillance.

A Pseudomonas aeruginosa-Derived Particulate Vaccine Protects against P. aeruginosa Infection.

Despite numerous efforts to develop an effective vaccine against Pseudomonas aeruginosa, no vaccine has yet been approved for human use. This study investigates the utility of the P. aeruginosa inherently produced polyhydroxyalkanaote (PHA) inclusions and associated host-cell proteins (HCP) as a particulate vaccine platform. We further engineered PHA inclusions to display epitopes derived from the outer membrane proteins OprF/OprI/AlgE (Ag) or the type III secretion system translocator PopB. PHA and engineered PHA beads induced antigen-specific humoral, cell-mediated immune responses, anti-HCP and anti-polysaccharide Psl responses in mice. Antibodies mediated opsonophagocytic killing and serotype-independent protective immunity as shown by 100% survival upon challenge with P. aeruginosa in an acute pneumonia murine model. Vaccines were stable at 4 °C for at least one year. Overall, our data suggest that vaccination with subcellular empty PHA beads was sufficient to elicit multiple immune effectors that can prevent P. aeruginosa infection.

Molecular Analysis of the CYP11B2 Gene in 62 Patients with Hypoaldosteronism Due to Aldosterone Synthase Deficiency.

CONTEXT: Isolated congenital hypoaldosteronism presents in early infancy with symptoms including vomiting, severe dehydration, salt wasting, and failure to thrive. The main causes of this rare autosomal recessive disorder is pathogenic variants of the CYP11B2 gene leading to aldosterone synthase deficiency. OBJECTIVE: To investigate the presence of CYP11B2 pathogenic variants in a cohort of patients with a clinical, biochemical, and hormonal profile suggestive of aldosterone synthase deficiency. DESIGN: Clinical and molecular study. SETTING: Tertiary academic Children's Hospital, Center for Rare Pediatric Endocrine Diseases. PATIENTS AND METHODS: Sixty-two patients (56 unrelated patients and 6 siblings), with hypoaldosteronism and their parents, underwent CYP11B2 gene sequencing after its selective amplification against the highly homologous CYP11B1 gene. In silico analysis of the identified novel variants was carried out to evaluate protein stability and potential pathogenicity. RESULTS: CYP11B2 gene sequencing revealed that 62 patients carried a total of 12 different pathogenic CYP11B2 gene variants, 6 of which are novel. Importantly, 96% of the 56 patients carried the previously reported p.T185I variant either in homozygosity or in compound heterozygosity with another variant. The 6 novel variants detected were: p.M1I, p.V129M, p.R141Q, p.A165T, p.R448C, and the donor splice site variant of intron 8, c.1398 + 1G > A. CONCLUSION: Molecular diagnosis was achieved in 62 patients with aldosterone synthase deficiency, the largest cohort thus far reported. Six novel genetic variants were identified as possibly pathogenic, extending the spectrum of reported molecular defects of the CYP11B2 gene.

Genomic and epidemiological evidence of bacterial transmission from probiotic capsule to blood in ICU patients.

Probiotics are routinely administered to hospitalized patients for many potential indications1 but have been associated with adverse effects that may outweigh their potential benefits2-7. It is particularly alarming that probiotic strains can cause bacteremia8,9, yet direct evidence for an ancestral link between blood isolates and administered probiotics is lacking. Here we report a markedly higher risk of Lactobacillus bacteremia for intensive care unit (ICU) patients treated with probiotics compared to those not treated, and provide genomics data that support the idea of direct clonal transmission of probiotics to the bloodstream. Whole-genome-based phylogeny showed that Lactobacilli isolated from treated patients' blood were phylogenetically inseparable from Lactobacilli isolated from the associated probiotic product. Indeed, the minute genetic diversity among the blood isolates mostly mirrored pre-existing genetic heterogeneity found in the probiotic product. Some blood isolates also contained de novo mutations, including a non-synonymous SNP conferring antibiotic resistance in one patient. Our findings support that probiotic strains can directly cause bacteremia and adaptively evolve within ICU patients.

Progress Toward the Elusive Pseudomonas aeruginosa Vaccine.

Background: The gram-negative bacterial pathogen Pseudomonas aeruginosa causes a wide range of infections, mostly in hospitalized and immunocompromised patients, those with burns, surgical wounds, or combat-related wounds, and in people with cystic fibrosis. The increasing antibiotic resistance of P. aeruginosa confers a pressing need for vaccines, yet there are no P. aeruginosa vaccines approved for human use, and recent promising candidates have failed in large clinical trials. Discussion: In this review, we summarize recent clinical trials and pre-clinical studies of P. aeruginosa vaccines and provide a suggested framework for the makeup of a future successful vaccine. Murine models of infection suggest that antibodies, specifically opsonophagocytic killing antibodies (OPK), antitoxin antibodies, and anti-attachment antibodies, combined with T cell immunity, specifically TH17 responses, are needed for broad and potent protection against P. aeruginosa infection. A better understanding of the human immune response to P. aeruginosa infections, and to vaccine candidates, will eventually pave the way to a successful vaccine for this wily pathogen.

