Immune gene expression analysis indicates the potential of a self-amplifying Covid-19 mRNA vaccine.

Remarkable potency has been demonstrated for mRNA vaccines in reducing the global burden of the ongoing COVID-19 pandemic. An alternative form of the mRNA vaccine is the self-amplifying mRNA (sa-mRNA) vaccine, which encodes an alphavirus replicase that self-amplifies the full-length mRNA and SARS-CoV-2 spike (S) transgene. However, early-phase clinical trials of sa-mRNA COVID-19 vaccine candidates have questioned the potential of this platform to develop potent vaccines. We examined the immune gene response to a candidate sa-mRNA vaccine against COVID-19, ARCT-021, and compared our findings to the host response to other forms of vaccines. In blood samples from healthy volunteers that participated in a phase I/II clinical trial, greater induction of transcripts involved in Toll-like receptor (TLR) signalling, antigen presentation and complement activation at 1 day post-vaccination was associated with higher anti-S antibody titers. Conversely, transcripts involved in T-cell maturation at day 7 post-vaccination informed the magnitude of eventual S-specific T-cell responses. The transcriptomic signature for ARCT-021 vaccination strongly correlated with live viral vector vaccines, adjuvanted vaccines and BNT162b2 1 day post-vaccination. Moreover, the ARCT-021 signature correlated with day 7 YF17D live-attenuated vaccine transcriptomic responses. Altogether, our findings show that sa-mRNA vaccination induces innate immune responses that are associated with the development of adaptive immunity from other forms of vaccines, supporting further development of this vaccine platform for clinical application.

Oral and Gut Microbial Diversity and Immune Regulation in Patients with HIV on Antiretroviral Therapy.

Despite evidence of a chronic inflammatory phenotype in people living with HIV (PLWH) on antiretroviral therapy (ART), the role of oral microbiota in chronic immune activation has not been fully explored. We aimed to determine the relationship between oral and gut microbiome diversity and chronic systemic inflammation in ART-treated PLWH with prevalent severe periodontitis, an inflammatory condition commonly associated with HIV infection. We assessed bacterial and fungal communities at oral and gastrointestinal sites in a cohort (n = 52) of primarily postmenopausal women on ART using 16S rRNA and internal transcribed spacer (ITS) sequencing and measured cellular and soluble markers of inflammation and immune dysfunction. Linear mixed-effect regression and differential abundance analyses were used to associate clinical characteristics and immunological markers with bacterial and fungal diversity and community composition. Bacterial α-diversity in plaque, saliva, and gut was associated with different immunological markers, while mycobial diversity was not associated with soluble or cellular biomarkers of immune stimulation or T cell dysfunction. Furthermore, lipopolysaccharide-positive (LPS+) bacteria previously linked to inflammatory outcomes were enriched at oral sites in patients with severe periodontitis. Fungal α-diversity was reduced in plaque from teeth with higher clinical attachment loss, a marker of periodontitis, and in saliva and plaque from patients with a history of AIDS. Our results show that both bacterial and fungal oral microbiome communities likely play a role in chronic systemic immune activation in PLWH. Thus, interventions targeting both inflammation and the microbiome, particularly in the oral cavity, may be necessary to reduce chronic immune dysregulation in patients with HIV.IMPORTANCE A feedback loop between dysbiotic gut microbiota, increased translocation of microbial products such as lipopolysaccharide, and inflammation has been hypothesized to cause immune system dysfunction in early HIV infection. However, despite evidence of a chronic inflammatory phenotype in patients on antiretroviral therapy (ART), the role of oral microbiota in systemic immune activation and the relationship between oral and gut bacterial and fungal diversity have not been explored. Our study suggests a crucial role for oral bacterial and fungal communities in long-term systemic immune activation in patients on ART, expanding the current paradigm focused on gut bacteria. Our results indicate that interventions targeting both inflammation and microbial diversity are needed to mitigate oral inflammation-related comorbidities, particularly in HIV-positive patients. More broadly, these findings can bolster general models of microbiome-mediated chronic systemic immune activation and aid the development of precise microbiota-targeted interventions to reverse chronic inflammation.

Colonizing multidrug-resistant bacteria and the longitudinal evolution of the intestinal microbiome after liver transplantation.

