Evolutionary and functional history of the Escherichia coli K1 capsule.

Escherichia coli is a leading cause of invasive bacterial infections in humans. Capsule polysaccharide has an important role in bacterial pathogenesis, and the K1 capsule has been firmly established as one of the most potent capsule types in E. coli through its association with severe infections. However, little is known about its distribution, evolution and functions across the E. coli phylogeny, which is fundamental to elucidating its role in the expansion of successful lineages. Using systematic surveys of invasive E. coli isolates, we show that the K1-cps locus is present in a quarter of bloodstream infection isolates and has emerged in at least four different extraintestinal pathogenic E. coli (ExPEC) phylogroups independently in the last 500 years. Phenotypic assessment demonstrates that K1 capsule synthesis enhances E. coli survival in human serum independent of genetic background, and that therapeutic targeting of the K1 capsule re-sensitizes E. coli from distinct genetic backgrounds to human serum. Our study highlights that assessing the evolutionary and functional properties of bacterial virulence factors at population levels is important to better monitor and predict the emergence of virulent clones, and to also inform therapies and preventive medicine to effectively control bacterial infections whilst significantly lowering antibiotic usage.

Human CEACAM1 is targeted by a Streptococcus pyogenes adhesin implicated in puerperal sepsis pathogenesis.

Life-threatening bacterial infections in women after childbirth, known as puerperal sepsis, resulted in classical epidemics and remain a global health problem. While outbreaks of puerperal sepsis have been ascribed to Streptococcus pyogenes, little is known about disease mechanisms. Here, we show that the bacterial R28 protein, which is epidemiologically associated with outbreaks of puerperal sepsis, specifically targets the human receptor CEACAM1. This interaction triggers events that would favor the development of puerperal sepsis, including adhesion to cervical cells, suppression of epithelial wound repair and subversion of innate immune responses. High-resolution structural analysis showed that an R28 domain with IgI3-like fold binds to the N-terminal domain of CEACAM1. Together, these findings demonstrate that a single adhesin-receptor interaction can drive the pathogenesis of bacterial sepsis and provide molecular insights into the pathogenesis of one of the most important infectious diseases in medical history.

Group B Streptococcus Surface Protein ß: Structural Characterization of a Complement Factor H-Binding Motif and Its Contribution to Immune Evasion.

The ß protein from group B Streptococcus (GBS) is a ∼132-kDa, cell-surface exposed molecule that binds to multiple host-derived ligands, including complement factor H (FH). Many details regarding this interaction and its significance to immune evasion by GBS remain unclear. In this study, we identified a three-helix bundle domain within the C-terminal half of the B75KN region of ß as the major FH-binding determinant and determined its crystal structure at 2.5 Å resolution. Analysis of this structure suggested a role in FH binding for a loop region connecting helices α1 and α2, which we confirmed by mutagenesis and direct binding studies. Using a combination of protein cross-linking and mass spectrometry, we observed that B75KN bound to complement control protein (CCP)3 and CCP4 domains of FH. Although this binding site lies within a complement regulatory region of FH, we determined that FH bound by ß retained its decay acceleration and cofactor activities. Heterologous expression of ß by Lactococcus lactis resulted in recruitment of FH to the bacterial surface and a significant reduction of C3b deposition following exposure to human serum. Surprisingly, we found that FH binding by ß was not required for bacterial resistance to phagocytosis by neutrophils or killing of bacteria by whole human blood. However, loss of the B75KN region significantly diminished bacterial survival in both assays. Although our results show that FH recruited to the bacterial surface through a high-affinity interaction maintains key complement-regulatory functions, they raise questions about the importance of FH binding to immune evasion by GBS as a whole.

Analysis of Escherichia coli K1 Virulence Genes by Transposon-Directed Sequencing.

