AbstractPneumonia is the leading infectious disease killer worldwide and commonly requires admission to critical care. Despite its prevalence, the underpinning biology of severe pneumonia remains incompletely understood. We performed multifaceted assessments of bronchoalveolar transcriptome, cytokines, microbiology, and clinical features to biologically dissect a cohort of patients with suspected severe pneumonia. Our data revealed three lung-restricted transcriptionally defined severe pneumonia endotypes (termed ‘Pneumotypes’ (Pn)). All three Pneumotypes had comparable clinical presentations and severity of respiratory failure but critically had divergent outcomes. Pn1, the most common, was characterised by low alveolar cytokines, expanded tolerogenic macrophages and epithelial damage. Pn3 was characterised by neutrophil-monocyte infiltration, IL-6-STAT3 activation and longer duration of mechanical ventilation. Pn2 displayed the fastest resolution, exhibiting a balanced immune response and epithelial-endothelial repair signatures. Our work has identified mechanistically distinct phenotypes in the lungs of patients with suspected pneumonia and acute lung injury, providing new targets for personalised therapy.

Diarrhea is a leading cause of childhood morbidity in Africa. Outside of multi-country sentinels, of which there are none in Nigeria, few studies focus on bacterial etiology. We performed a case-control study among children under five years of age. Stool specimens were collected from 120 children with, and 357 without, diarrhea attending primary health clinics on the northern outskirts of Ibadan between November 2015 and August 2019. Up to ten E. coli isolates were obtained per specimen and at least three were whole genome-sequenced using Illumina technology. Genomes were assembled using SPAdes, quality evaluated using QUAST, and Virulencefinder was used to identify virulence genes. The microbiological quality of water from 14 wells within the study area was assessed using total and coliform counts. Diarrhoeagenic Escherichia coli (DEC) were isolated from 79 (65.8%) of cases and 217 (60.8%) control children. All DEC pathotypes except Shiga toxin-producing E. coli, a number of hybrid DEC pathotypes, Salmonella and Yersina spp. were detected but no pathogen showed association with disease (p>0.05). Enterotoxigenic E. coli were more commonly recovered from younger controls but exclusively detected in cases aged over nine months. Temporally-linked, highly similar enteroaggregative E. coli were isolated from children in different households in eight instances. No well water sample drawn in the study qualified as potable. Children in northern Ibadan are commonly colonized with DEC. Access to water and sanitation, and vaccines targeting the most abundant pathogens may be critical for protecting children from the less overt consequences of enteric pathogen carriage.

Resulting from impaired collagen turnover, fibrosis is a hallmark of adipose tissue (AT) dysfunction and obesity-associated insulin resistance (IR). Prolidase, also known as peptidase D (PEPD), plays a vital role in collagen turnover by degrading proline-containing dipeptides but its specific functional relevance in AT is unknown. Here we show that in human and mouse obesity, PEPD expression and activity decrease in AT, and PEPD is released into the systemic circulation, which promotes fibrosis and AT IR. Loss of the enzymatic function of PEPD by genetic ablation or pharmacological inhibition causes AT fibrosis in mice. In addition to its intracellular enzymatic role, secreted extracellular PEPD protein enhances macrophage and adipocyte fibro-inflammatory responses via EGFR signalling, thereby promoting AT fibrosis and IR. We further show that decreased prolidase activity is coupled with increased systemic levels of PEPD that act as a pathogenic trigger of AT fibrosis and IR. Thus, PEPD produced by macrophages might serve as a biomarker of AT fibro-inflammation and could represent a therapeutic target for AT fibrosis and obesity-associated IR and type 2 diabetes. Obesity-associated AT fibro-inflammation and metabolic disturbances are linked to PEPD activity and PEPD extracellular levels.

