Last data update: Apr 29, 2024. (Total: 46658 publications since 2009)
Records 1-23 (of 23 Records) |
Query Trace: Willby M[original query] |
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Large community outbreak of legionnaires disease potentially associated with a cooling tower - Napa County, California, 2022
Grossmann NV , Milne C , Martinez MR , Relucio K , Sadeghi B , Wiley EN , Holland SN , Rutschmann S , Vugia DJ , Kimura A , Crain C , Akter F , Mukhopadhyay R , Crandall J , Shorrock M , Smith JC , Prasad N , Kahn R , Barskey AE , Lee S , Willby MJ , Kozak-Muiznieks NA , Lucas CE , Henderson KC , Hamlin JAP , Yang E , Clemmons NS , Ritter T , Henn J . MMWR Morb Mortal Wkly Rep 2023 72 (49) 1315-1320 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. |
Notes from the Field: Legionnaires disease in a U.S. traveler after staying in a private vacation rental house in the U.S. Virgin Islands - United States, February 2022
Mac VV , Labgold K , Moline HL , Smith JC , Carroll J , Clemmons N , Edens C , Ellis B , Harrison C , Henderson KC , Ishaq MK , Kozak-Muiznieks NA , Kunz J , Lawrence M , Lucas CE , Walker HL , Willby MJ , Ellis EM . MMWR Morb Mortal Wkly Rep 2023 72 (20) 564-565 On February 1, 2022, the U.S. Virgin Islands (USVI) Department of Health (VIDOH) was notified of a confirmed case of Legionnaires disease in an adult U.S. resident (Figure). The patient, a man aged 55 years, returned to his U.S. state of residence from leisure travel in USVI on January 22 and developed a cough, shortness of breath, and fatigue on January 23. On January 29, he was hospitalized for shortness of breath and received a positive SARS-CoV-2 test result at admission. The combination of the patient’s symptoms and recent travel history prompted administration of a urinary antigen test (UAT) for Legionnaires disease specific to Legionella pneumophila serogroup 1 (Lp1); a positive result was returned on January 31. Inpatient treatment administered for COVID-19 pneumonia and Legionnaires disease included remdesivir, oral levofloxacin, oral and intravenous steroid therapy, and as-needed use of a bronchodilator inhaler and an expectorant. Remdesivir was discontinued during inpatient treatment because of elevated liver enzymes. The patient recovered and was discharged on February 2. |
Discovery of substituted benzyloxy-benzylamine inhibitors of acetyltransferase Eis and their anti-mycobacterial activity
Pang AH , Green KD , Chandrika NT , Garzan A , Punetha A , Holbrook SYL , Willby MJ , Posey JE , Tsodikov OV , Garneau-Tsodikova S . Eur J Med Chem 2022 242 114698 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 (IC(50) 1M) 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 mc(2) 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
Punetha A , Green KD , Garzan A , Thamban Chandrika N , Willby MJ , Pang AH , Hou C , Holbrook SYL , Krieger K , Posey JE , Parish T , Tsodikov OV , Garneau-Tsodikova S . RSC Med Chem 2021 12 (11) 1894-1909 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 IC(50) = 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 (IC(50) ≤ 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. |
Molecular evaluation of fluoroquinolone resistance in serial Mycobacterium tuberculosis isolates from individuals diagnosed with multidrug-resistant tuberculosis.