Transposon-directed insertion site sequencing (TraDIS) combines random transposon mutagenesis and massively parallel sequencing to shed light on bacterial gene function on a genome-wide scale and in a high-throughput manner. The technique has proven to be successful in the determination of the fitness contribution of every gene under specific conditions both in vitro and in vivo. In this contribution, we describe the procedure used for the identification of Escherichia coli K1 genes essential for in vitro growth, survival in pooled human serum and gastrointestinal colonisation in a rodent model of neonatal invasive infection. TraDIS has broad application for systems-level analysis of a wide range of pathogenic, commensular and saprophytic bacteria.

Chemical Emissions from Cured and Uncured 3D-Printed Ventilator Patient Circuit Medical Parts.

Medical shortages during the COVID-19 pandemic saw numerous efforts to 3D print personal protective equipment and treatment supplies. There is, however, little research on the potential biocompatibility of 3D-printed parts using typical polymeric resins as pertaining to volatile organic compounds (VOCs), which have specific relevance for respiratory circuit equipment. Here, we measured VOCs emitted from freshly printed stereolithography (SLA) replacement medical parts using proton transfer reaction mass spectrometry and infrared differential absorption spectroscopy, and particulates using a scanning mobility particle sizer. We observed emission factors for individual VOCs ranging from ∼0.001 to ∼10 ng cm-3 min-1. Emissions were heavily dependent on postprint curing and mildly dependent on the type of SLA resin. Curing reduced the emission of all observed chemicals, and no compounds exceeded the recommended dose of 360 µg/d. VOC emissions steadily decreased for all parts over time, with an average e-folding time scale (time to decrease to 1/e of the starting value) of 2.6 ± 0.9 h.

Bacterial protein domains with a novel Ig-like fold target human CEACAM receptors.

Streptococcus agalactiae, also known as group B Streptococcus (GBS), is the major cause of neonatal sepsis in humans. A critical step to infection is adhesion of bacteria to epithelial surfaces. GBS adhesins have been identified to bind extracellular matrix components and cellular receptors. However, several putative adhesins have no host binding partner characterised. We report here that surface-expressed ß protein of GBS binds to human CEACAM1 and CEACAM5 receptors. A crystal structure of the complex showed that an IgSF domain in ß represents a novel Ig-fold subtype called IgI3, in which unique features allow binding to CEACAM1. Bioinformatic assessment revealed that this newly identified IgI3 fold is not exclusively present in GBS but is predicted to be present in adhesins from other clinically important human pathogens. In agreement with this prediction, we found that CEACAM1 binds to an IgI3 domain found in an adhesin from a different streptococcal species. Overall, our results indicate that the IgI3 fold could provide a broadly applied mechanism for bacteria to target CEACAMs.

Early risk assessment for COVID-19 patients from emergency department data using machine learning.

Since its emergence in late 2019, the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a pandemic with more than 55 million reported cases and 1.3 million estimated deaths worldwide. While epidemiological and clinical characteristics of COVID-19 have been reported, risk factors underlying the transition from mild to severe disease among patients remain poorly understood. In this retrospective study, we analysed data of 879 confirmed SARS-CoV-2 positive patients admitted to a two-site NHS Trust hospital in London, England, between January 1st and May 26th, 2020, with a majority of cases occurring in March and April. We extracted anonymised demographic data, physiological clinical variables and laboratory results from electronic healthcare records (EHR) and applied multivariate logistic regression, random forest and extreme gradient boosted trees. To evaluate the potential for early risk assessment, we used data available during patients' initial presentation at the emergency department (ED) to predict deterioration to one of three clinical endpoints in the remainder of the hospital stay: admission to intensive care, need for invasive mechanical ventilation and in-hospital mortality. Based on the trained models, we extracted the most informative clinical features in determining these patient trajectories. Considering our inclusion criteria, we have identified 129 of 879 (15%) patients that required intensive care, 62 of 878 (7%) patients needing mechanical ventilation, and 193 of 619 (31%) cases of in-hospital mortality. Our models learned successfully from early clinical data and predicted clinical endpoints with high accuracy, the best model achieving area under the receiver operating characteristic (AUC-ROC) scores of 0.76 to 0.87 (F1 scores of 0.42-0.60). Younger patient age was associated with an increased risk of receiving intensive care and ventilation, but lower risk of mortality. Clinical indicators of a patient's oxygen supply and selected laboratory results, such as blood lactate and creatinine levels, were most predictive of COVID-19 patient trajectories. Among COVID-19 patients machine learning can aid in the early identification of those with a poor prognosis, using EHR data collected during a patient's first presentation at ED. Patient age and measures of oxygenation status during ED stay are primary indicators of poor patient outcomes.

