• donor calls
• infectious disease
• organ evaluation

• Humans
• Surveys and Questionnaires
• Organ Transplantation/adverse effects
• Tissue Donors
• Tissue and Organ Procurement/methods
• Communicable Diseases
• Donor Selection/standards
• Donor Selection/methods
• Transplant Recipients/statistics & numerical data

• Adult
• Humans
• Child
• United States/epidemiology
• Staphylococcus aureus
• Retrospective Studies
• Organ Transplantation/adverse effects
• Staphylococcal Infections/diagnosis
• Staphylococcal Infections/epidemiology
• Bacteremia/diagnosis
• Bacteremia/epidemiology
• Risk Factors

• antibiotic prophylaxis
• liver transplant
• multi-drug-resistant organisms (MDRO)
• post liver transplant infections
• preventive measures

• Humans
• Methicillin-Resistant Staphylococcus aureus
• Staphylococcal Infections
• Multilocus Sequence Typing
• Phylogeny
• Health Personnel

• R01 AI121146 NIAID NIH HHS
• R25 GM055036 NIGMS NIH HHS
• U19 AI110820 NIAID NIH HHS

• Carbapenem-resistant Klebsiella pneumoniae
• Donor-derived infection
• Kidney transplantation
• Possible donor-derived carbapenem-resistant Klebsiella pneumoniae
• Preservation fluid

• bacteria
• donor-derived infection
• multidrug-resistant organisms

• Drug Resistance, Multiple, Bacterial
• Humans
• Organ Transplantation/adverse effects
• Retrospective Studies
• Tissue Donors
• Transplant Recipients

• K01-AI137317 NIH HHS
• K24 AI080942 NIAID NIH HHS
• L30 AI133754 NIAID NIH HHS
• K24-AI080942 NIH HHS
• UM1 AI104681 NIAID NIH HHS
• U54 CK000163 NCEZID CDC HHS
• K01 AI137317 NIAID NIH HHS

• Comparative genomics
• Complete genome assembly
• Methicillin-resistant
• Multi-drug resistance
• Nanopore and illumina sequencing
• Ocular infection
• Plasmid Tn552 transposon
• Staphylococcus aureus

• Infectious disease
• Innate immunity

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital.

Whole-genome sequencing (WGS) of Staphylococcus aureus is increasingly used as part of infection prevention practices. In this study, we established a long-read technology-based WGS screening program of all first-episode methicillin-resistant Staphylococcus aureus (MRSA) blood infections at a major urban hospital. A survey of 132 MRSA genomes assembled from long reads enabled detailed characterization of an outbreak lasting several months of a CC5/ST105/USA100 clone among 18 infants in a neonatal intensive care unit (NICU). Available hospital-wide genome surveillance data traced the origins of the outbreak to three patients admitted to adult wards during a 4-month period preceding the NICU outbreak. The pattern of changes among complete outbreak genomes provided full spatiotemporal resolution of its progression, which was characterized by multiple subtransmissions and likely precipitated by equipment sharing between adults and infants. Compared to other hospital strains, the outbreak strain carried distinct mutations and accessory genetic elements that impacted genes with roles in metabolism, resistance, and persistence. This included a DNA recognition domain recombination in the hsdS gene of a type I restriction modification system that altered DNA methylation. Transcriptome sequencing (RNA-Seq) profiling showed that the (epi)genetic changes in the outbreak clone attenuated agr gene expression and upregulated genes involved in stress response and biofilm formation. Overall, our findings demonstrate the utility of long-read sequencing for hospital surveillance and for characterizing accessory genomic elements that may impact MRSA virulence and persistence.

• MRSA
• NICU outbreak
• genome analysis

• Methicillin-resistant Staphylococcus aureus
• bacteremia
• pneumonia
• prevention
• solid organ transplantation
• treatment

• MRSA
• antimicrobial resistance
• evolution
• virulence

• Animals
• Anti-Bacterial Agents/pharmacology
• Child
• Chlorhexidine/pharmacology
• Community-Acquired Infections/drug therapy
• Community-Acquired Infections/microbiology
• Genome, Bacterial/genetics
• Humans
• Methicillin-Resistant Staphylococcus aureus/drug effects
• Methicillin-Resistant Staphylococcus aureus/genetics
• Mice
• Microbial Sensitivity Tests/methods
• Mupirocin/pharmacology
• Phylogeny
• Plasmids/genetics
• Staphylococcal Infections/drug therapy
• Staphylococcal Infections/microbiology
• Virulence/genetics

• R01 AI099394 NIAID NIH HHS
• S10 OD018522 NIH HHS
• R01 AI103268 NIAID NIH HHS
• R01 AI105129 NIAID NIH HHS
• HHSN272201400019C NIAID NIH HHS
• T32 AI007180 NIAID NIH HHS
• S10 OD023423 NIH HHS
• KL2 TR001435 NCATS NIH HHS
• K08 AI101005 NIAID NIH HHS
• MR/R015600/1 Medical Research Council
• R01 AI119145 NIAID NIH HHS
• T32 AI007647 NIAID NIH HHS
• R01 AI137526 NIAID NIH HHS

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality.