Background: S. Typhi “H58/4.3.1”, a single dominant MDR-lineage has emerged and spread throughout Asia and Africa over the last 30 years. A large global study speculated South Asia might be the site of original emergence of 4.3.1. Through an ongoing national-wide network of Surveillance of Enteric Fever in India (SEFI), we aim to study the genomic signatures of S. Typhi across different settings. We undertook this study to determine the genotypes of S. Typhi isolated from community (Tier-1) and Hospital (Tier-3) settings in a Southern India region. Methods and materials: A total of 193 and 200 S. Typhi isolated from Tier 1 (Jan-2017 to Sep-2019) and Tier 3 (Nov-2017 to Sep-2019) as part of the network was included. Susceptibility testing was done for most common antimicrobials. At present, whole genome sequencing was done for 30 strains each from Tier-1 and 3. SNP and comparative genomic analysis of Tier 1 and 3 was done to infer difference in the genomic signatures. Geospatial mapping was generated for Tier 1 strains for cluster analysis to infer transmission-dynamic events. Results: Susceptibility rates to first-line agents were highest among community strains with no MDR, whereas 8 MDR strains were isolated from hospital cases. Ciprofloxacin resistance was seen in 5% (9/193) of community isolates compared to the high rates 32% (63/200) among hospital isolates. Notably, genomic analysis showed the dominance of a single H8-lineage 4.3.1.2 (30/30) in community isolates. However, hospital isolates had broader distribution such as 4.3.1.2 (18), 4.3.1.1 (7), 4.3.1.3 (1) and 2.2 and 3.3 respectively. Within the community collection, two sub groups with respect to QRDR profile with S83Y (SG-I) and S83F (SG-II) were found. Interestingly, all SG-II (17/30) appeared to be a single strain with no SNPs differences, being circulated in a defined community setting over 3 months. Conclusion: The baseline data generated here provides multiple insights in terms of a single clade 4.3.1.2 dominating in a community setting, while diverse genotypes seen among typhoid cases attending a tertiary care hospital. However, phylo-geographical analysis of all the isolates would provide a clear picture of the ongoing transmission dynamics between two settings.

[This corrects the article DOI: 10.1371/journal.ppat.1002776.].

26 Recognition of Influenza A virus (IAV) by the innate immune system triggers pathways that 27 restrict viral replication, activates innate immune cells, and regulates adaptive immunity. 28 However, excessive innate immune activation can exaggerate disease. The pathways 29 promoting excessive activation are incompletely understood, with limited experimental 30 models to investigate mechanisms driving influenza-induced inflammation in humans. 31 Interferon regulatory factor (IRF5) is a transcription factor that plays important roles in 32 induction of cytokines after viral sensing. In an in vivo model of IAV infection, IRF5 33 deficiency reduced IAV-driven immune pathology and associated inflammatory cytokine 34 production, specifically reducing cytokine-producing myeloid cell populations in Irf5 -/- mice, 35 but not impacting type 1 IFN production or virus replication. Using cytometry by time-of- 36 flight (CyTOF), we identified that human lung IRF5 expression was highest in cells of the 37 myeloid lineage. To investigate the role of IRF5 in mediating human inflammatory responses 38 by myeloid cells to IAV, we employed human induced pluripotent stem cells (hIPSCs) with 39 biallelic mutations in IRF5 , demonstrating for the first time iPS-derived dendritic cells (iPS- 40 DCs) with biallelic mutations can be used to investigate regulation of human virus-induced 41 immune responses. Using this technology, we reveal that IRF5 deficiency in human DCs, or 42 macrophages, corresponded with reduced virus-induced inflammatory cytokine production, 43 with IRF5 acting downstream of TLR7 and, possibly, RIG-I after viral sensing. Thus, IRF5 44 acts as a regulator of myeloid cell inflammatory cytokine production during IAV infection in 45 mice and humans, and drives immune-mediated viral pathogenesis independently of type 1 46 IFN and virus replication. 47 The inflammatory response to Influenza A virus (IAV) participates in infection control but 52 contributes to disease severity. After viral detection intracellular pathways are 53 activated, initiating cytokine production, but these pathways are incompletely 54 understood. We show that interferon regulatory factor 5 (IRF5) mediates IAV-induced 55 inflammation and, in mice, drives pathology. This was independent of antiviral type 1 IFN 56 and virus replication, implying that IRF5 could be specifically targeted to treat influenza- 57 induced inflammation. We show for the first time that human iPSC technology can be 58 exploited in genetic studies of virus-induced immune responses. Using this technology, we 59 deleted IRF5 in human myeloid cells. These IRF5-deficient cells exhibited impaired 60 influenza-induced cytokine production and revealed that IRF5 acts downstream of Toll-like 61 receptor 7 and possibly retinoic acid-inducible gene-I. Our data demonstrate the importance 62 of IRF5 in influenza-induced inflammation, suggesting genetic variation in the IRF5 gene 63 may influence host susceptibility to viral diseases.