Willby M , Chopra P , Lemmer D , Klein K , Dalton TL , Engelthaler DM , Cegielski P , Posey JE . Antimicrob Agents Chemother 2020 65 (1) Fluoroquinolones (FQ) are crucial components of multidrug-resistant tuberculosis (MDR TB) treatment. Varying levels of resistance are associated with specific mutations within the Quinolone Resistance Determining Region (QRDR) of gyrA We sequenced the QRDR from serial isolates of MDR TB patients in the Preserving Effective TB Treatment Study (PETTS) with baseline (FQ(R)) or acquired FQ resistance (FQ(ACQR)) using the IonTorrent™ Personal Genome Machine to a depth of 10,000x and reported single nucleotide polymorphisms in ≥1% of reads. FQ(R) isolates harbored 15 distinct alleles with 1.3 (max=6) on average per isolate. Eighteen alleles were identified in FQ(ACQR) isolates with an average of 1.6 (max=9) per isolate. Isolates from 78% of FQ(ACQR) individuals had mutant alleles identified within 6 months of treatment initiation. Asp94Gly followed by Ala90Val were the predominant alleles in initial FQ-resistant isolates. Seventy-seven percent (36/47) of FQ(ACQR) group patients had isolates with FQ resistance alleles prior to changes to the FQ component of their treatment. Unlike individuals treated initially with other FQs, none of the 21 individuals treated initially with levofloxacin developed genotypic or phenotypic FQ resistance, although, country of residence is likely a contributing factor since 69% of these individuals were from a single country. Initial detection of phenotypic and genotypic resistance occurred simultaneously for most; however, phenotypic resistance occurred earlier in isolates harboring mixtures of very low-abundance (<1% of reads) alleles while genotypic resistance often occurred earlier for low-level resistance-associated alleles. Understanding factors influencing acquisition and evolution of FQ resistance could reveal strategies for improved treatment success. |
Structure-guided optimization of inhibitors of acetyltransferase Eis from Mycobacterium tuberculosis
Punetha A , Ngo HX , Holbrook SYL , Green KD , Willby MJ , Bonnett SA , Krieger K , Dennis EK , Posey JE , Parish T , Tsodikov OV , Garneau-Tsodikova S . ACS Chem Biol 2020 15 (6) 1581-1594 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
Ngo HX , Green KD , Gajadeera CS , Willby MJ , Holbrook SYL , Hou C , Garzan A , Mayhoub AS , Posey JE , Tsodikov OV , Garneau-Tsodikova S . ACS Infect Dis 2018 4 (6) 1030-1040 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
Green KD , Biswas T , Pang AH , Willby MJ , Reed MS , Stuchlik O , Pohl J , Posey JE , Tsodikov OV , Garneau-Tsodikova S . Biochemistry 2018 57 (5) 781-790 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 Apparent Cell-free M. tuberculosis DNA from Plasma.
Click ES , Murithi W , Ouma GS , McCarthy K , Willby M , Musau S , Alexander H , Pevzner E , Posey J , Cain KP . Sci Rep 2018 8 (1) 645 New diagnostics are needed to improve clinicians' ability to detect tuberculosis (TB) disease in key populations such as children and persons living with HIV and to rapidly detect drug resistance. Circulating cell-free DNA (ccfDNA) in plasma is a diagnostic target in new obstetric and oncologic applications, but its utility for diagnosing TB is not known. Here we show that Mycobacterium tuberculosis complex DNA can be detected in plasma of persons with sputum smear-positive TB, even in the absence of mycobacteremia. Among 40 participants with bacteriologically-confirmed smear-positive TB disease who had plasma tested by quantitative PCR (qPCR), 18/40 (45%) had a positive result on at least one triplicate reaction. Our results suggest that plasma DNA may be a useful target for improving clinicians' ability to diagnose TB. We anticipate these findings to be the starting point for optimized methods of TB ccfDNA testing and sequence-based diagnostic applications such as molecular detection of drug resistance. |
Detection of Mycobacterium tuberculosis pncA mutations by the NIPRO Genoscholar(™)·PZA-TB II as compared to conventional sequencing.
Willby MJ , Wijkander M , Havumaki J , Johnson K , Werngren J , Hoffner S , Denkinger CM , Posey JE . Antimicrob Agents Chemother 2017 62 (1) Pyrazinamide (PZA) is a standard component of first-line treatment regimens for Mycobacterium tuberculosis (Mtb) and is included in treatment regimens for drug-resistant Mtb whenever possible. It is therefore imperative that susceptibility to PZA be reliably assessed prior to 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, NIPRO GenoscholarPZA-TB II, was developed for the detection of mutations within the pncA gene including the promoter region likely to lead to PZA resistance. The sensitivity and specificity of this assay was evaluated by two independent laboratories using a combined total of 249 strains with mutations in pncA and its promoter as well as 21 strains with wild-type pncA Overall, the assay showed good sensitivity (93.2%, 95%CI 89.3, 95.8) and moderate specificity (91.2%, 95%CI 77.0, 97.0) for the identification of Mtb predicted to be resistant to PZA based upon 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
Garzan A , Willby MJ , Ngo HX , Gajadeera CS , Green KD , Holbrook SY , Hou C , Posey JE , Tsodikov OV , Garneau-Tsodikova S . ACS Infect Dis 2017 3 (4) 302-309 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. |
