Large Community Outbreak of Legionnaires Disease Potentially Associated with a Cooling Tower - Napa County, California, 2022.

Legionnaires disease is a serious infection acquired by inhalation of water droplets from human-made building water systems that contain Legionella bacteria. On July 11 and 12, 2022, Napa County Public Health (NCPH) in California received reports of three positive urinary antigen tests for Legionella pneumophila serogroup 1 in the town of Napa. By July 21, six Legionnaires disease cases had been confirmed among Napa County residents, compared with a baseline of one or two cases per year. NCPH requested assistance from the California Department of Public Health (CDPH) and CDC to aid in the investigations. Close temporal and geospatial clustering permitted a focused environmental sampling strategy of high-risk facilities which, coupled with whole genome sequencing results from samples and investigation of water system maintenance, facilitated potential linking of the outbreak with an environmental source. NCPH, with technical support from CDC and CDPH, instructed and monitored remediation practices for all environmental locations that tested positive for Legionella. The investigation response to this community outbreak illustrates the importance of interdisciplinary collaboration by public health agencies, laboratory support, timely communication with the public, and cooperation of managers of potentially implicated water systems. Timely identification of possible sources, sampling, and remediation of any facility testing positive for Legionella is crucial to interrupting further transmission.

Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity.

A clinically significant mechanism of tuberculosis resistance to the aminoglycoside kanamycin (KAN) is its acetylation catalyzed by upregulated Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. In search for inhibitors of Eis, we discovered an inhibitor with a substituted benzyloxy-benzylamine scaffold. A structure-activity relationship study of 38 compounds in this structural family yielded highly potent (IC50 ∼ 1 µM) Eis inhibitors, which did not inhibit other acetyltransferases. Crystal structures of Eis in complexes with three of the inhibitors showed that the inhibitors were bound in the aminoglycoside binding site of Eis, consistent with the competitive mode of inhibition, as established by kinetics measurements. When tested in Mtb cultures, two inhibitors (47 and 55) completely abolished resistance to KAN of the highly KAN-resistant strain Mtb mc2 6230 K204, likely due to Eis inhibition as a major mechanism. Thirteen of the compounds were toxic even in the absence of KAN to Mtb and other mycobacteria, but not to non-mycobacteria or to mammalian cells. This, yet unidentified mechanism of toxicity, distinct from Eis inhibition, will merit future studies along with further development of these molecules as anti-mycobacterial agents.

Structure-based design of haloperidol analogues as inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis to overcome kanamycin resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a deadly bacterial disease. Drug-resistant strains of Mtb make eradication of TB a daunting task. Overexpression of the enhanced intracellular survival (Eis) protein by Mtb confers resistance to the second-line antibiotic kanamycin (KAN). Eis is an acetyltransferase that acetylates KAN, inactivating its antimicrobial function. Development of Eis inhibitors as KAN adjuvant therapeutics is an attractive path to forestall and overcome KAN resistance. We discovered that an antipsychotic drug, haloperidol (HPD, 1), was a potent Eis inhibitor with IC50 = 0.39 ± 0.08 µM. We determined the crystal structure of the Eis-haloperidol (1) complex, which guided synthesis of 34 analogues. The structure-activity relationship study showed that in addition to haloperidol (1), eight analogues, some of which were smaller than 1, potently inhibited Eis (IC50 ≤ 1 µM). Crystal structures of Eis in complexes with three potent analogues and droperidol (DPD), an antiemetic and antipsychotic, were determined. Three compounds partially restored KAN sensitivity of a KAN-resistant Mtb strain K204 overexpressing Eis. The Eis inhibitors generally did not exhibit cytotoxicity against mammalian cells. All tested compounds were modestly metabolically stable in human liver microsomes, exhibiting 30-60% metabolism over the course of the assay. While direct repurposing of haloperidol as an anti-TB agent is unlikely due to its neurotoxicity, this study reveals potential approaches to modifying this chemical scaffold to minimize toxicity and improve metabolic stability, while preserving potent Eis inhibition.