Genes on the Move: In Vitro Transduction of Antimicrobial Resistance Genes between Human and Canine Staphylococcal Pathogens.

Transmission of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) between people and pets, and their co-carriage, are well-described. Potential exchange of antimicrobial resistance (AMR) genes amongst these staphylococci was investigated in vitro through endogenous bacteriophage-mediated transduction. Bacteriophages were UV-induced from seven donor isolates of canine (MRSP) and human (MRSA) origin, containing tet(M), tet(K), fusB or fusC, and lysates filtered. Twenty-seven tetracycline- and fusidic acid- (FA-) susceptible recipients were used in 122 donor-recipient combinations (22 tetracycline, 100 FA) across 415 assays (115 tetracycline, 300 FA). Bacteriophage lysates were incubated with recipients and presumed transductants quantified on antimicrobial-supplemented agar plates. Tetracycline resistance transduction from MRSP and MRSA to methicillin-susceptible S. pseudintermedius (MSSP) was confirmed by PCR in 15/115 assays. No FA-resistance transfer occurred, confirmed by negative fusB/fusC PCR, but colonies resulting from FA assays had high MICs (≥32 mg/L) and showed mutations in fusA, two at a novel position (F88L), nine at H457[Y/N/L]. Horizontal gene transfer of tetracycline-resistance confirms that resistance genes can be shared between coagulase-positive staphylococci from different hosts. Cross-species AMR transmission highlights the importance of good antimicrobial stewardship across humans and veterinary species to support One Health.

Increase in COVID-19 inpatient survival following detection of Thromboembolic and Cytokine storm risk from the point of admission to hospital by a near real time Traffic-light System (TraCe-Tic)

Abstract Introduction Our goal was to evaluate if traffic-light driven personalized care for COVID-19 was associated with improved survival in acute hospital settings. Methods Discharge outcomes were evaluated before and after prospective implementation of a real-time dashboard with feedback to ward-based clinicians. Thromboembolism categories were "medium-risk" (D-dimer >1000 ng/mL or CRP >200 mg/L); "high-risk" (D-dimer >3000 ng/mL or CRP >250 mg/L) or "suspected" (D-dimer >5000 ng/mL). Cytokine storm risk was categorized by ferritin. Results 939/1039 COVID-19 positive patients (median age 67 years, 563/939 (60%) male) completed hospital encounters to death or discharge by 21st May 2020. Thromboembolism flag criteria were reached by 568/939 (60.5%), including 238/275 (86.6%) of the patients who died, and 330/664 (49.7%) of the patients who survived to discharge, p < 0.0001. Cytokine storm flag criteria were reached by 212 (22.6%) of admissions, including 80/275 (29.1%) of the patients who died, and 132/664 (19.9%) of the patients who survived, p < 0.0001. The maximum thromboembolism flag discriminated completed encounter mortality (no flag: 37/371 [9.97%] died; medium-risk: 68/239 [28.5%]; high-risk: 105/205 [51.2%]; and suspected thromboembolism: 65/124 [52.4%], p < 0.0001). Flag criteria were reached by 535 consecutive COVID-19 positive patients whose hospital encounter completed before traffic-light introduction: 173/535 (32.3% [95% confidence intervals 28.0, 36.0]) died. For the 200 consecutive admissions after implementation of real-time traffic light flags, 46/200 (23.0% [95% confidence intervals 17.1, 28.9]) died, p = 0.013. Adjusted for age and sex, the probability of death was 0.33 (95% confidence intervals 0.30, 0.37) before traffic light implementation, 0.22 (0.17, 0.27) after implementation, p < 0.001. In subgroup analyses, older patients, males, and patients with hypertension (p ≤ 0.01), and/or diabetes (p = 0.05) derived the greatest benefit from admission under the traffic light system. Conclusion Personalized early interventions were associated with a 33% reduction in early mortality. We suggest benefit predominantly resulted from early triggers to review/enhance anticoagulation management, without exposing lower-risk patients to potential risks of full anticoagulation therapy.

