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USA300 has remained the dominant community and healthcare associated methicillin-resistantStaphylococcus aureus(MRSA) clone in the United States and in northern South America for at least the past 20 years. In this time, it has experienced epidemic spread in both of these locations. However, its pre-epidemic evolutionary history and origins are incompletely understood. Large sequencing databases, such as NCBI, PATRIC, and Staphopia, contain clues to the early evolution of USA300 in the form of sequenced genomes of USA300 isolates that are representative of lineages that diverged prior to the establishment of the South American (SAE) and North American (NAE) epidemics. In addition, historical isolates collected prior to the emergence of epidemics can help reconstruct early events in the history of this lineage. Here, we take advantage of the accrued, publicly available data, as well as two newly sequenced pre-epidemic historical isolates from 1996, and a very early diverging ACME-negative NAE genome to understand the pre-epidemic evolution of USA300. We use database mining techniques to emphasize genomes similar to pre-epidemic isolates, with the goal of reconstructing the early molecular evolution of the USA300 lineage. Phylogenetic analysis with these genomes confirms that the North American Epidemic and South American Epidemic USA300 lineages diverged from a most recent common ancestor around 1970 with high confidence, and it also pinpoints the independent acquisition events of the of the ACME and COMER loci with greater precision than in previous studies. We solidify evidence for a North American origin of the USA300 lineage and identify multiple introductions of USA300 into South America from North America. Notably, we describe a third major USA300 clade (the pre-epidemic branching clade; PEB1) consisting of both MSSA and MRSA isolates circulating around the world that diverged from the USA300 lineage prior to the establishment of the South American and North American epidemics. We present a detailed analysis of specific sequence characteristics of each of the major clades, and present diagnostic positions that can be used to classify new genomes.

The emergence of drug-resistant tuberculosis is a significant global health issue. The presence of heteroresistant

Drug-resistant Neisseria gonorrhoeae is a serious global health concern. New drugs are needed that can overcome existing drug resistance and limit the development of new resistances.

Patients with burn injuries are at high risk for infectious complications, and infections are the most common cause of death after the first 72 h of hospitalization. Hospital-acquired infections caused by multidrug resistant (MDR) Gram-negative bacteria (GNB) in this population are concerning. Here, we evaluated carriage with MDR GNB in patients in a large tertiary-care burn intensive care unit. Twenty-nine patients in the burn unit were screened for intestinal carriage. Samples were cultured on selective media. Median time from admission to the burn unit to first sample collection was 9 days (IQR 5 – 17 days). In 21 (72%) patients, MDR GNB were recovered; the most common bacterial species isolated was Pseudomonas aeruginosa, which was found in 11/29 (38%) of patients. Two of these patients later developed bloodstream infections with P. aeruginosa. Transmission of KPC-31-producing ST22 Citrobacter freundii was detected. Samples from two patients grew genetically similar C. freundii isolates that were resistant to ceftazidime-avibactam. On analysis of whole-genome sequencing, bla(KPC-31) was part of a Tn4401b transposon that was present on two different plasmids in each C. freundii isolate. Plasmid curing experiments showed that removal of both copies of bla(KPC-31) was required to restore susceptibility to ceftazidime-avibactam. In summary, MDR GNB colonization is common in burn patients and patient-to-patient transmission of highly resistant GNB occurs. These results emphasize the ongoing need for infection prevention and antimicrobial stewardship efforts in this highly vulnerable population.

New variants of SARS-CoV-2 with potential for enhanced transmission, replication, and immune evasion capabilities continue to emerge causing reduced vaccine efficacy and/or treatment failure. As of January 2021, the WHO has defined five ‘variants of concern’ (VOC): B.1.1.7 (Alpha, α), B.1.351 (Beta, β), P.1 (Gamma, γ), B.1.617.2 (Delta, δ), and B.1.1.529 (Omicron, o). To provide a therapeutic option for the treatment of COVID-19 and variants, Nirmatrelvir, the antiviral component of PAXLOVID™, an oral outpatient treatment recently authorized for conditional or emergency use treatment of COVID-19, was developed to inhibit SARS-CoV-2 replication. Nirmatrelvir (PF-07321332) is a specific inhibitor of coronavirus main protease (Mpro, also referred to as 3CLpro), with potent antiviral activity against several human coronaviruses, including SARS-CoV-2, SARS-CoV, and MERS (Owen et al, Science 2021. doi: 10.1126/science.abl4784). Here, we evaluated PF-07321332 against the five SARS-CoV-2 VOC (α, β, γ, δ,, o) and two Variants of Interest or VOI, C.37 (λ) and B.1.621 (μ), using qRT-PCR in VeroE6 cells lacking the P-glycoprotein (Pgp) multidrug transporter gene (VeroE6 P-gp knockout cells). Nirmatrelvir potently inhibited USA-WA1/2020 strain, and α, β, γ, λ, δ, μ, and o variants in VeroE6 P-gp knockout cells with mean EC50 values 38.0 nM, 41.0 nM, 127.2 nM, 24.9 nM, 21.2 nM, 15.9 nM, 25.7 nM and 16.2 nM, respectively. Sequence analysis of the Mpro encoded by the variants showed ~100% identity of active site amino acid sequences, reflecting the essential role of Mpro during viral replication leading to ability of Nirmatrelvir to exhibit potent activity across all the variants.

