Faculty Bibliography

Gastrointestinal (GI) colonization by methicillin-resistant Staphylococcus aureus (MRSA) is associated with a high risk of transmission and invasive disease in vulnerable populations. The immune and microbial factors that permit GI colonization remain unknown. Male sex is correlated with enhanced Staphylococcus aureus nasal carriage, skin and soft tissue infections, and bacterial sepsis. Here, we established a mouse model of sexual dimorphism during GI colonization by MRSA. Our results show that in contrast to male mice that were susceptible to persistent colonization, female mice rapidly cleared MRSA from the GI tract following oral inoculation in a manner dependent on the gut microbiota. This colonization resistance displayed by female mice was mediated by an increase in IL-17A+ CD4+ T cells (Th17) and dependent on neutrophils. Ovariectomy of female mice increased MRSA burden, but gonadal female mice that have the Y chromosome retained enhanced Th17 responses and colonization resistance. Our study reveals a novel intersection between sex and gut microbiota underlying colonization resistance against a major widespread pathogen.

Pathogenic Enterobacter species are of increasing clinical concern due to the multidrug-resistant nature of these bacteria, including resistance to carbapenem antibiotics. Our understanding of Enterobacter virulence is limited, hindering the development of new prophylactics and therapeutics targeting infections caused by Enterobacter species. In this study, we assessed the virulence of contemporary clinical Enterobacter hormaechei isolates in a mouse model of intraperitoneal infection and used comparative genomics to identify genes promoting virulence. Through mutagenesis and complementation studies, we found two porin-encoding genes, ompC and ompD, to be required for E. hormaechei virulence. These porins imported clinically relevant carbapenems into the bacteria, and thus loss of OmpC and OmpD desensitized E. hormaechei to the antibiotics. Our genomic analyses suggest porin-related genes are frequently mutated in E. hormaechei, perhaps due to the selective pressure of antibiotic therapy during infection. Despite the importance of OmpC and OmpD during infection of immunocompetent hosts, we found the two porins to be dispensable for virulence in a neutropenic mouse model. Moreover, porin loss provided a fitness advantage during carbapenem treatment in an ex vivo human whole blood model of bacteremia. Our data provide experimental evidence of pathogenic Enterobacter species gaining antibiotic resistance via loss of porins and argue antibiotic therapy during infection of immunocompromised patients is a conducive environment for the selection of porin mutations enhancing the multidrug-resistant profile of these pathogens.

BACKGROUND:Bloodstream infections (BSI) are associated with high mortality rates, particularly when caused by resistant pathogens. Reducing the delay in diagnosis and initiation of appropriate treatment is crucial for improving clinical outcomes. The implementation of polymerase chain reaction (PCR) tests in the diagnostic process offers a promising approach to achieving quicker identification of pathogens, thereby potentially reducing mortality associated with BSI. METHODS:BSI, for which diagnostic protocol has been unchanged. RESULTS:(VRE) BSI and 384 with MSSA BSI. The mean 30-day mortality risk difference in the period post-intervention estimated in our difference-in-differences model was -6.03 per 100 (95% CI: -10.35 to -1.7), with event study plots suggesting minimal deviation from parallel trends in the pre-treatment period. CONCLUSIONS:Findings suggest that introduction of BCID2 PCR testing for enterococcal bloodstream infections (BSI) may be associated with a reduction in mortality, however, interpretation of the effects must be approached with caution given the relative imprecision of estimates. Further research with larger samples is essential to establish a definitive conclusion on the impact of rapid PCR testing on mortality in BSI. This is an innovative approach using causal methods to evaluate interventions aimed at the improvement of infection control and antimicrobial treatment strategies.

The antimicrobial activity of histones was discovered in the 1940s, but their mechanism of action is not fully known. Here we show that methicillin-resistant Staphylococcus aureus (MRSA) is susceptible to histone H1 (H1), even in the presence of divalent cations and serum. Through selective evolution and a genome-wide screen of a transposon library, as well as physiological and pharmacological experiments, we elucidated how H1 kills MRSA. We show that H1 first binds to wall teichoic acids with high affinity. Once bound, H1 requires a potentiated membrane and a metabolically active bacterium to permeabilize the membrane and enter the cell. Upon entry, H1 accumulates intracellularly, in close association with the bacterial DNA. Of note, anti-H1 antibodies inhibit neutrophil extracellular trap killing of MRSA. Moreover, H1 colocalizes with bacterial DNA in abscess samples of MRSA-infected patients, suggesting a role for H1 in combating MRSA in vivo.