Development and implementation of a COVID-19 near real-time traffic light system in an acute hospital setting.

Common causes of death in COVID-19 due to SARS-CoV-2 include thromboembolic disease, cytokine storm and adult respiratory distress syndrome (ARDS). Our aim was to develop a system for early detection of disease pattern in the emergency department (ED) that would enhance opportunities for personalised accelerated care to prevent disease progression. A single Trust's COVID-19 response control command was established, and a reporting team with bioinformaticians was deployed to develop a real-time traffic light system to support clinical and operational teams. An attempt was made to identify predictive elements for thromboembolism, cytokine storm and ARDS based on physiological measurements and blood tests, and to communicate to clinicians managing the patient, initially via single consultants. The input variables were age, sex, and first recorded blood pressure, respiratory rate, temperature, heart rate, indices of oxygenation and C-reactive protein. Early admissions were used to refine the predictors used in the traffic lights. Of 923 consecutive patients who tested COVID-19 positive, 592 (64%) flagged at risk for thromboembolism, 241/923 (26%) for cytokine storm and 361/923 (39%) for ARDS. Thromboembolism and cytokine storm flags were met in the ED for 342 (37.1%) patients. Of the 318 (34.5%) patients receiving thromboembolism flags, 49 (5.3% of all patients) were for suspected thromboembolism, 103 (11.1%) were high-risk and 166 (18.0%) were medium-risk. Of the 89 (9.6%) who received a cytokine storm flag from the ED, 18 (2.0% of all patients) were for suspected cytokine storm, 13 (1.4%) were high-risk and 58 (6.3%) were medium-risk. Males were more likely to receive a specific traffic light flag. In conclusion, ED predictors were used to identify high proportions of COVID-19 admissions at risk of clinical deterioration due to severity of disease, enabling accelerated care targeted to those more likely to benefit. Larger prospective studies are encouraged.

Increase in COVID-19 inpatient survival following detection of Thromboembolic and Cytokine storm risk from the point of admission to hospital by a near real time Traffic-light System (TraCe-Tic).

INTRODUCTION: Our goal was to evaluate if traffic-light driven personalized care for COVID-19 was associated with improved survival in acute hospital settings. METHODS: Discharge outcomes were evaluated before and after prospective implementation of a real-time dashboard with feedback to ward-based clinicians. Thromboembolism categories were "medium-risk" (D-dimer >1000ng/mL or CRP >200mg/L); "high-risk" (D-dimer >3000ng/mL or CRP >250mg/L) or "suspected" (D-dimer >5000ng/mL). Cytokine storm risk was categorized by ferritin. RESULTS: 939/1039 COVID-19 positive patients (median age 67 years, 563/939 (60%) male) completed hospital encounters to death or discharge by 21st May 2020. Thromboembolism flag criteria were reached by 568/939 (60.5%), including 238/275 (86.6%) of the patients who died, and 330/664 (49.7%) of the patients who survived to discharge, p<0.0001. Cytokine storm flag criteria were reached by 212 (22.6%) of admissions, including 80/275 (29.1%) of the patients who died, and 132/664 (19.9%) of the patients who survived, p<0.0001. The maximum thromboembolism flag discriminated completed encounter mortality (no flag: 37/371 [9.97%] died; medium-risk: 68/239 [28.5%]; high-risk: 105/205 [51.2%]; and suspected thromboembolism: 65/124 [52.4%], p<0.0001). Flag criteria were reached by 535 consecutive COVID-19 positive patients whose hospital encounter completed before traffic-light introduction: 173/535 (32.3% [95% confidence intervals 28.0, 36.0]) died. For the 200 consecutive admissions after implementation of real-time traffic light flags, 46/200 (23.0% [95% confidence intervals 17.1, 28.9]) died, p=0.013. Adjusted for age and sex, the probability of death was 0.33 (95% confidence intervals 0.30, 0.37) before traffic light implementation, 0.22 (0.17, 0.27) after implementation, p<0.001. In subgroup analyses, older patients, males, and patients with hypertension (p≤0.01), and/or diabetes (p=0.05) derived the greatest benefit from admission under the traffic light system. CONCLUSION: Personalized early interventions were associated with a 33% reduction in early mortality. We suggest benefit predominantly resulted from early triggers to review/enhance anticoagulation management, without exposing lower-risk patients to potential risks of full anticoagulation therapy.

