Faculty Bibliography

Asymptomatic carriers of antimicrobial-resistant organisms (AMROs) can unwittingly transmit these pathogens in hospitals, contributing to the burden of healthcare-associated infections (HAIs). Surveillance in hospitals can involve different types of observations; however, a framework to coherently synthesize these datasets to identify AMRO carriers is lacking. Here, we develop a new inference framework combining a data-driven mechanistic transmission model and multimodal observations from clinical cultures, electronic health records, patient mobility, and genomic data. Using extensive simulated outbreaks, we validate the inference framework for AMROs with various levels of community importation and hospital transmission and evaluate the utility of different combinations of data sources. Inference results show that using multimodal observations consistently improves the accuracy in identifying AMRO carriers. We apply the inference framework to carbapenem-resistant Klebsiella pneumoniae (CRKP) at an urban quaternary care hospital in New York City, United States and find that the addition of even sparsely sampled genome sequence data to patient characteristics supports more accurate identification of CRKP carriers. Model simulations suggest that inference-guided targeted isolation leads to a greater reduction of AMRO burdens compared to alternative, heuristic approaches. Thus, the synergistic effect of utilizing multimodal observations for estimating AMRO carriage risk may inform improved interventions in hospital settings.

Rapid molecular assays guiding treatment of methicillin-resistant Staphylococcus aureus (MRSA) detect SCCmec (Xpert) or the SCCmec-orfX junction (BCID2). Sequence variation in this region can disrupt primer binding, yielding false-negative results. Investigation of a missed bloodstream infection linked escape to a CRISPR-Cas-associated SCCmec variant, leading to identification of 64 variants from 45 patients-2% of 2,432 screened. Misdiagnosis was restricted to clonal complex 5, a hospital-associated lineage; 11 of 40 SCCmec/junctions evaded detection by BCID2 or Xpert. Variants had mecA instability and circulated in healthcare settings. Our findings reveal a unique escape mechanism and underscore a threat to diagnostic accuracy.

We recently described the evolution of a community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) USA300 variant responsible for an outbreak of skin and soft tissue infections. Acquisition of a mosaic version of the Φ11 prophage (mΦ11) that increases skin abscess size was an early step in CA-MRSA adaptation that primed the successful spread of the clone. The present report shows how prophage mΦ11 exerts its effect on virulence for skin infection without encoding a known toxin or fitness genes. Abscess size and skin inflammation were associated with DNA methylase activity of an mΦ11-encoded adenine methyltransferase (designated pamA). pamA increased expression of fibronectin-binding protein A (fnbA; FnBPA), and inactivation of fnbA eliminated the effect of pamA on abscess virulence without affecting strains lacking pamA. Thus, fnbA is a pamA-specific virulence factor. Mechanistically, pamA was shown to promote biofilm formation in vivo in skin abscesses, a phenotype linked to FnBPA's role in biofilm formation. Collectively, these data reveal a critical mechanism-epigenetic regulation of staphylococcal gene expression-by which phage can regulate virulence to drive adaptive leaps by S. aureus.

Staphylococcus aureus is a global health concern, resulting in significant disease burden in both hospital and community settings. To establish infection, the bacteria must contend with a multitude of host defense mechanisms, including "nutritional immunity", in which nutrients are sequestered away from invading pathogens. Importantly, S. aureus requires iron for growth during infection, which it acquires through the lysis of erythrocytes (hemolysis). HlgAB, a secreted bi-component pore forming toxin, contributes to the ability of S. aureus to lyse erythrocytes to release heme iron. HlgAB consists of two subunits, the S-subunit HlgA and the F-subunit HlgB. Prior work has shown that the hemolytic activity of HlgAB is dependent on the binding of HlgA to the host receptor Duffy Antigen Receptor for Chemokines (DARC). Here we show that HlgB binds the surface of erythrocytes independently of DARC or HlgA. Our comparative genomic analysis reveals high conservation of hlgA and hlgB genes across S. aureus lineages. By performing structure-function studies, we identified a series of loops within the rim domain of HlgB that are required for the binding of HlgB to erythrocytes and erythrocyte lysis by HlgAB. The importance of HlgB-mediated host targeting was validated in a tissue culture model of S. aureus-mediated lysis of primary human erythrocytes, in an in vivo murine model of intoxication, and during in vivo systemic infection. Altogether, these findings expand our mechanistic insights into how S. aureus overcomes nutritional immunity, and the role of HlgB in S. aureus pathophysiology.

The T300A substitution in ATG16L1 associated with Crohn's disease impairs autophagy, yet up to 50% of humans are heterozygous for this allele. Here, we demonstrate that heterozygosity for the analogous substitution in mice (Atg16L1^T316A), but not homozygosity, protects against lethal Salmonella enterica Typhimurium infection. One copy of Atg16L1^T316A was sufficient to enhance cytokine production through inflammasome activation, which was necessary for protection. In contrast, two copies of Atg16L1^T316A inhibited the autophagy-related process of LC3-associated phagocytosis (LAP) and increased susceptibility. Macrophages from human donors heterozygous for ATG16L1^T300A displayed elevated inflammasome activation while homozygosity impaired LAP, similar to mice. These results clarify how the T300A substitution impacts ATG16L1 function and suggest it can be beneficial to heterozygous carriers, providing an explanation for its prevalence within the human population.

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.

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