Infections by multidrug-resistant bacteria (MDRB) remain a leading cause of morbidity and mortality after liver transplantation (LT). Gut dysbiosis characteristic of end-stage liver disease may predispose patients to intestinal MDRB colonization and infection, in turn exacerbating dysbiosis. However, relationships between MDRB colonization and dysbiosis after LT remain unclear. We prospectively recruited 177 adult patients undergoing LT at a single tertiary care center. 16 S V3-V4 rRNA sequencing was performed on 723 fecal samples collected pre-LT and periodically until one-year post-LT to test whether MDRB colonization was associated with decreased microbiome diversity. In multivariate linear mixed-effect models, MDRB colonization predicts reduced Shannon α-diversity, after controlling for underlying liver disease, antibiotic exposures, and clinical complications. Importantly, pre-LT microbial markers predict subsequent colonization by MDRB. Our results suggest MDRB colonization as a major, previously unrecognized, marker of persistent dysbiosis. Therapeutic approaches accounting for microbial and clinical factors are needed to address post-transplant microbiome health.

Genomic Surveillance Reveals Diversity of Multidrug-Resistant Organism Colonization and Infection: A Prospective Cohort Study in Liver Transplant Recipients.

Background: Multidrug-resistant organisms (MDROs) are an important cause of morbidity and mortality after solid organ transplantation. We aimed to characterize MDRO colonization dynamics and infection in liver transplant (LT) recipients through innovative use of active surveillance and whole-genome sequencing (WGS). Methods: We prospectively enrolled consecutive adult patients undergoing LT from March 2014 to March 2016. Fecal samples were collected at multiple timepoints from time of enrollment to 12 months posttransplant. Samples were screened for carbapenem-resistant Enterobacteriaceae (CRE), Enterobacteriaceae resistant to third-generation cephalosporins (Ceph-RE), and vancomycin-resistant enterococci. We performed WGS of CRE and selected Ceph-RE isolates. We also collected clinical data including demographics, transplant characteristics, and infection data. Results: We collected 998 stool samples and 119 rectal swabs from 128 patients. MDRO colonization was detected in 86 (67%) patients at least once and was significantly associated with subsequent MDRO infection (0 vs 19.8%, P = .002). Child-Turcotte-Pugh score at LT and duration of post-LT hospitalization were independent predictors of both MDRO colonization and infection. Temporal dynamics differed between MDROs with respect to onset of colonization, clearance, and infections. We detected an unexpected diversity of CRE colonizing isolates and previously unrecognized transmission that spanned Ceph-RE and CRE phenotypes, as well as a cluster of mcr-1-producing isolates. Conclusions: Active surveillance and WGS showed that MDRO colonization is a highly dynamic and complex process after LT. Understanding that complexity is crucial for informing decisions regarding MDRO infection control, use of therapeutic decolonization, and empiric treatment regimens.

Detection of mcr-1-Carrying Escherichia coli Causing Bloodstream Infection in a New York City Hospital: Avian Origins, Human Concerns?

The spread of mcr-1 in the United States remains poorly defined. mcr-1-producing Escherichia coli that also carried blaSHV-12 was detected in a hospitalized patient. No additional cases were identified during screening of 801 Gram-negative isolates. Genomic sequencing identified an IncX4 mcr-1- harboring plasmid and ST117 clonal background associated with avian pathogenic E coli.

The Surface Protein SdrF Mediates Staphylococcus epidermidis Adherence to Keratin.

Background: Staphylococcus epidermidis, a major component of skin flora, is an opportunist, often causing prosthetic device infections. A family of structurally related proteins mediates staphylococcal attachment to host tissues, contributing to the success of S. epidermidis as a pathogen. We examined the ability of the surface protein SdrF to adhere to keratin, a major molecule expressed on the skin surface. Methods: A heterologous Lactococcus lactis expression system was used to express SdrF and its ligand-binding domains. Adherence to keratin types 1 and 10, human foreskin keratinocytes, and nasal epithelial cells was examined. Results: SdrF bound human keratins 1 and 10 and adhered to keratinocytes and epithelial cells. Binding involved both the A and B domains. Anti-SdrF antibodies reduced adherence of S. epidermidis to keratin and keratinocytes. RNA interference reduced keratin synthesis in keratinocytes and, as a result, SdrF adherence. Direct force measurements using atomic force microscopy showed that SdrF mediates bacterial adhesion to keratin 10 through strong and weak bonds involving the A and B regions; strong adhesion was primarily mediated by the A region. Conclusions: These studies demonstrate that SdrF mediates adherence to human keratin and suggest that SdrF may facilitate S. epidermidis colonization of the skin.