Leukocyte Immunoglobulin-Like Receptors (LILRs) on Human Neutrophils: Modulators of Infection and Immunity.

Neutrophils have a crucial role in defense against microbes. Immune receptors allow neutrophils to sense their environment, with many receptors functioning to recognize signs of infection and to promote antimicrobial effector functions. However, the neutrophil response must be tightly regulated to prevent excessive inflammation and tissue damage, and regulation is achieved by expression of inhibitory receptors that can raise activation thresholds. The leukocyte immunoglobulin-like receptor (LILR) family contain activating and inhibitory members that can up- or down-regulate immune cell activity. New ligands and functions for LILR continue to emerge. Understanding the role of LILR in neutrophil biology is of general interest as they can activate and suppress antimicrobial responses of neutrophils and because several human pathogens exploit these receptors for immune evasion. This review focuses on the role of LILR in neutrophil biology. We focus on the current knowledge of LILR expression on neutrophils, the known functions of LILR on neutrophils, and how these receptors may contribute to shaping neutrophil responses during infection.

The Orphan Immune Receptor LILRB3 Modulates Fc Receptor-Mediated Functions of Neutrophils.

Neutrophils are critical to the generation of effective immune responses and for killing invading microbes. Paired immune receptors provide important mechanisms to modulate neutrophil activation thresholds and effector functions. Expression of the leukocyte Ig-like receptor (LILR)A6 (ILT8/CD85b) and LILRB3 (ILT5/CD85a) paired-receptor system on human neutrophils has remained unclear because of the lack of specific molecular tools. Additionally, there is little known of their possible functions in neutrophil biology. The objective of this study was to characterize expression of LILRA6/LILRB3 receptors during human neutrophil differentiation and activation, and to assess their roles in modulating Fc receptor-mediated effector functions. LILRB3, but not LILRA6, was detected in human neutrophil lysates following immunoprecipitation by mass spectrometry. We demonstrate high LILRB3 expression on the surface of resting neutrophils and release from the surface following neutrophil activation. Surface expression was recapitulated in a human PLB-985 cell model of neutrophil-like differentiation. Continuous ligation of LILRB3 inhibited key IgA-mediated effector functions, including production of reactive oxygen species, phagocytic uptake, and microbial killing. This suggests that LILRB3 provides an important checkpoint to control human neutrophil activation and their antimicrobial effector functions during resting and early-activation stages of the neutrophil life cycle.

Loss of Trefoil Factor 2 Sensitizes Rat Pups to Systemic Infection with the Neonatal Pathogen Escherichia coli K1.

Gastrointestinal (GI) colonization of 2-day-old (P2) rat pups with Escherichia coli K1 results in translocation of the colonizing bacteria across the small intestine, bacteremia, and invasion of the meninges, with animals frequently succumbing to lethal infection. Infection, but not colonization, is strongly age dependent; pups become progressively less susceptible to infection over the P2-to-P9 period. Colonization leads to strong downregulation of the gene encoding trefoil factor 2 (Tff2), preventing maturation of the protective mucus barrier in the small intestine. Trefoil factors promote mucosal homeostasis. We investigated the contribution of Tff2 to protection of the neonatal rat from E. coli K1 bacteremia and tissue invasion. Deletion of tff2, using clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9, sensitized P9 pups to E. coli K1 bacteremia. There were no differences between tff2-/- homozygotes and the wild type with regard to the dynamics of GI colonization. Loss of the capacity to elaborate Tff2 did not impact GI tract integrity or the thickness of the small-intestinal mucus layer but, in contrast to P9 wild-type pups, enabled E. coli K1 bacteria to gain access to epithelial surfaces in the distal region of the small intestine and exploit an intracellular route across the epithelial monolayer to enter the blood circulation via the mesenteric lymphatic system. Although primarily associated with the mammalian gastric mucosa, we conclude that loss of Tff2 in the developing neonatal small intestine enables the opportunistic neonatal pathogen E. coli K1 to enter the compromised mucus layer in the distal small intestine prior to systemic invasion and infection.