Cryptococcus neoformans ( C. neoformans var. grubii ) is an environmentally acquired pathogen causing 181,000 HIV-associated deaths each year. We sequenced 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. The phylogeny of C. neoformans reveals a recent exponential population expansion, consistent with the increase in the number of susceptible hosts. In our study population, this expansion has been driven by three sub-clades of the C. neoformans VNIa lineage; VNIa-4, VNIa-5 and VNIa-93. These three sub-clades account for 91% of clinical isolates sequenced in our study. Combining the genome data with clinical information, we find that the VNIa-93 sub-clade, the most common sub-clade in Uganda and Malawi, was associated with better outcomes than VNIa-4 and VNIa-5, which predominate in Southeast Asia. This study lays the foundation for further work investigating the dominance of VNIa-4, VNIa-5 and VNIa-93 and the association between lineage and clinical phenotype.

ABSTRACT Nontyphoidal Salmonella (NTS), particularly Salmonella enterica serovar Typhimurium, is among the leading etiologic agents of bacterial enterocolitis globally and a well-characterized cause of invasive disease (iNTS) in sub-Saharan Africa. In contrast, S. Typhimurium is poorly defined in Southeast Asia, a known hot spot for zoonotic disease with a recently described burden of iNTS disease. Here, we aimed to add insight into the epidemiology and potential impact of zoonotic transfer and antimicrobial resistance (AMR) in S. Typhimurium associated with iNTS and enterocolitis in Vietnam. We performed whole-genome sequencing and phylogenetic reconstruction on 85 human (enterocolitis, carriage, and iNTS) and 113 animal S. Typhimurium isolates isolated in Vietnam. We found limited evidence for the zoonotic transmission of S. Typhimurium. However, we describe a chain of events where a pandemic monophasic variant of S. Typhimurium (serovar I: 4,[5], 12: i: sequence type 34 [ST34]) has been introduced into Vietnam, reacquired a phase 2 flagellum, and acquired an IncHI2 multidrug-resistant plasmid. Notably, these novel biphasic ST34 S. Typhimurium variants were significantly associated with iNTS in Vietnamese HIV-infected patients. Our study represents the first characterization of novel iNTS organisms isolated outside sub-Saharan Africa and outlines a new pathway for the emergence of alternative Salmonella variants into susceptible human populations.

Infections with carbapenemase-producing Enterobacteriaceae (CPE) and vancomycin-resistant enterococci (VRE) are associated with increased morbidity and mortality, but the carriage rates of CRE and VRE among hospital inpatients are unknown. A point-prevalence survey was conducted to determine CPE and VRE carriage rates in hospitalized adults. Eight hundred and eighteen of 960 (85.2%) adult inpatients were invited to participate in the study. Of these, 595 patients (72.7%) consented and provided specimens. Of 540 samples tested, none were positive for CPE. One hundred and thirty of 540 (24.1%) samples were VRE positive, and 34 of 40 (85%) of wards had cases. Universal screening for CPE may not be cost-effective in low-prevalence settings, but targeted screening of high-risk patients should continue. The optimal screening strategy for VRE remains to be determined, as universal screening and isolation is not feasible in the study setting.