Reduced Vancomycin Susceptibility of Methicillin-Susceptible Staphylococcus aureus Has No Significant Impact on Mortality but Results in an Increase in Complicated Infection.

Methicillin-susceptible Staphylococcus aureus (MSSA) bloodstream infections (BSIs) often lead to severe complications despite the availability of effective antibiotics. It remains unclear whether elevated vancomycin MICs are associated with worse outcomes. We conducted a 2-year retrospective cohort study (n = 252) of patients with MSSA BSIs at a tertiary care hospital. We defined reduced vancomycin susceptibility (RVS) as a Microscan MIC of 2 mg/liter. All strains were genotyped (spa) and assessed for agr functionality. Multivariable logistic regression models were used to examine the impact of RVS phenotype and strain genotype on 30-day all-cause mortality and complicated bacteremia (metastatic spread, endovascular infection, or duration ≥3 days). One-third of patients (84/252) were infected with RVS isolates. RVS Infections were more frequently associated with metastatic or embolic sites of infection (36% versus 17%, P < 0.001), and endovascular infection (26% versus 12%, P = 0.004). These infections occurred more often in patients with fewer underlying comorbidities (Charlson comorbidity index of ≥3 [73% versus 88%, P = 0.002]). Genotyping identified 127 spa-types and 14 Spa-clonal complexes (Spa-CCs). Spa-CC002 and Spa-CC008 were more likely to exhibit the RVS phenotype versus other Spa-CCs (OR = 2.2, P < 0.01). The RVS phenotype was not significantly associated with 30-day mortality; however, it was associated with complicated bacteremia (adjusted odds ratio of 2.35 [range, 1.26 to 4.37]; P = 0.007) in adjusted analyses. The association of RVS strains with complicated infection and fewer underlying comorbidities suggests the phenotype as a potential marker of strain virulence in MSSA BSIs. The RVS phenotype itself was not a significant predictor of mortality in this patient cohort. Further studies are necessary to explore this host-pathogen relationship.

Evolutionary Dynamics of Pandemic Methicillin-Sensitive Staphylococcus aureus ST398 and Its International Spread via Routes of Human Migration.

Methicillin-susceptible Staphylococcus aureus (MSSA) accounts for the majority of S. aureus infections globally, and yet surprisingly little is known about its clonal evolution. We applied comparative whole-genome sequencing (WGS) analyses to epidemiologically and geographically diverse ST398-MSSA, a pandemic lineage affecting both humans and livestock. Bayesian phylogenetic analysis predicted divergence of human-associated ST398-MSSA ~40 years ago. Isolates from Midwestern pigs and veterinarians differed substantially from those in New York City (NYC). Pig ST398 strains contained a large region of recombination representing imports from multiple sequence types (STs). Phylogeographic analyses supported the spread of ST398-MSSA along local cultural and migratory links between parts of the Caribbean, North America, and France, respectively. Applying pairwise single-nucleotide polymorphism (SNP) distances as a measure of genetic relatedness between isolates, we observed that ST398 not only clustered in households but also frequently extended across local social networks. Isolates collected from environmental surfaces reflected the full diversity of colonizing individuals, highlighting their potentially critical role as reservoirs for transmission and diversification. Strikingly, we observed high within-host SNP variability compared to our previous studies on the dominant methicillin-resistant Staphylococcus aureus (MRSA) clone USA300. Our data indicate that the dynamics of colonization, persistence, and transmission differ substantially between USA300-MRSA and ST398-MSSA. Taken together, our study reveals local and international routes of transmission for a major MSSA clone, indicating key impacts of recombination and mutation on genetic diversification and highlighting important ecological differences from epidemic USA300. Our study demonstrates extensive local and international routes of transmission for a major MSSA clone despite the lack of substantial antibiotic resistance. IMPORTANCE: Unlike methicillin-resistant Staphylococcus aureus (MRSA), surprisingly little is known about the clonal evolution of methicillin-susceptible S. aureus (MSSA), although these strains account for the majority of S. aureus infections. To better understand how MSSA spreads and becomes established in communities, we applied comparative bacterial whole-genome sequencing to pandemic ST398-MSSA, a clone of clinical importance affecting humans and livestock in different geographic regions. Phylogeographic analyses identified that ST398-MSSA spread along local cultural and migratory links between parts of the Caribbean, North America, and France, respectively. We observed high within-host SNP variability compared to our previous studies on the dominant MRSA clone USA300. Our data indicate that the dynamics of colonization, persistence, and transmission differ substantially between USA300 MRSA and ST398 MSSA.