Molecular basis determining species specificity for TLR2 inhibition by staphylococcal superantigen-like protein 3 (SSL3).

Staphylococcus aureus is a versatile opportunistic pathogen, causing disease in human and animal species. Its pathogenicity is linked to the ability of S. aureus to secrete immunomodulatory molecules. These evasion proteins bind to host receptors or their ligands, resulting in inhibitory effects through high affinity protein-protein interactions. Staphylococcal evasion molecules are often species-specific due to differences in host target proteins between species. We recently solved the crystal structure of murine TLR2 in complex with immunomodulatory molecule staphylococcal superantigen-like protein 3 (SSL3), which revealed the essential residues within SSL3 for TLR2 inhibition. In this study we aimed to investigate the molecular basis of the interaction on the TLR2 side. The SSL3 binding region on murine TLR2 was compared to that of other species through sequence alignment and homology modeling, which identified interspecies differences. To examine whether this resulted in altered SSL3 activity on the corresponding TLR2s, bovine, equine, human, and murine TLR2 were stably expressed in HEK293T cells and the ability of SSL3 to inhibit TLR2 was assessed. We found that SSL3 was unable to inhibit bovine TLR2. Subsequent loss and gain of function mutagenesis showed that the lack of inhibition is explained by the absence of two tyrosine residues in bovine TLR2 that play a prominent role in the SSL3-TLR2 interface. We found no evidence for the existence of allelic SSL3 variants that have adapted to the bovine host. Thus, within this paper we reveal the molecular determinants of the TLR2-SSL3 interaction which adds to our understanding of staphylococcal host specificity.

Genome-Wide Identification by Transposon Insertion Sequencing of Escherichia coli K1 Genes Essential for In Vitro Growth, Gastrointestinal Colonizing Capacity, and Survival in Serum.

Escherichia coli K1 strains are major causative agents of invasive disease of newborn infants. The age dependency of infection can be reproduced in neonatal rats. Colonization of the small intestine following oral administration of K1 bacteria leads rapidly to invasion of the blood circulation; bacteria that avoid capture by the mesenteric lymphatic system and evade antibacterial mechanisms in the blood may disseminate to cause organ-specific infections such as meningitis. Some E. coli K1 surface constituents, in particular the polysialic acid capsule, are known to contribute to invasive potential, but a comprehensive picture of the factors that determine the fully virulent phenotype has not emerged so far. We constructed a library and constituent sublibraries of ∼775,000 Tn5 transposon mutants of E. coli K1 strain A192PP and employed transposon-directed insertion site sequencing (TraDIS) to identify genes required for fitness for infection of 2-day-old rats. Transposon insertions were lacking in 357 genes following recovery on selective agar; these genes were considered essential for growth in nutrient-replete medium. Colonization of the midsection of the small intestine was facilitated by 167 E. coli K1 gene products. Restricted bacterial translocation across epithelial barriers precluded TraDIS analysis of gut-to-blood and blood-to-brain transits; 97 genes were required for survival in human serum. This study revealed that a large number of bacterial genes, many of which were not previously associated with systemic E. coli K1 infection, are required to realize full invasive potential.IMPORTANCEEscherichia coli K1 strains cause life-threatening infections in newborn infants. They are acquired from the mother at birth and colonize the small intestine, from where they invade the blood and central nervous system. It is difficult to obtain information from acutely ill patients that sheds light on physiological and bacterial factors determining invasive disease. Key aspects of naturally occurring age-dependent human infection can be reproduced in neonatal rats. Here, we employ transposon-directed insertion site sequencing to identify genes essential for the in vitro growth of E. coli K1 and genes that contribute to the colonization of susceptible rats. The presence of bottlenecks to invasion of the blood and cerebrospinal compartments precluded insertion site sequencing analysis, but we identified genes for survival in serum.