UNLABELLED:isolates with low intrinsic virulence. IMPORTANCE/OBJECTIVE:infection.

Dehydration and malnutrition are common and often underdiagnosed in hospital settings. Multidrug-resistant bacterial infections result in more than 35,000 deaths a year in nosocomial patients. The effect of temporal dietary and water restriction (DWR) on susceptibility to multidrug-resistant pathogens is unknown. We report that DWR markedly increased susceptibility to systemic infection by ESKAPE pathogens. Using a murine bloodstream model of methicillin-resistant Staphylococcus aureus infection, we show that DWR leads to significantly increased mortality and morbidity. DWR causes increased bacterial burden, severe pathology, and increased numbers of phagocytes in the kidney. DWR appears to alter the functionality of these phagocytes and is therefore unable to control infection. Mechanistically, we show that DWR impairs the ability of macrophages to phagocytose multiple bacterial pathogens and efferocytose apoptotic neutrophils. Together, this work highlights the crucial impact that diet and hydration play in protecting against infection.

Efflux pump antiporters confer drug resistance to bacteria by coupling proton import with the expulsion of antibiotics from the cytoplasm. Despite efforts there remains a lack of understanding as to how acid/base chemistry drives drug efflux. Here, we uncover the proton-coupling mechanism of the Staphylococcus aureus efflux pump NorA by elucidating structures in various protonation states of two essential acidic residues using cryo-EM. Protonation of Glu222 and Asp307 within the C-terminal domain stabilized the inward-occluded conformation by forming hydrogen bonds between the acidic residues and a single helix within the N-terminal domain responsible for occluding the substrate binding pocket. Remarkably, deprotonation of both Glu222 and Asp307 is needed to release interdomain tethering interactions, leading to opening of the pocket for antibiotic entry. Hence, the two acidic residues serve as a "belt and suspenders" protection mechanism to prevent simultaneous binding of protons and drug that enforce NorA coupling stoichiometry and confer antibiotic resistance.

The agr quorum-sensing system links Staphylococcus aureus metabolism to virulence, in part by increasing bacterial survival during exposure to lethal concentrations of H₂O₂, a crucial host defense against S. aureus. We now report that protection by agr surprisingly extends beyond post-exponential growth to the exit from stationary phase when the agr system is no longer turned on. Thus, agr can be considered a constitutive protective factor. Deletion of agr resulted in decreased ATP levels and growth, despite increased rates of respiration or fermentation at appropriate oxygen tensions, suggesting that Δagr cells undergo a shift towards a hyperactive metabolic state in response to diminished metabolic efficiency. As expected from increased respiratory gene expression, reactive oxygen species (ROS) accumulated more in the agr mutant than in wild-type cells, thereby explaining elevated susceptibility of Δagr strains to lethal H₂O₂ doses. Increased survival of wild-type agr cells during H₂O₂ exposure required sodA, which detoxifies superoxide. Additionally, pretreatment of S. aureus with respiration-reducing menadione protected Δagr cells from killing by H₂O₂. Thus, genetic deletion and pharmacologic experiments indicate that agr helps control endogenous ROS, thereby providing resilience against exogenous ROS. The long-lived 'memory' of agr-mediated protection, which is uncoupled from agr activation kinetics, increased hematogenous dissemination to certain tissues during sepsis in ROS-producing, wild-type mice but not ROS-deficient (Cybb

Organisms determine the transcription rates of thousands of genes through a few modes of regulation that recur across the genome¹. In bacteria, the relationship between the regulatory architecture of a gene and its expression is well understood for individual model gene circuits^2,3. However, a broader perspective of these dynamics at the genome scale is lacking, in part because bacterial transcriptomics has hitherto captured only a static snapshot of expression averaged across millions of cells⁴. As a result, the full diversity of gene expression dynamics and their relation to regulatory architecture remains unknown. Here we present a novel genome-wide classification of regulatory modes based on the transcriptional response of each gene to its own replication, which we term the transcription-replication interaction profile (TRIP). Analysing single-bacterium RNA-sequencing data, we found that the response to the universal perturbation of chromosomal replication integrates biological regulatory factors with biophysical molecular events on the chromosome to reveal the local regulatory context of a gene. Whereas the TRIPs of many genes conform to a gene dosage-dependent pattern, others diverge in distinct ways, and this is shaped by factors such as intra-operon position and repression state. By revealing the underlying mechanistic drivers of gene expression heterogeneity, this work provides a quantitative, biophysical framework for modelling replication-dependent expression dynamics.