Staphylococcus epidermidis Protection Against Staphylococcus aureus Colonization in People Living With Human Immunodeficiency Virus in an Inner-City Outpatient Population: A Cross-Sectional Study.

BACKGROUND: People living with human immunodeficiency virus (PLWH) have been disproportionally affected by methicillin-resistant Staphylococcus aureus (MRSA) colonization and infection, in particular by clones USA300 and USA500. However, the contribution of epidemiological, bacterial, and immunological risk factors to the excess of S aureus in PLWH remain incompletely understood. METHODS: In this cross-sectional study, we determined the prevalence and molecular epidemiology of S aureus colonization in 93 PLWH attending an urban human immunodeficiency virus (HIV) clinic. Participants completed a structured interview assessing demographic information and risk factors for MRSA. Swabs were obtained from the nose, throat, and groin and cultured for S aureus and Staphylococcus epidermidis. RESULTS: Most participants had well controlled HIV infection (89, 96% CD4 >200). Thirty-six (39%) individuals were colonized with S aureus at 1 or more body sites, including 6 (6%) with MRSA. Regular gym use was a risk factor for S aureus but not MRSA carriage. In contrast, S epidermidis was present in almost all individuals (n = 84, 90%), predominantly in the nares (n = 66, 71%). Using generalized estimating equation models, we observed that the odds of S aureus colonization were significantly and drastically reduced when S epidermidis was detected (P = .0001). After controlling for site, gender, and age, we identified that the odds of S aureus colonization were 80% less if S epidermidis was present (adjusted odds ratio, 0.20; 95% confidence interval, .09-.45; P < .0001). CONCLUSIONS: Taken together, we observed a lower prevalence of S aureus and MRSA colonization than has been previously reported in PLWH. In this cohort, colonization with S epidermidis was protective against S aureus colonization.

Peripheral Intravenous Catheter Placement Is an Underrecognized Source of Staphylococcus aureus Bloodstream Infection.

Few studies have focused on the risks of peripheral intravenous catheters (PIVs) as sources for Staphylococcus aureus bacteremia (SAB), a life-threatening complication. We identified 34 PIV-related infections (7.6%) in a cohort of 445 patients with SAB. Peripheral intravenous catheter-related SAB was associated with significantly longer bacteremia duration and thrombophlebitis at old PIV sites rather than current PIVs.

Association of Environmental Contamination in the Home With the Risk for Recurrent Community-Associated, Methicillin-Resistant Staphylococcus aureus Infection.