Immune evasion by a staphylococcal inhibitor of myeloperoxidase.

Staphylococcus aureus is highly adapted to its host and has evolved many strategies to resist opsonization and phagocytosis. Even after uptake by neutrophils, S. aureus shows resistance to killing, which suggests the presence of phagosomal immune evasion molecules. With the aid of secretome phage display, we identified a highly conserved protein that specifically binds and inhibits human myeloperoxidase (MPO), a major player in the oxidative defense of neutrophils. We have named this protein "staphylococcal peroxidase inhibitor" (SPIN). To gain insight into inhibition of MPO by SPIN, we solved the cocrystal structure of SPIN bound to a recombinant form of human MPO at 2.4-Å resolution. This structure reveals that SPIN acts as a molecular plug that prevents H2O2 substrate access to the MPO active site. In subsequent experiments, we observed that SPIN expression increases inside the neutrophil phagosome, where MPO is located, compared with outside the neutrophil. Moreover, bacteria with a deleted gene encoding SPIN showed decreased survival compared with WT bacteria after phagocytosis by neutrophils. Taken together, our results demonstrate that S. aureus secretes a unique proteinaceous MPO inhibitor to enhance survival by interfering with MPO-mediated killing.

Postnatal development of the small intestinal mucosa drives age-dependent, regio-selective susceptibility to Escherichia coli K1 infection.

The strong age dependency of neonatal systemic infection with Escherichia coli K1 can be replicated in the neonatal rat. Gastrointestinal (GI) colonization of two-day-old (P2) rats leads to invasion of the blood within 48 h of initiation of colonization; pups become progressively less susceptible to infection over the P2-P9 period. We show that, in animals colonized at P2 but not at P9, E. coli K1 bacteria gain access to the enterocyte surface in the mid-region of the small intestine and translocate through the epithelial cell monolayer by an intracellular pathway to the submucosa. In this region of the GI tract, the protective mucus barrier is poorly developed but matures to full thickness over P2-P9, coincident with the development of resistance to invasion. At P9, E. coli K1 bacteria are physically separated from villi by the mucus layer and their numbers controlled by mucus-embedded antimicrobial peptides, preventing invasion of host tissues.

Pathoadaptive Mutations of Escherichia coli K1 in Experimental Neonatal Systemic Infection.

Although Escherichia coli K1 strains are benign commensals in adults, their acquisition at birth by the newborn may result in life-threatening systemic infections, most commonly sepsis and meningitis. Key features of these infections, including stable gastrointestinal (GI) colonization and age-dependent invasion of the bloodstream, can be replicated in the neonatal rat. We previously increased the capacity of a septicemia isolate of E. coli K1 to elicit systemic infection following colonization of the small intestine by serial passage through two-day-old (P2) rat pups. The passaged strain, A192PP (belonging to sequence type 95), induces lethal infection in all pups fed 2-6 x 106 CFU. Here we use whole-genome sequencing to identify mutations responsible for the threefold increase in lethality between the initial clinical isolate and the passaged derivative. Only four single nucleotide polymorphisms (SNPs), in genes (gloB, yjgV, tdcE) or promoters (thrA) involved in metabolic functions, were found: no changes were detected in genes encoding virulence determinants associated with the invasive potential of E. coli K1. The passaged strain differed in carbon source utilization in comparison to the clinical isolate, most notably its inability to metabolize glucose for growth. Deletion of each of the four genes from the E. coli A192PP chromosome altered the proteome, reduced the number of colonizing bacteria in the small intestine and increased the number of P2 survivors. This work indicates that changes in metabolic potential lead to increased colonization of the neonatal GI tract, increasing the potential for translocation across the GI epithelium into the systemic circulation.