Structure-Guided Optimization of Inhibitors of Acetyltransferase Eis from Mycobacterium tuberculosis.

The enhanced intracellular survival (Eis) protein of Mycobacterium tuberculosis (Mtb) is a versatile acetyltransferase that multiacetylates aminoglycoside antibiotics abolishing their binding to the bacterial ribosome. When overexpressed as a result of promoter mutations, Eis causes drug resistance. In an attempt to overcome the Eis-mediated kanamycin resistance of Mtb, we designed and optimized structurally unique thieno[2,3-d]pyrimidine Eis inhibitors toward effective kanamycin adjuvant combination therapy. We obtained 12 crystal structures of enzyme-inhibitor complexes, which guided our rational structure-based design of 72 thieno[2,3-d]pyrimidine analogues divided into three families. We evaluated the potency of these inhibitors in vitro as well as their ability to restore the activity of kanamycin in a resistant strain of Mtb, in which Eis was upregulated. Furthermore, we evaluated the metabolic stability of 11 compounds in vitro. This study showcases how structural information can guide Eis inhibitor design.

Potent 1,2,4-Triazino[5,6 b]indole-3-thioether Inhibitors of the Kanamycin Resistance Enzyme Eis from Mycobacterium tuberculosis.

A common cause of resistance to kanamycin (KAN) in tuberculosis is overexpression of the enhanced intracellular survival (Eis) protein. Eis is an acetyltransferase that multiacetylates KAN and other aminoglycosides, rendering them unable to bind the bacterial ribosome. By high-throughput screening, a series of substituted 1,2,4-triazino[5,6 b]indole-3-thioether molecules were identified as effective Eis inhibitors. Herein, we purchased 17 and synthesized 22 new compounds, evaluated their potency, and characterized their steady-state kinetics. Four inhibitors were found not only to inhibit Eis in vitro, but also to act as adjuvants of KAN and partially restore KAN sensitivity in a Mycobacterium tuberculosis KAN-resistant strain in which Eis is upregulated. A crystal structure of Eis in complex with a potent inhibitor and CoA shows that the inhibitors bind in the aminoglycoside binding site snugly inserted into a hydrophobic cavity. These inhibitors will undergo preclinical development as novel KAN adjuvant therapies to treat KAN-resistant tuberculosis.

Acetylation by Eis and Deacetylation by Rv1151c of Mycobacterium tuberculosis HupB: Biochemical and Structural Insight.

Bacterial nucleoid-associated proteins (NAPs) are critical to genome integrity and chromosome maintenance. Post-translational modifications of bacterial NAPs appear to function similarly to their better studied mammalian counterparts. The histone-like NAP HupB from Mycobacterium tuberculosis (Mtb) was previously observed to be acetylated by the acetyltransferase Eis, leading to genome reorganization. We report biochemical and structural aspects of acetylation of HupB by Eis. We also found that the SirT-family NAD+-dependent deacetylase Rv1151c from Mtb deacetylated HupB in vitro and characterized the deacetylation kinetics. We propose that activities of Eis and Rv1151c could regulate the acetylation status of HupB to remodel the mycobacterial chromosome in response to environmental changes.

Detection of Mycobacterium tuberculosis pncA Mutations by the Nipro Genoscholar PZA-TB II Assay Compared to Conventional Sequencing.

Pyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosis and is included in treatment regimens for drug-resistant M. tuberculosis whenever possible. Therefore, it is imperative that susceptibility to PZA be assessed reliably prior to the initiation of therapy. Currently available growth-based PZA susceptibility tests are time-consuming, and results can be inconsistent. Molecular tests have been developed for most first-line antituberculosis drugs; however, a commercial molecular test is not yet available for rapid detection of PZA resistance. Recently, a line probe assay, the Nipro Genoscholar PZA-TB II assay, was developed for the detection of mutations within the pncA gene, including the promoter region, that are likely to lead to PZA resistance. The sensitivity and specificity of this assay were evaluated by two independent laboratories, using a combined total of 249 strains with mutations in pncA or its promoter and 21 strains with wild-type pncA Overall, the assay showed good sensitivity (93.2% [95% confidence interval, 89.3 to 95.8%]) and moderate specificity (91.2% [95% confidence interval, 77.0 to 97.0%]) for the identification of M. tuberculosis strains predicted to be resistant to PZA on the basis of the presence of mutations (excluding known PZA-susceptible mutations) in the pncA coding region or promoter. The assay shows promise for the molecular prediction of PZA resistance.

Combating Enhanced Intracellular Survival (Eis)-Mediated Kanamycin Resistance of Mycobacterium tuberculosis by Novel Pyrrolo[1,5-a]pyrazine-Based Eis Inhibitors.

Tuberculosis (TB) remains one of the leading causes of mortality worldwide. Hence, the identification of highly effective antitubercular drugs with novel modes of action is crucial. In this paper, we report the discovery and development of pyrrolo[1,5-a]pyrazine-based analogues as highly potent inhibitors of the Mycobacterium tuberculosis (Mtb) acetyltransferase enhanced intracellular survival (Eis), whose up-regulation causes clinically observed resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN). We performed a structure-activity relationship (SAR) study to optimize these compounds as potent Eis inhibitors both against purified enzyme and in mycobacterial cells. A crystal structure of Eis in complex with one of the most potent inhibitors reveals that the compound is bound to Eis in the AG binding pocket, serving as the structural basis for the SAR. These Eis inhibitors have no observed cytotoxicity to mammalian cells and are promising leads for the development of innovative AG adjuvant therapies against drug-resistant TB.

Discovery and Optimization of Two Eis Inhibitor Families as Kanamycin Adjuvants against Drug-Resistant M. tuberculosis.

Drug-resistant tuberculosis (TB) is a global threat and innovative approaches such as using adjuvants of anti-TB therapeutics are required to combat it. High-throughput screening yielded two lead scaffolds of inhibitors of Mycobacterium tuberculosis (Mtb) acetyltransferase Eis, whose upregulation causes resistance to the anti-TB drug kanamycin (KAN). Chemical optimization on these scaffolds resulted in potent Eis inhibitors. One compound restored the activity of KAN in a KAN-resistant Mtb strain. Model structures of Eis-inhibitor complexes explain the structure-activity relationship.

Sulfonamide-Based Inhibitors of Aminoglycoside Acetyltransferase Eis Abolish Resistance to Kanamycin in Mycobacterium tuberculosis.

A two-drug combination therapy where one drug targets an offending cell and the other targets a resistance mechanism to the first drug is a time-tested, yet underexploited approach to combat or prevent drug resistance. By high-throughput screening, we identified a sulfonamide scaffold that served as a pharmacophore to generate inhibitors of Mycobacterium tuberculosis acetyltransferase Eis, whose upregulation causes resistance to the aminoglycoside (AG) antibiotic kanamycin A (KAN) in Mycobacterium tuberculosis. Rational systematic derivatization of this scaffold to maximize Eis inhibition and abolish the Eis-mediated KAN resistance of M. tuberculosis yielded several highly potent agents. A crystal structure of Eis in complex with one of the most potent inhibitors revealed that the inhibitor bound Eis in the AG-binding pocket held by a conformationally malleable region of Eis (residues 28-37) bearing key hydrophobic residues. These Eis inhibitors are promising leads for preclinical development of innovative AG combination therapies against resistant TB.

Potent Inhibitors of Acetyltransferase Eis Overcome Kanamycin Resistance in Mycobacterium tuberculosis.