Staphylococcus aureus persistently colonizes the nasopharynx in humans, which increases the risk for invasive diseases, such as skin infection and bacteremia. Nasal colonization triggers IgG responses against staphylococcal surface antigens; however, these antibodies cannot prevent subsequent colonization or disease. Here, we describe S. aureus WU1, a multilocus sequence type 88 (ST88) isolate that persistently colonizes the nasopharynx in mice. We report that staphylococcal protein A (SpA) is required for persistence of S. aureus WU1 in the nasopharynx. Compared to animals colonized by wild-type S. aureus , mice colonized with the Δ spa variant mount increased IgG responses against staphylococcal colonization determinants. Immunization of mice with a nontoxigenic SpA variant, which cannot cross-link B cell receptors and divert antibody responses, elicits protein A-neutralizing antibodies that promote IgG responses against colonizing S. aureus and diminish pathogen persistence. IMPORTANCE Staphylococcus aureus persistently colonizes the nasopharynx in about one-third of the human population, thereby promoting community- and hospital-acquired infections. Antibiotics are currently used for decolonization of individuals at increased risk of infection. However, the efficacy of antibiotics is limited by recolonization and selection for drug-resistant strains. Here, we propose a model of how staphylococcal protein A (SpA), a B cell superantigen, modifies host immune responses during colonization to support continued persistence of S. aureus in the nasopharynx. We show that this mechanism can be thwarted by vaccine-induced anti-SpA antibodies that promote IgG responses against staphylococcal antigens and diminish colonization.

Mycobacterium tuberculosis (Mtb) executes a plethora of immune-evasive mechanisms, which contribute to its pathogenesis, limited efficacy of current therapy, and the emergence of drug-resistant strains. This has led to resurgence in attempts to develop new therapeutic strategies/targets against tuberculosis (TB). We show that Mtb down-regulates sirtuin 1 (SIRT1), a nicotinamide adenine dinucleotide (NAD+)–dependent deacetylase, in monocytes/macrophages, TB animal models, and TB patients with active disease. Activation of SIRT1 reduced intracellular growth of drug-susceptible and drug-resistant strains of Mtb and induced phagosome-lysosome fusion and autophagy in a SIRT1-dependent manner. SIRT1 activation dampened Mtb-mediated persistent inflammatory responses via deacetylation of RelA/p65, leading to impaired binding of RelA/p65 on the promoter of inflammatory genes. In Mtb-infected mice, the use of SIRT1 activators ameliorated lung pathology, reduced chronic inflammation, and enhanced efficacy of anti-TB drug. Mass cytometry–based high-dimensional analysis revealed that SIRT1 activation mediated modulation of lung myeloid cells in Mtb-infected mice. Myeloid cell–specific SIRT1 knockout mice display increased inflammatory responses and susceptibility to Mtb infection. Collectively, these results provide a link between SIRT1 activation and TB pathogenesis and indicate a potential of SIRT1 activators in designing an effective and clinically relevant host-directed therapies for TB.

Cases of tuberculosis identified during 1992-1994 through an active tuberculosis surveillance network among six hospitals that serve New York City (the TBNetwork) were analyzed according to the occupational status of the patients. Clinical data were obtained by review of medical records, and restriction fragment length polymorphism (RFLP) typing of Mycobacterium tuberculosis isolates was performed. No known nosocomial outbreaks of tuberculosis occurred at these hospitals in the study period. Occupational status was known for 142 of 201 patients whose isolates were available for strain typing. Patients infected by organisms with a clustered strain typing pattern, as determined by RFLP analysis, were presumed to have recently acquired disease. RFLP typing revealed that isolates from 13 (65%) of 20 health care workers and 50 (41%) of 122 non-health care workers had a clustered RFLP pattern. The strains infecting eight (89%) of nine health care workers seropositive for human immunodeficiency virus (HIV) had a clustered RFLP pattern. Multivariate analysis of 75 patients with known HIV and occupational status revealed that HIV status (P = .03) and health care worker status (P = .02; RR = 2.77) were independent risk factors for a clustered RFLP strain. These findings suggest that many of the apparently sporadic cases of tuberculosis among health care workers may be due to unrecognized occupational transmission.