Therapy for bacteremia caused by Staphylococcus aureus is often ineffective, even when treatment conditions are optimal according to experimental protocols. Adapted subclones, such as those bearing mutations that attenuate agr-mediated virulence activation, are associated with persistent infection and patient mortality. To identify additional alterations in agr-defective mutants, we sequenced and assembled the complete genomes of clone pairs from colonizing and infected sites of several patients in whom S. aureus demonstrated a within-host loss of agr function. We report that events associated with agr inactivation result in agr-defective blood and nares strain pairs that are enriched in mutations compared to pairs from wild-type controls. The random distribution of mutations between colonizing and infecting strains from the same patient, and between strains from different patients, suggests that much of the genetic complexity of agr-defective strains results from prolonged infection or therapy-induced stress. However, in one of the agr-defective infecting strains, multiple genetic changes resulted in increased virulence in a murine model of bloodstream infection, bypassing the mutation of agr and raising the possibility that some changes were selected. Expression profiling correlated the elevated virulence of this agr-defective mutant to restored expression of the agr-regulated ESAT6-like type VII secretion system, a known virulence factor. Thus, additional mutations outside the agr locus can contribute to diversification and adaptation during infection by S. aureus agr mutants associated with poor patient outcomes.

• Staphylococcus aureus
• gene regulation
• genome analysis

Staphylococcus aureus is an opportunistic pathogen that causes a range of serious infections associated with significant morbidity, by strains increasingly resistant to antibiotics. However, to date all candidate vaccines have failed to induce protective immune responses in humans. We need a more comprehensive understanding of the antigenic targets important in the context of human infection. To investigate infection-associated immune responses, patients were sampled at initial presentation and during convalescence from three types of clinical infection; skin and soft tissue infection (SSTI), prosthetic joint infection (PJI) and pediatric hematogenous osteomyelitis (PHO). Reactivity of serum IgG was tested with an array of recombinant proteins, representing over 2,652 in-vitro-translated open reading frames (ORFs) from a community-acquired methicillin-resistant S. aureus USA300 strain. High-level reactivity was demonstrated for 104 proteins with serum IgG in all patient samples. Overall, high-level IgG-reactivity was most commonly directed against a subset of secreted proteins. Although based on limited surveys, we found subsets of S. aureus proteins with differential reactivity with serum samples from patients with different clinical syndromes. Together, our studies have revealed a hierarchy within the diverse proteins of the S. aureus “immunome”, which will help to advance efforts to develop protective immunotherapeutic agents.

• bacteremia
• liver transplantation
• methicillin-resistant Staphylococcus aureus
• morbidity
• mortality
• risk factors

Staphylococcus aureus is a Gram-positive opportunistic pathogen that causes superficial and invasive infections in the hospital and community. High mortality from infection emphasizes the need for improved methods for prevention and treatment. Although S. aureus possesses an arsenal of virulence factors that contribute to evasion of host defenses, few studies have examined long-term humoral and B-cell responses. Adults with acute-phase skin and soft tissue infections were recruited; blood samples were obtained; and S. aureus isolates, including methicillin-resistant strains, were subjected to genomic sequence analysis. In comparisons of acute-phase sera with convalescent-phase sera, a minority (37.5%) of patients displayed 2-fold or greater increases in antibody titers against three or more S. aureus antigens, whereas nearly half exhibited no changes, despite the presence of toxin genes in most infecting strains. Moreover, enhanced antibody responses waned over time, which could reflect a defect in B-cell memory or long-lived plasma cells. However, memory B cells reactive with a range of S. aureus antigens were prevalent at both acute-phase and convalescent-phase time points. While some memory B cells exhibited toxin-specific binding, those cross-reactive with structurally related leucocidin subunits were dominant across patients, suggesting the targeting of conserved epitopes. Memory B-cell reactivity correlated with serum antibody levels for selected S. aureus exotoxins, suggesting a relationship between the cellular and humoral compartments. Overall, although there was no global defect in the representation of anti-S. aureus memory B cells, there was evidence of restrictions in the range of epitopes recognized, which may suggest potential therapeutic approaches for augmenting host defenses.