Sulfonamide-based inhibitors of aminoglycoside acetyltransferase Eis abolish resistance to kanamycin in Mycobacterium tuberculosis
Garzan A , Willby MJ , Green KD , Gajadeera CS , Hou C , Tsodikov OV , Posey JE , Garneau-Tsodikova S . J Med Chem 2016 59 (23) 10619-10628 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. |
Discovery and optimization of two Eis inhibitor families as kanamycin adjuvants against drug-resistant M. tuberculosis
Garzan A , Willby MJ , Green KD , Tsodikov OV , Posey JE , Garneau-Tsodikova S . ACS Med Chem Lett 2016 7 (12) 1219-1221 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. |
Potent inhibitors of acetyltransferase Eis overcome kanamycin resistance in Mycobacterium tuberculosis
Willby MJ , Green KD , Gajadeera CS , Hou C , Tsodikov OV , Posey JE , Garneau-Tsodikova S . ACS Chem Biol 2016 11 (6) 1639-46 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 123000-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. |
Respiratory viruses associated hospitalization among children aged <5 years in Bangladesh: 2010-2014
Homaira N , Luby SP , Hossain K , Islam K , Ahmed M , Rahman M , Rahman Z , Paul RC , Bhuiyan MU , Brooks WA , Sohel BM , Banik KC , Widdowson MA , Willby M , Rahman M , Bresee J , Ramirez KS , Azziz-Baumgartner E . PLoS One 2016 11 (2) e0147982 BACKGROUND: We combined hospital-based surveillance and health utilization survey data to estimate the incidence of respiratory viral infections associated hospitalization among children aged < 5 years in Bangladesh. METHODS: Surveillance physicians collected respiratory specimens from children aged <5 years hospitalized with respiratory illness and residing in the primary hospital catchment areas. We tested respiratory specimens for respiratory syncytial virus, parainfluenza viruses, human metapneumovirus, influenza, adenovirus and rhinoviruses using rRT-PCR. During 2013, we conducted a health utilization survey in the primary catchment areas of the hospitals to determine the proportion of all hospitalizations for respiratory illness among children aged <5 years at the surveillance hospitals during the preceding 12 months. We estimated the respiratory virus-specific incidence of hospitalization by dividing the estimated number of hospitalized children with a laboratory confirmed infection with a respiratory virus by the population aged <5 years of the catchment areas and adjusted for the proportion of children who were hospitalized at the surveillance hospitals. RESULTS: We estimated that the annual incidence per 1000 children (95% CI) of all cause associated respiratory hospitalization was 11.5 (10-12). The incidences per 1000 children (95% CI) per year for respiratory syncytial virus, parainfluenza, adenovirus, human metapneumovirus and influenza infections were 3(2-3), 0.5(0.4-0.8), 0.4 (0.3-0.6), 0.4 (0.3-0.6), and 0.4 (0.3-0.6) respectively. The incidences per 1000 children (95%CI) of rhinovirus-associated infections among hospitalized children were 5 (3-7), 2 (1-3), 1 (0.6-2), and 3 (2-4) in 2010, 2011, 2012 and 2013, respectively. CONCLUSION: Our data suggest that respiratory viruses are associated with a substantial burden of hospitalization in children aged <5 years in Bangladesh. |
Multiorgan WU polyomavirus infection in bone marrow transplant recipient
Siebrasse EA , Nguyen NL , Willby MJ , Erdman DD , Menegus MA , Wang D . Emerg Infect Dis 2016 22 (1) 24-31 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. |
Rv3351c, a Mycobacterium tuberculosis gene that affects bacterial growth and alveolar epithelial cell viability.
Pavlicek RL , Fine-Coulson K , Gupta T , Quinn FD , Posey JE , Willby M , Castro-Garza J , Karls RK . Can J Microbiol 2015 61 (12) 1-10 Despite the interactions known to occur between various lower respiratory tract pathogens and alveolar epithelial cells (AECs), few reports examine factors influencing the interplay between Mycobacterium tuberculosis bacilli and AECs during infection. Importantly, in vitro studies have demonstrated that the M. tuberculosis hbha and esxA gene products HBHA and ESAT6 directly or indirectly influence AEC survival. In this report, we identify Rv3351c as another M. tuberculosis gene that impacts the fate of both the pathogen and AEC host. Intracellular replication of an Rv3351c mutant in the human AEC type II pneumocyte cell line A549 was markedly reduced relative to the complemented mutant and parent strain. Deletion of Rv3351c diminished the release of lactate dehydrogenase and decreased uptake of trypan blue vital stain by host cells infected with M. tuberculosis bacilli, suggesting attenuated cytotoxic effects. Interestingly, an isogenic hbha mutant displayed reductions in AEC killing similar to those observed for the Rv3351c mutant. This opens the possibility that multiple M. tuberculosis gene products interact with AECs. We also observed that Rv3351c aids intracellular replication and survival of M. tuberculosis in macrophages. This places Rv3351c in the same standing as HBHA and ESAT6, which are important factors in AECs and macrophages. Defining the mechanism(s) by which Rv3351c functions to aid pathogen survival within the host may lead to new drug or vaccine targets. |
Mycobacterium tuberculosis pncA polymorphisms that do not confer pyrazinamide resistance at a breakpoint concentration of 100 µg/ml in MGIT.