The adherens junctions control susceptibility to Staphylococcus aureus α-toxin.

Staphylococcus aureus is both a transient skin colonizer and a formidable human pathogen, ranking among the leading causes of skin and soft tissue infections as well as severe pneumonia. The secreted bacterial α-toxin is essential for S. aureus virulence in these epithelial diseases. To discover host cellular factors required for α-toxin cytotoxicity, we conducted a genetic screen using mutagenized haploid human cells. Our screen identified a cytoplasmic member of the adherens junctions, plekstrin-homology domain containing protein 7 (PLEKHA7), as the second most significantly enriched gene after the known α-toxin receptor, a disintegrin and metalloprotease 10 (ADAM10). Here we report a new, unexpected role for PLEKHA7 and several components of cellular adherens junctions in controlling susceptibility to S. aureus α-toxin. We find that despite being injured by α-toxin pore formation, PLEKHA7 knockout cells recover after intoxication. By infecting PLEKHA7(-/-) mice with methicillin-resistant S. aureus USA300 LAC strain, we demonstrate that this junctional protein controls disease severity in both skin infection and lethal S. aureus pneumonia. Our results suggest that adherens junctions actively control cellular responses to a potent pore-forming bacterial toxin and identify PLEKHA7 as a potential nonessential host target to reduce S. aureus virulence during epithelial infections.

Staphylococcal Esx proteins modulate apoptosis and release of intracellular Staphylococcus aureus during infection in epithelial cells.

The opportunistic pathogen Staphylococcus aureus is one of the major causes of health care-associated infections. S. aureus is primarily an extracellular pathogen, but it was recently reported to invade and replicate in several host cell types. The ability of S. aureus to persist within cells has been implicated in resistance to antimicrobials and recurrent infections. However, few staphylococcal proteins that mediate intracellular survival have been identified. Here we examine if EsxA and EsxB, substrates of the ESAT-6-like secretion system (Ess), are important during intracellular S. aureus infection. The Esx proteins are required for staphylococcal virulence, but their functions during infection are unclear. While isogenic S. aureus esxA and esxB mutants were not defective for epithelial cell invasion in vitro, a significant increase in early/late apoptosis was observed in esxA mutant-infected cells compared to wild-type-infected cells. Impeding secretion of EsxA by deleting C-terminal residues of the protein also resulted in a significant increase of epithelial cell apoptosis. Furthermore, cells transfected with esxA showed an increased protection from apoptotic cell death. A double mutant lacking both EsxA and EsxB also induced increased apoptosis but, remarkably, was unable to escape from cells as efficiently as the single mutants or the wild type. Thus, using in vitro models of intracellular staphylococcal infection, we demonstrate that EsxA interferes with host cell apoptotic pathways and, together with EsxB, mediates the release of S. aureus from the host cell.

A novel point mutation in the KCNJ5 gene causing primary hyperaldosteronism and early-onset autosomal dominant hypertension.

CONTEXT: Aldosterone production in the adrenal zona glomerulosa is mainly regulated by angiotensin II, [K(+)], and ACTH. Genetic deletion of subunits of K(+)-selective leak (KCNK) channels TWIK-related acid sensitive K(+)-1 and/or TWIK-related acid sensitive K(+)-3 in mice results in primary hyperaldosteronism, whereas mutations in the KCNJ5 (potassium inwardly rectifying channel, subfamily J, member 5) gene are implicated in primary hyperaldosteronism and, in certain cases, in autonomous glomerulosa cell proliferation in humans. OBJECTIVE: The objective of the study was to investigate the role of KCNK3, KCNK5, KCNK9, and KCNJ5 genes in a family with primary hyperaldosteronism and early-onset hypertension. PATIENTS AND METHODS: Two patients, a mother and a daughter, presented with severe primary hyperaldosteronism, bilateral massive adrenal hyperplasia, and early-onset hypertension refractory to medical treatment. Genomic DNA was isolated and the exons of the entire coding regions of the above genes were amplified and sequenced. Electrophysiological studies were performed to determine the effect of identified mutation(s) on the membrane reversal potentials. RESULTS: Sequencing of the KCNJ5 gene revealed a single, heterozygous guanine to thymine (G → T) substitution at nucleotide position 470 (n.G470T), resulting in isoleucine (I) to serine (S) substitution at amino acid 157 (p.I157S). This mutation results in loss of ion selectivity, cell membrane depolarization, increased Ca(2+) entry in adrenal glomerulosa cells, and increased aldosterone synthesis. Sequencing of the KCNK3, KCNK5, and KCNK9 genes revealed no mutations in our patients. CONCLUSIONS: These findings explain the pathogenesis in a subset of patients with severe hypertension and implicate loss of K(+) channel selectivity in constitutive aldosterone production.