A major cause of tuberculosis (TB) resistance to the aminoglycoside kanamycin (KAN) is the Mycobacterium tuberculosis (Mtb) acetyltransferase Eis. Upregulation of this enzyme is responsible for inactivation of KAN through acetylation of its amino groups. A 123 000-compound high-throughput screen (HTS) yielded several small-molecule Eis inhibitors that share an isothiazole S,S-dioxide heterocyclic core. These were investigated for their structure-activity relationships. Crystal structures of Eis in complex with two potent inhibitors show that these molecules are bound in the conformationally adaptable aminoglycoside binding site of the enzyme, thereby obstructing binding of KAN for acetylation. Importantly, we demonstrate that several Eis inhibitors, when used in combination with KAN against resistant Mtb, efficiently overcome KAN resistance. This approach paves the way toward development of novel combination therapies against aminoglycoside-resistant TB.

Multiorgan WU Polyomavirus Infection in Bone Marrow Transplant Recipient.

WU polyomavirus (WUPyV) was detected in a bone marrow transplant recipient with severe acute respiratory distress syndrome who died in 2001. Crystalline lattices of polyomavirus-like particles were observed in the patient's lung by electron microscopy. WUPyV was detected in the lung and other tissues by real-time quantitative PCR and identified in the lung and trachea by immunohistochemistry. A subset of WUPyV-positive cells in the lung had morphologic features of macrophages. Although the role of WUPyV as a human pathogen remains unclear, these results clearly demonstrate evidence for infection of respiratory tract tissues in this patient.

Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG.

Owing to the rise in drug resistance in tuberculosis combined with the global spread of its causative pathogen, Mycobacterium tuberculosis (Mtb), innovative anti mycobacterial agents are urgently needed. Recently, we developed a novel primase-pyrophosphatase assay and used it to discover inhibitors of an essential Mtb enzyme, primase DnaG (Mtb DnaG), a promising and unexplored potential target for novel antituberculosis chemotherapeutics. Doxorubicin, an anthracycline antibiotic used as an anticancer drug, was found to be a potent inhibitor of Mtb DnaG. In this study, we investigated both inhibition of Mtb DnaG and the inhibitory activity against in vitro growth of Mtb and M. smegmatis (Msm) by other anthracyclines, daunorubicin and idarubicin, as well as by less cytotoxic DNA intercalators: aloe-emodin, rhein and a mitoxantrone derivative. Generally, low-µM inhibition of Mtb DnaG by the anthracyclines was correlated with their low-µM minimum inhibitory concentrations. Aloe-emodin displayed threefold weaker potency than doxorubicin against Mtb DnaG and similar inhibition of Msm (but not Mtb) in the mid-µM range, whereas rhein (a close analog of aloe-emodin) and a di-glucosylated mitoxantrone derivative did not show significant inhibition of Mtb DnaG or antimycobacterial activity. Taken together, these observations strongly suggest that several clinically used anthracyclines and aloe-emodin target mycobacterial primase, setting the stage for a more extensive exploration of this enzyme as an antibacterial target.

Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis.

As the prevalence of multidrug-resistant and extensively drug-resistant tuberculosis strains continues to rise, so does the need to develop accurate and rapid molecular tests to complement time-consuming growth-based drug susceptibility testing. Performance of molecular methods relies on the association of specific mutations with phenotypic drug resistance and while considerable progress has been made for resistance detection of first-line antituberculosis drugs, rapid detection of resistance for second-line drugs lags behind. The rrs A1401G allele is considered a strong predictor of cross-resistance between the three second-line injectable drugs, capreomycin (CAP), kanamycin, and amikacin. However, discordance is often observed between the rrs A1401G mutation and CAP resistance, with up to 40% of rrs A1401G mutants being classified as CAP susceptible. We measured the MICs to CAP in 53 clinical isolates harboring the rrs A1401G mutation and found that the CAP MICs ranged from 8 µg/ml to 40 µg/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 µg/ml. These data support the results of prior studies, which suggest that the critical concentration of CAP (10 µg/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP-resistant strains and suggest that a larger percentage of resistant isolates could be identified by lowering the critical concentration. These data also suggest that differences in resistance levels among clinical isolates are possibly due to second site or compensatory mutations located elsewhere in the genome.