IMPORTANCE: The role of environmental contamination in recurrent Staphylococcus aureus infections within households and its potential effect on intervention strategies has been debated recently. OBJECTIVE: To assess whether household environmental contamination increases the risk for recurrent infection among individuals with a community-associated methicillin-resistant S aureus (MRSA) infection. DESIGN, SETTING, AND PARTICIPANTS: This cohort study was conducted from November 1, 2011, to June 30, 2014, in the Columbia University Medical Center catchment area. All patients within 72 hours of presentation with skin or soft-tissue infections and blood, urine, or sputum cultures positive for MRSA were identified. Two hundred sixty-two patients met study inclusion criteria; 83 of these (31.7%) agreed to participate (index patients) with 214 household members. Participants were followed up for 6 months, and 62 of the 83 households (74.7%) completed follow-up. Participants and researchers were blinded to exposure status throughout the study. Follow-up was completed on June 30, 2014, and data were assessed from July 1, 2014, to February 19, 2016. EXPOSURE: Concordant environmental contamination, defined as having an isolate with the identical staphylococcal protein A and staphylococcal chromosomal cassette mec type or antibiogram type as the index patient's clinical isolate, present on 1 or more environmental surfaces at the time of a home visit to the index patient after infection. MAIN OUTCOMES AND MEASURES: Index recurrent infection, defined as any self-reported infection among the index patients during follow-up. RESULTS: One patient did not complete any follow-up. Of the remaining 82 index patients, 53 (64.6%) were female and 59 (72.0%) were Hispanic. The mean age was 30 (SD, 20; range, 1-79) years. Forty-nine of 61 MRSA infections where the clinical isolate could be obtained (80.3%) were due to the epidemic strain USA300. Among the 82 households in which a patient had an index MRSA infection, the clinical isolate was present in the environment in 20 (24.4%) and not found in 62 (75.6%). Thirty-five patients (42.7%) reported a recurrent infection during follow-up, of whom 15 (42.9%) required hospitalization. Thirteen recurrent infections were from the 20 households (65.0%) with and 22 were from the 62 households (35.5%) without environmental contamination (P = .04). Environmental contamination increased the rate of index recurrent infection (incident rate ratio, 2.05; 95% CI, 1.03-4.10; P = .04). CONCLUSIONS AND RELEVANCE: Household environmental contamination was associated with an increased rate of recurrent infection. Environmental decontamination should be considered as a strategy to prevent future MRSA infections, particularly among households where an infection has occurred.

Population Structure of Klebsiella pneumoniae Causing Bloodstream Infections at a New York City Tertiary Care Hospital: Diversification of Multidrug-Resistant Isolates.

Despite the growing importance of carbapenem-resistant Klebsiella pneumoniae (CRKP), the clonal relationships between CRKP and antibiotic-susceptible isolates remain unclear. We compared the genetic diversity and clinical features of CRKP, third-generation and/or fourth-generation cephalosporin-resistant (Ceph-R) K. pneumoniae, and susceptible K. pneumoniae isolates causing bloodstream infections at a tertiary care hospital in New York City between January 2012 and July 2013. Drug susceptibilities were determined with the Vitek 2 system. Isolates underwent multilocus sequence typing and PCR sequencing of the wzi and blaKPC genes. Clinical and microbiological data were extracted from patient records and correlated with molecular data. Among 223 patients, we identified 272 isolates. Of these, 194 were susceptible, 30 Ceph-R, and 48 CRKP, belonging to 144 sequence types (STs). Susceptible (127 STs) and Ceph-R (20 STs) isolates were highly diverse. ST258 dominated CRKP strains (12 STs, with 63% ST258). There was minimal overlap in STs between resistance groups. The blaKPC-3 gene (30%) was restricted to ST258/wzi154, whereas blaKPC-2 (70%) was observed for several wzi allele types. CRKP infections occurred more frequently among solid organ transplant (31%) and dialysis (17%) patients. Mortality rates were high overall (28%) and highest among CRKP-infected patients (59%). In multivariable analyses, advanced age, comorbidities, and disease severity were significant predictors of 30-day mortality rates, whereas the K. pneumoniae susceptibility phenotype was not. Among CRKP infections, we observed a borderline significant association of increased mortality rates with ST258 and the wzi154 allele. Although the clonal spread of ST258 continues to contribute substantially to the dissemination of CRKP, non-ST258 strains appear to be evolving. Further investigations into the mechanisms promoting CRKP diversification and the effects of clonal backgrounds on outcomes are warranted.

Emergence of sequence type 398 as a community- and healthcare-associated methicillin-susceptible staphylococcus aureus in northern Manhattan.

The methicillin-susceptible Staphylococcus aureus (MSSA) clone sequence type (ST) 398 has increasingly been identified as a pathogen in diverse geographic settings, yet its epidemiology remains incompletely understood. In this case-control study of MSSA infections, we identified ST398 MSSA as both a major community- and hospital-associated MSSA pathogen in the Dominican neighborhood of northern Manhattan.

Molecular characterization of methicillin-susceptible Staphylococcus aureus clinical isolates in the United States, 2004 to 2010.