The contribution of B-cell memory and long-term antibody responses to host defenses against S. aureus exotoxins remains poorly understood. Our studies confirmed that infection did not commonly lead to enhanced long-term humoral responses. Whereas circulating memory B cells against S. aureus secreted exotoxins were prevalent, they were dominated by cross-reactivity with structurally related leucocidin subunits, consistent with recognition of conserved epitopes. These findings also provide the first evidence of a relationship between the reactivity of antistaphylococcal circulating memory B cells and serum antibody levels. In general, infection was not associated with a global defect in B-cell memory for S. aureus secreted factors, and responses were highly dominated by cross-reactivity to structurally related exotoxins, which arguably may alone be suboptimal in providing host defenses. Our studies illuminate aspects of the S. aureus-host relationship that may better inform strategies for the development of an effective protective vaccine.

• MRSA
• Staphylococcus aureus
• antibodies
• host response
• host-pathogen interactions
• leukocidins
• memory B cells
• pore-forming toxins
• skin and soft tissue infection (SSTI)
• superantigens

• Bacteria
• Donation after cardiac death donor
• Infection
• Liver transplant
• Multidrug resistant
• Transmission

• Adult
• Allografts/microbiology
• Anti-Bacterial Agents/therapeutic use
• Bacterial Infections/drug therapy
• Bacterial Infections/epidemiology
• Bacterial Infections/microbiology
• Bacterial Infections/transmission
• China/epidemiology
• Drug Resistance, Multiple, Bacterial
• End Stage Liver Disease/surgery
• Enterobacter aerogenes/isolation & purification
• Enterobacter aerogenes/physiology
• Female
• Graft Survival
• Gram-Positive Bacteria/isolation & purification
• Humans
• Klebsiella pneumoniae/isolation & purification
• Klebsiella pneumoniae/physiology
• Liver/microbiology
• Liver/surgery
• Liver Transplantation/adverse effects
• Liver Transplantation/methods
• Male
• Retrospective Studies
• Tissue Donors
• Tissue and Organ Harvesting/adverse effects
• Young Adult

• bacterial infections
• disease transmission, infectious
• kidney transplantation
• mycoses
• unrelated donors

• Adolescent
• Adult
• Bacterial Infections/transmission
• Child
• Child, Preschool
• China
• Death
• Female
• Humans
• Kidney Transplantation
• Male
• Middle Aged
• Mycoses/transmission
• Risk Factors
• Tissue Donors
• Treatment Outcome
• Young Adult

• MRSA
• Staphylococcus aureus
• genome
• host range
• pathogenesis

Kubes et al. show that methicillin-resistant Staphylococcus aureus (MRSA) survive and proliferate inside Kupffer cells. Intracellular MRSA is resistant to neutrophil-killing and antibiotics treatment and, when released into the circulation, can infect other organs.

Methicillin-resistant Staphylococcus aureus (MRSA) bacteremia is reaching epidemic proportions causing morbidity, mortality, and chronic disease due to relapses, suggesting an intracellular reservoir. Using spinning-disk confocal intravital microscopy to track MRSA-GFP in vivo, we identified that within minutes after intravenous infection MRSA is primarily sequestered and killed by intravascular Kupffer cells (KCs) in the liver. However, a minority of the Staphylococci overcome the KC’s antimicrobial defenses. These bacteria survive and proliferate for many days within this intracellular niche, where they remain undetected by recruited neutrophils. Over time, the KCs lyse, releasing bacteria into the circulation, enabling dissemination to other organs such as the kidneys. Vancomycin, the antibiotic of choice to treat MRSA bacteremia, could not penetrate the KCs to eradicate intracellular MRSA. However, based on the intravascular location of these specific macrophages, we designed a liposomal formulation of vancomycin that is efficiently taken up by KCs and diminished the intracellular MRSA. Targeting the source of the reservoir dramatically protected the liver but also dissemination to other organs, and prevented mortality. This vancomycin formulation strategy could help treat patients with Staphylococcal bacteremia without a need for novel antibiotics by targeting the previously inaccessible intracellular reservoir in KCs.

• MRSA clones
• MRSA epidemiology
• Whole-genome sequencing

• Evolution
• MRSA
• USA300

• Adolescent
• Adult
• Aged
• Aged, 80 and over
• Child
• Child, Preschool
• Community-Acquired Infections/epidemiology
• Community-Acquired Infections/microbiology
• Evolution, Molecular
• Genome, Bacterial/genetics
• Humans
• Infant
• Methicillin-Resistant Staphylococcus aureus/classification
• Methicillin-Resistant Staphylococcus aureus/genetics
• Middle Aged
• Molecular Epidemiology
• Phylogeny
• Staphylococcal Infections/epidemiology
• Staphylococcal Infections/microbiology
• United States/epidemiology
• Whole Genome Sequencing
• Young Adult