AbstractC. neoformansvar.grubii(C. neoformans) is an environmentally acquired pathogen causing 181 000 HIV-associated deaths each year. We used whole genome sequencing (WGS) to characterise 699 isolates, primarilyC. neoformansfrom HIV-infected patients, from 5 countries in Asia and Africa. We found that 91% of our clinical isolates belonged to one of three highly clonal sub-clades of VNIa, which we have termed VNIa-4, VNIa-5 and VNIa-93. Parsimony analysis revealed frequent, long distance transmissions ofC. neoformans; international transmissions took place on 13% of VNIa-4 branches, and intercontinental transmissions on 7% of VNIa-93 branches. The median length of within sub-clade internal branches was 3-6 SNPs, while terminal branches were 44.5-77.5 SNPs. The short median internal branches were partly driven by the large number (12-15% of internal branches) of polytomies in the within-sub-clade trees. To simultaneously explain our observation of no apparent molecular clock, short internal branches and frequent polytomies we hypothesise thatC. neoformansVNIa spends much of its time in the environment in a quiescent state, while, when it is sampled, it has almost always undergone an extended period of growth. Infections with VNIa-93 were associated with a significantly reduced risk of death by 10 weeks compared with infections with VNIa-4 (Hazard Ratio = 0.45, p = 0.003). We detected a recombination in the mitochondrial sequence of VNIa-5, suggesting that mitochondria could be involved in the propensity of this sub-clade to infect HIV-uninfected patients. These data highlight the insight into the biology and epidemiology of pathogenic fungi which can be gained from WGS data.

C. neoformans var. grubii ( C. neoformans ) is an environmentally acquired pathogen causing 181 000 HIV-associated deaths each year. We used whole genome sequencing (WGS) to characterise 699 isolates, primarily C. neoformans from HIV-infected patients, from 5 countries in Asia and Africa. We found that 91% of our clinical isolates belonged to one of three highly clonal sub-clades of VNIa, which we have termed VNIa-4, VNIa-5 and VNIa-93. Parsimony analysis revealed frequent, long distance transmissions of C. neoformans ; international transmissions took place on 13% of VNIa-4 branches, and intercontinental transmissions on 7% of VNIa-93 branches. The median length of within sub-clade internal branches was 3-6 SNPs, while terminal branches were 44.5-77.5 SNPs. The short median internal branches were partly driven by the large number (12-15% of internal branches) of polytomies in the within-sub-clade trees. To simultaneously explain our observation of no apparent molecular clock, short internal branches and frequent polytomies we hypothesise that C. neoformans VNIa spends much of its time in the environment in a quiescent state, while, when it is sampled, it has almost always undergone an extended period of growth. Infections with VNIa-93 were associated with a significantly reduced risk of death by 10 weeks compared with infections with VNIa-4 (Hazard Ratio = 0.45, p = 0.003). We detected a recombination in the mitochondrial sequence of VNIa-5, suggesting that mitochondria could be involved in the propensity of this sub-clade to infect HIV-uninfected patients. These data highlight the insight into the biology and epidemiology of pathogenic fungi which can be gained from WGS data.

NK cells were found to be recruited in a temporally controlled manner to the nasal-associated lymphoid tissue and the cervical lymph nodes of mice after intranasal immunization with Ag85B-early secreted antigenic target 6kDa from Mycobacterium tuberculosis mixed with Escherichia coli heat-labile toxin as adjuvant. These NK cells were activated and secreted a diverse range of cytokines and other immunomodulators. Using Ab depletion targeting anti-asialo GM1, we found evidence for altered trafficking, impaired activation, and cytokine secretion of dendritic cells, macrophages, and neutrophils in immunized NK cell-depleted mice compared with control animals. Analysis of Ag-specific immune responses revealed an attenuated Ab and cytokine response in immunized NK cell-depleted animals. Systemic administration of rIL-6 but not rIFN-γ significantly restored immune responses in mice depleted of NK cells. In conclusion, cytokine production, particularly IL-6, via NK cells and NK cell-activated immune populations plays an important role in the establishment of local innate immune responses and the consequent development of adaptive immunity after mucosal immunization.