While much is known about the geographic distribution of different clonal types of methicillin-resistant Staphylococcus aureus (MRSA), few studies have assessed the molecular epidemiology of methicillin-susceptible S. aureus (MSSA), despite its continued clinical importance. In each U.S. Census region, reference laboratories collected successive MSSA isolates from patients with invasive or superficial staphylococcal infections for use in the Tigecycline Evaluation and Surveillance Trial. All isolates from the periods of 2004 to 2005 and 2009 to 2010 underwent antimicrobial susceptibility testing and characterization of their staphylococcal protein A (spa) type. Of the 708 isolates analyzed, 274 spa types were identified and divided into 15 genetic clusters. The most common clones were spa t002 (n = 63, 8.9%) and t008 (n = 56, 7.9%). While the distribution of the predominant spa types did not differ by U.S. Census region or time period, spa t008 was nearly twice as common in community skin and soft tissue infections than in nosocomial bloodstream infections (11.1% versus 5.6%, respectively; P = 0.008). Despite such differences, both community and nosocomial settings had diverse staphylococcal clonal types representing all major spa clusters. In contrast to those of MRSA, MSSA infectious isolates show wide genetic diversity without clear geographical or temporal clustering. Notably, the prevalent MSSA strains (spa t002 and spa t008) are analogous to the predominant MRSA clones, further demonstrating the importance of both lineages.

High prevalence of colonization with Staphylococcus aureus clone USA300 at multiple body sites among sexually transmitted disease clinic patients: an unrecognized reservoir.

Extranasal colonization is increasingly recognized as an important reservoir for Staphylococcus aureus among high-risk populations. We conducted a cross-sectional study of multiple body site colonization among 173 randomly selected STD clinic patients in Baltimore, Maryland. Staphylococcal carriage at extranasal sites, including the oropharynx, groin, rectum, and genitals, was common among study subjects. The USA300 clone was particularly associated with multiple sites of colonization compared with non-USA300 strains (p = .01). Given their high burden of multi-site colonization and confluence of established staphylococcal risk factors, STD clinic patients may represent a community-based reservoir for S. aureus and be well suited for innovative infection control initiatives.

Identification of a highly transmissible animal-independent Staphylococcus aureus ST398 clone with distinct genomic and cell adhesion properties.

UNLABELLED: A methicillin-resistant Staphylococcus aureus (MRSA) clone known as ST398 has emerged as a major cause of acute infections in individuals who have close contact with livestock. More recently, the emergence of an animal-independent ST398 methicillin-sensitive S. aureus (MSSA) clone has been documented in several countries. However, the limited surveillance of MSSA has precluded an accurate assessment of the global spread of ST398 and its clinical relevance. Here we provide evidence that ST398 is a frequent source of MSSA infections in northern Manhattan and is readily transmitted between individuals in households. This contrasts with the limited transmissibility of livestock-associated ST398 (LA-ST398) MRSA strains between humans. Our whole-genome sequence analysis revealed that the chromosome of the human-associated ST398 MSSA clone is smaller than that of the LA-ST398 MRSA reference strain S0385, due mainly to fewer mobile genetic elements (MGEs). In contrast, human ST398 MSSA isolates harbored the prophage ϕ3 and the human-specific immune evasion cluster (IEC) genes chp and scn. While most of the core genome was conserved between the human ST398 MSSA clone and S0385, these strains differed substantially in their repertoire and composition of intact adhesion genes. These genetic changes were associated with significantly enhanced adhesion of human ST398 MSSA isolates to human skin keratinocytes and keratin. We propose that the human ST398 MSSA clone can spread independent of animal contact using an optimized repertoire of MGEs and adhesion molecules adapted to transmission among humans. IMPORTANCE: Staphylococcus aureus strains have generally been considered to be species specific. However, cross-species transfers of S. aureus clones, such as ST398 methicillin-resistant S. aureus (MRSA), from swine to humans have been reported. Recently, we observed the emergence of ST398 methicillin-susceptible S. aureus (MSSA) as a colonizing strain of humans in northern Manhattan. Here we report that ST398 is a frequent cause of MSSA infections in this urban setting. The ST398 MSSA clone was readily transmitted within households, independent of animal contact. We discovered that human ST398 MSSA genomes were smaller than that of the LA-ST398 strain S0385 due to fewer mobile genetic elements. Human and LA-ST398 strains also differed in their composition of adhesion genes and their ability to bind to human skin keratinocytes, providing a potential mechanism of S. aureus host adaptation. Our findings illustrate the importance of implementing molecular surveillance of MSSA given the evidence for the rapid and clinically undetected spread of ST398 MSSA.