Multidrug-Resistant Methicillin-Resistant Staphylococcus aureus Associated with Bacteremia and Monocyte Evasion, Rio de Janeiro, Brazil.

We typed 600 methicillin-resistant Staphylococcus aureus (MRSA) isolates collected in 51 hospitals in the Rio de Janeiro, Brazil, metropolitan area during 2014-2017. We found that multiple new clonal complex (CC) 5 sequence types had replaced previously dominant MRSA lineages in hospitals. Whole-genome analysis of 208 isolates revealed an emerging sublineage of multidrug-resistant MRSA, sequence type 105, staphylococcal cassette chromosome mec II, spa t002, which we designated the Rio de Janeiro (RdJ) clone. Using molecular clock analysis, we hypothesized that this lineage began to expand in the Rio de Janeiro metropolitan area in 2009. Multivariate analysis supported an association between bloodstream infections and the CC5 lineage that includes the RdJ clone. Compared with other closely related isolates, representative isolates of the RdJ clone more effectively evaded immune function related to monocytic cells, as evidenced by decreased phagocytosis rate and increased numbers of viable unphagocytosed (free) bacteria after in vitro exposure to monocytes.

Copper Resistance Promotes Fitness of Methicillin-Resistant Staphylococcus aureus during Urinary Tract Infection.

Urinary tract infection (UTI) is one of the most common infectious conditions affecting people in the United States and around the world. Our knowledge of the host-pathogen interaction during UTI caused by Gram-positive bacterial uropathogens is limited compared to that for Gram-negative pathogens. Here, we investigated whether copper and the primary copper-containing protein, ceruloplasmin, are mobilized to urine during naturally occurring UTI caused by Gram-positive uropathogens in patients. Next, we probed the role of copper resistance in the fitness of methicillin-resistant Staphylococcus aureus (MRSA) during experimental UTI in a murine model. Our findings demonstrate that urinary copper and ceruloplasmin content are elevated during UTI caused by Enterococcus faecalis, S. aureus, S. epidermidis, and S. saprophyticus. MRSA strains successfully colonize the urinary tract of female CBA mice with selective induction of inflammation in the kidneys but not the bladder. MRSA mutants lacking CopL, a copper-binding cell surface lipoprotein, and the ACME genomic region containing copL, exhibit decreased fitness in the mouse urinary tract compared to parental strains. Copper sensitivity assays, cell-associated copper and iron content, and bioavailability of iron during copper stress demonstrate that homeostasis of copper and iron is interlinked in S. aureus. Importantly, relative fitness of the MRSA mutant lacking the ACME region is further decreased in mice that receive supplemental copper compared to the parental strain. In summary, copper is mobilized to the urinary tract during UTI caused by Gram-positive pathogens, and copper resistance is a fitness factor for MRSA during UTI. IMPORTANCE Urinary tract infection (UTI) is an extremely common infectious condition affecting people throughout the world. Increasing antibiotic resistance in pathogens causing UTI threatens our ability to continue to treat patients in the clinics. Better understanding of the host-pathogen interface is critical for development of novel interventional strategies. Here, we sought to elucidate the role of copper in host-Staphylococcus aureus interaction during UTI. Our results reveal that copper is mobilized to the urine as a host response in patients with UTI. Our findings from the murine model of UTI demonstrate that copper resistance is involved in the fitness of methicillin-resistant S. aureus (MRSA) during interaction with the host. We also establish a critical link between adaptation to copper stress and iron homeostasis in S. aureus.

Local Diversification of Methicillin- Resistant Staphylococcus aureus ST239 in South America After Its Rapid Worldwide Dissemination.

The global spread of specific clones of methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem, and understanding the dynamics of geographical spread requires worldwide surveillance. Over the past 20 years, the ST239 lineage of MRSA has been recognized as an emerging clone across the globe, with detailed studies focusing on isolates from Europe and Asia. Less is known about this lineage in South America, and, particularly, Brazil where it was the predominant lineage of MRSA in the early 1990s to 2000s. To gain a better understanding about the introduction and spread of ST239 MRSA in Brazil we undertook a comparative phylogenomic analysis of ST239 genomes, adding seven completed, closed Brazilian genomes. Brazilian ST239 isolates grouped in a subtree with those from South American, and Western, romance-language-speaking, European countries, here designated the South American clade. After an initial worldwide radiation in the 1960s and 1970s, we estimate that ST239 began to spread in South America and Brazil in approximately 1988. This clone demonstrates specific genomic changes that are suggestive of local divergence and adaptational change including agrC single-nucleotide polymorphisms variants, and a distinct pattern of virulence-associated genes (mainly the presence of the chp and the absence of sea and sasX). A survey of a geographically and chronologically diverse set of 100 Brazilian ST239 isolates identified this virulence genotype as the predominant pattern in Brazil, and uncovered an unexpectedly high prevalence of agr-dysfunction (30%). ST239 isolates from Brazil also appear to have undergone transposon (IS256) insertions in or near global regulatory genes (agr and mgr) that likely led to rapid reprogramming of bacterial traits. In general, the overall pattern observed in phylogenomic analyses of ST239 is of a rapid initial global radiation, with subsequent local spread and adaptation in multiple different geographic locations. Most ST239 isolates harbor the ardA gene, which we show here to have in vivo anti-restriction activity. We hypothesize that this gene may have improved the ability of this lineage to acquire multiple resistance genes and distinct virulence-associated genes in each local context. The allopatric divergence pattern of ST239 also may suggest strong selective pressures for specific traits in different geographical locations.