Human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls.

BACKGROUND: Human coronaviruses (HCoVs) have been detected in children with upper and lower respiratory symptoms, but little is known about their relationship with severe respiratory illness. OBJECTIVE: To compare the prevalence of HCoV species among children hospitalized for acute respiratory illness and/or fever (ARI/fever) with that among asymptomatic controls and to assess the severity of outcomes among hospitalized children with HCoV infection compared with other respiratory viruses. METHODS: From December 2003 to April 2004 and October 2004 to April 2005, we conducted prospective, population-based surveillance of children <5 years of age hospitalized for ARI/fever in 3 US counties. Asymptomatic outpatient controls were enrolled concurrently. Nasal/throat swabs were tested for HCoV species HKU1, NL63, 229E, and OC43 by real-time reverse-transcription polymerase chain reaction. Specimens from hospitalized children were also tested for other common respiratory viruses. Demographic and medical data were collected by parent/guardian interview and medical chart review. RESULTS: Overall, HCoV was detected in 113 (7.6%) of 1481 hospitalized children (83 [5.7%] after excluding 30 cases coinfected with other viruses) and 53 (7.1%) of 742 controls. The prevalence of HCoV or individual species was not significantly higher among hospitalized children than controls. Hospitalized children testing positive for HCoV alone tended to be less ill than those infected with other viruses, whereas those coinfected with HCoV and other viruses were clinically similar to those infected with other viruses alone. CONCLUSIONS: In this study of children hospitalized for ARI/fever, HCoV infection was not associated with hospitalization or with increased severity of illness.

HMW1 is required for stability and localization of HMW2 to the attachment organelle of Mycoplasma pneumoniae.

The cytoskeletal proteins HMW1 and HMW2 are components of the terminal organelle of the cell wall-less bacterium Mycoplasma pneumoniae. HMW1 is required for a tapered, filamentous morphology but exhibits accelerated turnover in the absence of HMW2. Here, we report that a reciprocal dependency exists between HMW1 and HMW2, with HMW2 subject to accelerated turnover with the loss of HMW1. Furthermore, the instability of HMW2 correlated with its failure to localize to the attachment organelle. The C-terminal domain of HMW1 is essential for both function and its accelerated turnover in the absence of HMW2. We constructed HMW1 deletion derivatives lacking portions of this domain and examined each for stability and function. The C-terminal 41 residues were particularly important for proper localization and function in cell morphology and P1 localization, but the entire C-terminal domain was required to stabilize HMW2. The significance of these findings in the context of attachment organelle assembly is considered.

Characterization of a Mycoplasma pneumoniae hmw3 mutant: implications for attachment organelle assembly.

The proteins required for adherence of the pathogen Mycoplasma pneumoniae to host respiratory epithelial cells are localized to a polar structure, the attachment organelle. A number of these proteins have been characterized functionally by analysis of noncytadhering mutants, and many are components of the mycoplasma cytoskeleton. Mutations in some cytadherence-associated proteins have pleiotropic effects, including decreased stability of other proteins, loss of adherence and motility, and abnormal morphology. The function of protein HMW3, a component of the attachment organelle, has been difficult to discern due to lack of an appropriate mutant. In this paper, we report that loss of HMW3 resulted in decreased levels and more diffuse localization of cytoskeletal protein P65, subtle changes in morphology, inability to cluster the adhesin P1 consistently at the terminal organelle, reduced cytadherence, and, in some cells, an atypical electron-dense core in the attachment organelle. This phenotype suggests a role for HMW3 in the architecture and stability of the attachment organelle.