Avian pathogenic Escherichia coli (APEC) causes respiratory and systemic disease in poultry. Sequencing of a multilocus sequence type 95 (ST95) serogroup O1 strain previously indicated that APEC resembles E. coli causing extraintestinal human dis-eases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains (cid:1) 7122 and IMT2125) and compared them to each other and to the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to human ST23 ETEC than to APEC O1, indicating that separation of pathotypes on the basis of their extraintestinal or diarrheagenic nature is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2,185 signature-tagged transposon mutants of (cid:1) 7122 following intra-air sac inoculation of turkeys. This procedure identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for the virulence of (cid:1) 7122 and were conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli strains adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for the evolution of E. coli strains adapted to cause avian or human disease via acquisition of distinct virulence genes. Statisticalanalysis. Bacterialcountsinthelung,liver,andspleenwere analyzedfortheeffectofmutationbymeansofaone-wayF-testafterlog 10 transformation of the data using GraphPad Prism, version 5.04 (Graph-Pad Software, Inc., La Jolla, CA). Subsequent pairwise comparisons with the wild type were performed, and P values of (cid:1) 0.05 were taken to be significant.

Salmonella enterica 19 respectively. A unique feature of typhoid infection is asymptomatic carriage within the 20 gallbladder, which is linked with S . Typhi transmission. Despite this, S . Typhi responses to 21 bile have been poorly studied. RNA-Seq of S . Typhi Ty2 and a clinical S . Typhi isolate 22 belonging to the globally dominant H58 lineage (129-0238), as well as S . Typhimurium 23 14028, revealed that 249, 389 and 453 genes respectively were differentially expressed in the 24 presence of 3% bile compared to control cultures lacking bile. fad genes, the actP-acs 25 operon, and putative sialic acid uptake and metabolism genes (t1787-t1790) were upregulated 26 in all strains following bile exposure, which may represent adaptation to the small intestine 27 environment. Genes within the Salmonella pathogenicity island 1 (SPI-1), encoding a type 28 IIII secretion system (T3SS), and motility genes were significantly upregulated in both S . 29 Typhi strains in bile, but downregulated in S . Typhimurium. Western blots of the SPI-1 30 proteins SipC, SipD, SopB and SopE validated the gene expression data. Consistent with this, 31 bile significantly increased S . Typhi HeLa cell invasion whilst S . Typhimurium invasion was 32 significantly repressed. Protein stability assays demonstrated that in S . Typhi the half-life of 33 HilD, the dominant regulator of SPI-1, is three times longer in the presence of bile; this 34 increase in stability was independent of the acetyltransferase Pat. Overall, we found that S . 35 Typhi exhibits a specific response to bile, especially with regards to virulence gene 36 expression, which could impact pathogenesis and transmission. aim this study to compare global bile responses between S. Typhi and S . Typhimurium isolates, which might explain differences in pathogenesis and reveal 93 processes for the carrier state.

Antimicrobial resistance (AMR) is a global public health threat. Emergence of AMR occurs naturally, but can also be selected for by antimicrobial exposure in clinical and veterinary medicine. Despite growing worldwide attention to AMR, there are substantial limitations in our understanding of the burden, distribution and determinants of AMR at the population level. We highlight the importance of population-based approaches to assess the association between antimicrobial use and AMR in humans and animals. Such approaches are needed to improve our understanding of the development and spread of AMR in order to inform strategies for the prevention, detection and management of AMR, and to support the sustainable use of antimicrobials in healthcare.