Culture-Independent Analysis of Pediatric Bronchoalveolar Lavage Specimens.

RATIONALE: The clinical utility of culture-independent testing of pediatric BAL specimens is unknown. In addition, the variability of the pediatric pulmonary microbiome with patient characteristics is not well understood. OBJECTIVES: To compare testing with 16S rRNA gene-based sequencing to conventional cultures of BAL specimens in children Methods: Study subjects were not more than 22 years old and underwent BAL from May 2013 to August 2015 as part of clinical care. DNA extracted from BAL specimens was used for 16S rRNA gene-based analysis, and results were compared with routine cultures from the same samples. Indices of microbial diversity and relative taxon abundances were compared on the basis of subject characteristics (diagnosis and antibiotic use). RESULTS: From 81 participants (male, 51%; median age, 9 yr), 89 samples were collected. The 16S rRNA genes of 77 samples (86.5%) from 70 subjects were successfully analyzed. These 70 subjects included 23 with cystic fibrosis, 19 who were immunocompromised, and 28 who were nonimmunocompromised. Of 68 organisms identified in culture, 16S rRNA gene-based analyses detected corresponding taxa in 66 (97.1%) and also identified potentially clinically significant organisms missed by cultures (e.g., Staphylococcus, Legionella, and Pseudomonas). Taxa that varied significantly with diagnosis and antibiotic use included Veillonella, Corynebacterium, Haemophilus, and Streptococcus. The microbiota of cystic fibrosis samples was less diverse. A "core" group of 15 taxa present in all three diagnosis groups was identified. CONCLUSIONS: Culture-independent analysis was concordant with routine cultures and showed the potential to detect noncultured pathogens. Although culture-independent testing identified relative changes in organism abundance associated with clinical characteristics, distinct microbiome profiles associated with disease states were not identified.

Lambda Interferon Restructures the Nasal Microbiome and Increases Susceptibility to Staphylococcus aureus Superinfection.

UNLABELLED: Much of the morbidity and mortality associated with influenza virus respiratory infection is due to bacterial coinfection with pathogens that colonize the upper respiratory tract such as methicillin-resistant Staphylococcus aureus (MRSA) and Streptococcus pneumoniae. A major component of the immune response to influenza virus is the production of type I and III interferons. Here we show that the immune response to infection with influenza virus causes an increase and restructuring of the upper respiratory microbiota in wild-type (WT) mice but not in Il28r(-/-) mutant mice lacking the receptor for type III interferon. Mice lacking the IL-28 receptor fail to induce STAT1 phosphorylation and expression of its regulator, SOCS1. Il28r(-/-) mutant mice have increased expression of interleukin-22 (IL-22), as well as Ngal and RegIIIγ, in the nasal cavity, the source of organisms that would be aspirated to cause pneumonia. Proteomic analysis reveals changes in several cytoskeletal proteins that contribute to barrier function in the nasal epithelium that may contribute to the effects of IL-28 signaling on the microbiota. The importance of the effects of IL-28 signaling in the pathogenesis of MRSA pneumonia after influenza virus infection was confirmed by showing that WT mice nasally colonized before or after influenza virus infection had significantly higher levels of infection in the upper airways, as well as significantly greater susceptibility to MRSA pneumonia than Il28r(-/-) mutant mice did. Our results suggest that activation of the type III interferon in response to influenza virus infection has a major effect in expanding the upper airway microbiome and increasing susceptibility to lower respiratory tract infection. IMPORTANCE: S. aureus and influenza virus are important respiratory pathogens, and coinfection with these organisms is associated with significant morbidity and mortality. The ability of influenza virus to increase susceptibility to S. aureus infection is less well understood. We show here that influenza virus leads to a change in the upper airway microbiome in a type III interferon-dependent manner. Mice lacking the type III interferon receptor have altered STAT1 and IL-22 signaling. In coinfection studies, mice without the type III interferon receptor had significantly less nasal S. aureus colonization and subsequent pneumonia than infected WT mice did. This work demonstrates that type III interferons induced by influenza virus contribute to nasal colonization and pneumonia due to S. aureus superinfection.

Parallel Epidemics of Community-Associated Methicillin-Resistant Staphylococcus aureus USA300 Infection in North and South America.