Recent reports have implicated community-associated methicillin-resistant Staphylococcus aureus (MRSA) as a cause of outbreaks in the neonatal intensive care unit (NICU). This study was conducted to determine whether community-associated MRSA caused such an outbreak in our NICU and the extent of nasal colonization with MRSA among exposed babies and health care workers. MRSA recovered from infected and colonized babies were genotyped by pulse-field gel electrophoresis (PFGE). Infection control measures were intensified and included nasal screening for MRSA colonization of exposed babies and all new admissions to the NICU within 24 hours of delivery. PFGE type A was recovered from five infected infants and colonized 81% of the exposed infants. The colonization rate during the outbreak was 9.3% and was 1.9% during admission screening. No MRSA infection occurred during 12 months while admission screening was implemented. Hospital-associated MRSA was the dominant strain in this outbreak. Higher colonization rates occurred during the outbreak period.

A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis. As such, insertion sequence (IS) 6110, which is unique to members of the M. tuberculosis complex and is present in variable numbers and in discrete genomic locales among strains, has been extensively used in molecular epidemiologic studies. Genotyping, using IS6110-based restriction fragment length polymorphism (RFLP), was standardized by the international community, and this has facilitated inter- and intralaboratory comparison, thereby serving as a model system for subspeciation of M. tuberculosis. When IS6110-based RFLP was used in conjunction with conventional epidemiologic data, its utility was realized. In this chapter, we discuss the basic methodology for conducting IS6110-based RFLP and analyzing the resulting hybridization profiles.

Tuberculosis and malaria together result in an estimated 5 million deaths annually. The spread of multidrug resistance in the most pathogenic causative agents, Mycobacterium tuberculosis and Plasmodium falciparum, underscores the need to identify active compounds with novel inhibitory properties. Although genetically unrelated, both organisms use a type II fatty-acid synthase system. Enoyl acyl carrier protein reductase (ENR), a key type II enzyme, has been repeatedly validated as an effective antimicrobial target. Using high throughput inhibitor screens with a combinatorial library, we have identified two novel classes of compounds with activity against the M. tuberculosis and P. falciparum enzyme (referred to as InhA and PfENR, respectively). The crystal structure of InhA complexed with NAD+ and one of the inhibitors was determined to elucidate the mode of binding. Structural analysis of InhA with the broad spectrum antimicrobial triclosan revealed a unique stoichiometry where the enzyme contained either a single triclosan molecule, in a configuration typical of other bacterial ENR:triclosan structures, or harbored two triclosan molecules bound to the active site. Significantly, these compounds do not require activation and are effective against wild-type and drug-resistant strains of M. tuberculosis and P. falciparum. Moreover, they provide broader chemical diversity and elucidate key elements of inhibitor binding to InhA for subsequent chemical optimization.

Methicillin-resistant Staphylococcus aureus (MRSA) is an important cause of nosocomial infection. Outbreaks of infection caused by these pathogens are generally considered to be traceable to introduction of single strains into a hospital population. A large outbreak of bacteremic disease that recently occurred in our neonatal intensive care unit (11 episodes in 10 patients) involved 9 low birth weight infants and was associated with serious infection (4 episodes of meningitis). To determine the role of a single point source in this outbreak, isolates were characterized based on phenotypic and genotypic analyses. Phenotypic analysis included assessing hemolytic activity, phage typing, antimicrobial susceptibility testing and methicillin resistance population analysis. Genotypic analysis included assessment of plasmid profiles, dot-blot hybridization, restriction enzyme fragment pattern analysis and hybridization analysis of chromosomal DNA using a panel of staphylococcal gene probes. This analysis established that at least two distinct strains of MRSA were responsible for disease during this outbreak. This experience demonstrates the potential for MRSA to cause severe disease in the neonatal intensive care unit and indicates that the epidemiology of MRSA outbreaks is more complex than the spread of a single strain of bacteria.

To define the stability of methicillin-resistant Staphylococcus aureus (MRSA) in vivo, 22 isolates collected at one New York institution in 1989 and 1990 were studied. All 22 belonged to one of two distinct methicillin-resistant phenotypes (class 3 or 2), which were precisely identified as belonging to two distinct genotypes. Genotypic classification was based on restriction analysis of chromosomal DNA with EcoRI and HindIII and Southern analysis of ClaI digests using two DNA probes. One was specific for the mec gene; the other was specific for transposon Tn554. The findings suggest that the MRSA isolates studied were representative of two genetically distinct MRSA "clones," each with a unique strain-specific methicillin-resistant phenotype that is stable under the conditions of invasive disease, carriage, and spread from patient to patient.