• Wellcome Trust
• 098051 Wellcome Trust

• Donor-derived infections
• Multidrug-resistant bacteria
• Solid organ transplant

Staphylococcus aureus is an important model organism and pathogen. This S. aureus proteome overview details shared and specific proteins and selected virulence-relevant protein complexes from representative strains of all three major clades. To determine the strain distribution and major clades we used a refined strain comparison combining ribosomal RNA, MLST markers, and looking at highly-conserved regions shared between strains. This analysis shows three sub-clades (A–C) for S. aureus. As calculations are complex and strain annotation is quite time consuming we compare here key representatives of each clade with each other: model strains COL, USA300, Newman, and HG001 (clade A), model strain N315 and Mu50 (clade B) and ED133 and MRSA252 (clade C). We look at these individual proteomes and compare them to a background of 64 S. aureus strains. There are overall 13,284 S. aureus proteins not part of the core proteome which are involved in different strain-specific or more general complexes requiring detailed annotation and new experimental data to be accurately delineated. By comparison of the eight representative strains, we identify strain-specific proteins (e.g., 18 in COL, 105 in N315 and 44 in Newman) that characterize each strain and analyze pathogenicity islands if they contain such strain-specific proteins. We identify strain-specific protein repertoires involved in virulence, in cell wall metabolism, and phosphorylation. Finally we compare and analyze protein complexes conserved and well-characterized among S. aureus (a total of 103 complexes), as well as predict and analyze several individual protein complexes, including structure modeling in the three clades.

• Staphylococcus aureus
• model strain
• protein complexes
• proteome

Infections are an important cause of morbidity and mortality in solid organ transplant recipients. Consequently, infection prevention is an essential component of any organ transplant program. Given their frequent and often prolonged contact with the healthcare system, solid organ transplant recipients are at high risk for healthcare-associated infections, including those caused by antibiotic-resistant organisms. In this chapter we review several different healthcare-associated infections of importance to transplant recipients, including those caused by bacterial, viral, and fungal organisms. We also describe infection prevention and control strategies applicable to this patient population. These practices focus on clinical interventions and environmental controls designed to prevent the spread of potentially pathogenic organisms in the healthcare setting. We also describe post-exposure interventions applicable to solid organ transplant recipients exposed to potential pathogens in order to reduce their risk of subsequent infection.

The surface capsular polysaccharide (CP) is a virulence factor that has been used as an antigen in several successful vaccines against bacterial pathogens. A vaccine has not yet been licensed against Staphylococcus aureus, although two multicomponent vaccines that contain CP antigens are in clinical trials. In this study, we evaluated CP production in USA300 methicillin-resistant S. aureus (MRSA) isolates that have become the predominant community-associated MRSA clones in the United States. We found that all 167 USA300 MRSA and 50 USA300 methicillin-susceptible S. aureus (MSSA) isolates were CP negative (CP⁻). Moreover, all 16 USA500 isolates, which have been postulated to be the progenitor lineage of USA300, were also CP⁻. Whole-genome sequence analysis of 146 CP⁻ USA300 MRSA isolates revealed they all carry a cap5 locus with 4 conserved mutations compared with strain Newman. Genetic complementation experiments revealed that three of these mutations (in the cap5 promoter, cap5D nucleotide 994, and cap5E nucleotide 223) ablated CP production in USA300 and that Cap5E75 Asp, located in the coenzyme-binding domain, is essential for capsule production. All but three USA300 MSSA isolates had the same four cap5 mutations found in USA300 MRSA isolates. Most isolates with a USA500 pulsotype carried three of these four USA300-specific mutations, suggesting the fourth mutation occurred in the USA300 lineage. Phylogenetic analysis of the cap loci of our USA300 isolates as well as publicly available genomes from 41 other sequence types revealed that the USA300-specific cap5 mutations arose sequentially in S. aureus in a common ancestor of USA300 and USA500 isolates.

The USA300 MRSA clone emerged as a community-associated pathogen in the United States nearly 20 years ago. Since then, it has rapidly disseminated and now causes health care-associated infections. This study shows that the CP-negative (CP⁻) phenotype has persisted among USA300 isolates and is a universal and characteristic trait of this highly successful MRSA lineage. It is important to note that a vaccine consisting solely of CP antigens would not likely demonstrate high efficacy in the U.S. population, where about half of MRSA isolates comprise USA300. Moreover, conversion of a USA300 strain to a CP-positive (CP⁺) phenotype is unlikely in vivo or in vitro since it would require the reversion of 3 mutations. We have also established that USA300 MSSA isolates and USA500 isolates are CP⁻ and provide new insight into the evolution of the USA300 and USA500 lineages.