BACKGROUND: The community-associated methicillin-resistant Staphylococcus aureus (CA-MRSA) epidemic in the United States is attributed to the spread of the USA300 clone. An epidemic of CA-MRSA closely related to USA300 has occurred in northern South America (USA300 Latin-American variant, USA300-LV). Using phylogenomic analysis, we aimed to understand the relationships between these 2 epidemics. METHODS: We sequenced the genomes of 51 MRSA clinical isolates collected between 1999 and 2012 from the United States, Colombia, Venezuela, and Ecuador. Phylogenetic analysis was used to infer the relationships and times since the divergence of the major clades. RESULTS: Phylogenetic analyses revealed 2 dominant clades that segregated by geographical region, had a putative common ancestor in 1975, and originated in 1989, in North America, and in 1985, in South America. Emergence of these parallel epidemics coincides with the independent acquisition of the arginine catabolic mobile element (ACME) in North American isolates and a novel copper and mercury resistance (COMER) mobile element in South American isolates. CONCLUSIONS: Our results reveal the existence of 2 parallel USA300 epidemics that shared a recent common ancestor. The simultaneous rapid dissemination of these 2 epidemic clades suggests the presence of shared, potentially convergent adaptations that enhance fitness and ability to spread.

CD4+ T cells promote the pathogenesis of Staphylococcus aureus pneumonia.

We postulated that the activation of proinflammatory signaling by methicillin-resistant Staphylococcus aureus (MRSA) strain USA300 is a major factor in the pathogenesis of severe pneumonia and a target for immunomodulation. Local activation of T cells in the lung was a conserved feature of multiple strains of S. aureus, in addition to USA300. The pattern of Vß chain activation was consistent with known superantigens, but deletion of SelX or SEK and SEQ was not sufficient to prevent T-cell activation, indicating the participation of multiple genes. Using Rag2(-/-), Cd4(-/-), and Cd28(-/-) mice, we observed significantly improved clearance of MRSA from the airways and decreased lung pathology, compared with findings for wild-type controls. The improved outcome correlated with decreased production of proinflammatory cytokines (tumor necrosis factor, KC, interleukin 6, and interleukin 1ß). Our data suggest that T-cell-mediated hypercytokinemia induced by infection with MRSA strain USA300 contributes to pathogenesis and may be a therapeutic target for improving outcomes of this common infection in a clinical setting.

Evolution of hypervirulence by a MRSA clone through acquisition of a transposable element.

Staphylococcus aureus has evolved as a pathogen that causes a range of diseases in humans. There are two dominant modes of evolution thought to explain most of the virulence differences between strains. First, virulence genes may be acquired from other organisms. Second, mutations may cause changes in the regulation and expression of genes. Here we describe an evolutionary event in which transposition of an IS element has a direct impact on virulence gene regulation resulting in hypervirulence. Whole-genome analysis of a methicillin-resistant S. aureus (MRSA) strain USA500 revealed acquisition of a transposable element (IS256) that is absent from close relatives of this strain. Of the multiple copies of IS256 found in the USA500 genome, one was inserted in the promoter sequence of repressor of toxins (Rot), a master transcriptional regulator responsible for the expression of virulence factors in S. aureus. We show that insertion into the rot promoter by IS256 results in the derepression of cytotoxin expression and increased virulence. Taken together, this work provides new insight into evolutionary strategies by which S. aureus is able to modify its virulence properties and demonstrates a novel mechanism by which horizontal gene transfer directly impacts virulence through altering toxin regulation.

Genome Sequence of Bacterial Interference Strain Staphylococcus aureus 502A.

Staphylococcus aureus 502A was a strain used in bacterial interference programs during the 1960s and early 1970s. Infants were deliberately colonized with 502A with the goal of preventing colonization with more invasive strains. We present the completed genome sequence of this organism.

Emergence of the epidemic methicillin-resistant Staphylococcus aureus strain USA300 coincides with horizontal transfer of the arginine catabolic mobile element and speG-mediated adaptations for survival on skin.

UNLABELLED: The arginine catabolic mobile element (ACME) is the largest genomic region distinguishing epidemic USA300 strains of methicillin-resistant Staphylococcus aureus (MRSA) from other S. aureus strains. However, the functional relevance of ACME to infection and disease has remained unclear. Using phylogenetic analysis, we have shown that the modular segments of ACME were assembled into a single genetic locus in Staphylococcus epidermidis and then horizontally transferred to the common ancestor of USA300 strains in an extremely recent event. Acquisition of one ACME gene, speG, allowed USA300 strains to withstand levels of polyamines (e.g., spermidine) produced in skin that are toxic to other closely related S. aureus strains. speG-mediated polyamine tolerance also enhanced biofilm formation, adherence to fibrinogen/fibronectin, and resistance to antibiotic and keratinocyte-mediated killing. We suggest that these properties gave USA300 a major selective advantage during skin infection and colonization, contributing to the extraordinary evolutionary success of this clone. IMPORTANCE: Over the past 15 years, methicillin-resistant Staphylococcus aureus (MRSA) has become a major public health problem. It is likely that adaptations in specific MRSA lineages (e.g., USA300) drove the spread of MRSA across the United States and allowed it to replace other, less-virulent S. aureus strains. We suggest that one major factor in the evolutionary success of MRSA may have been the acquisition of a gene (speG) that allows S. aureus to evade the toxicity of polyamines (e.g., spermidine and spermine) that are produced in human skin. Polyamine tolerance likely gave MRSA multiple fitness advantages, including the formation of more-robust biofilms, increased adherence to host tissues, and resistance to antibiotics and killing by human skin cells.