Whitfield MG , Warren RM , Streicher EM , Sampson SL , Sirgel FA , van Helden PD , Mercante A , Willby M , Hughes K , Birkness K , Morlock G , van Rie A , Posey JE . J Clin Microbiol 2015 Sequencing of the Mycobacterium tuberculosis pncA gene allows for pyrazinamide susceptibility testing. We summarize data on pncA polymorphisms which do not confer resistance at a susceptibility breakpoint of 100 mug/ml pyrazinamide in MGIT within a cohort of isolates from South Africa and the US Centre for Disease Control. |
Correlation between GyrA substitutions and ofloxacin, levofloxacin, and moxifloxacin cross-resistance in Mycobacterium tuberculosis
Willby M , Sikes RD , Malik S , Metchock B , Posey JE . Antimicrob Agents Chemother 2015 59 (9) 5427-34 Newer generation fluoroquinolones moxifloxacin (MXF) and levofloxacin (LVX) are becoming more common components of TB treatment regimens. However, the critical concentrations for testing susceptibility of Mycobacterium tuberculosis to MXF and LVX are not yet well established. Additionally, the degree of cross-resistance between ofloxacin (OFX) and these newer fluoroquinolones has not been thoroughly investigated. In this study, minimal inhibitory concentrations (MICs) for MXF and LVX and susceptibility to the critical concentration of OFX were determined using the agar proportion method for 133 isolates of M. tuberculosis. Most isolates resistant to OFX had LVX MICs >1 mug/mL and MXF MICs >0.5mug/mL. The presence of mutations within the GyrA QRDR correlated well with increased MICs, and the level of LVX and MXF resistance was dependent on the specific GyrA mutation present. Substitutions Ala90Val, Asp94Ala and Asp94Tyr resulted in low-level MXF resistance (MICs >0.5 but ≤2 mug/mL) while other mutations led to MXF MIC >2mug/mL. Based on these results, a critical concentration of 1 mug/mL is suggested for LVX and 0.5 mug/ml for MXF drug susceptibility testing by agar proportion with reflex testing for MXF at 2 mug/mL. |
Disparities in capreomycin resistance levels associated with the rrs A1401G mutation in clinical isolates of Mycobacterium tuberculosis.
Reeves AZ , Campbell PJ , Willby MJ , Posey JE . Antimicrob Agents Chemother 2014 59 (1) 444-9 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 minimal inhibitory concentrations (MICs) to CAP in 53 clinical isolates harboring the A1401G mutation and found that the CAP MICs ranged from 8 mug/ml to 40 mug/ml. These results were drastically different from engineered A1401G mutants generated in isogenic Mycobacterium tuberculosis, which exclusively exhibited high-level CAP MICs of 40 mug/ml. These data support prior studies suggesting the critical concentration of CAP (10 mug/ml) used to determine resistance by indirect agar proportion may be too high to detect all CAP resistant strains and suggests 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. |
Antimycobacterial activity of DNA intercalator inhibitors of Mycobacterium tuberculosis primase DnaG.
Gajadeera C , Willby MJ , Green KD , Shaul P , Fridman M , Garneau-Tsodikova S , Posey JE , Tsodikov OV . J Antibiot (Tokyo) 2014 68 (3) 153-7 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-muM inhibition of Mtb DnaG by the anthracyclines was correlated with their low-muM 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-muM 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. |
New insights into fluoroquinolone resistance in Mycobacterium tuberculosis: functional genetic analysis of gyrA and gyrB mutations.
Malik S , Willby M , Sikes D , Tsodikov OV , Posey JE . PLoS One 2012 7 (6) e39754 Fluoroquinolone antibiotics are among the most potent second-line drugs used for treatment of multidrug-resistant tuberculosis (MDR TB), and resistance to this class of antibiotics is one criterion for defining extensively drug resistant tuberculosis (XDR TB). Fluoroquinolone resistance in Mycobacterium tuberculosis has been associated with modification of the quinolone resistance determining region (QRDR) of gyrA. Recent studies suggest that amino acid substitutions in gyrB may also play a crucial role in resistance, but functional genetic studies of these mutations in M. tuberculosis are lacking. In this study, we examined twenty six mutations in gyrase genes gyrA (seven) and gyrB (nineteen) to determine the clinical relevance and role of these mutations in fluoroquinolone resistance. Transductants or clinical isolates harboring T80A, T80A+A90G, A90G, G247S and A384V gyrA mutations were susceptible to all fluoroquinolones tested. The A74S mutation conferred low-level resistance to moxifloxacin but susceptibility to ciprofloxacin, levofloxacin and ofloxacin, and the A74S+D94G double mutation conferred cross resistance to all the fluoroquinolones tested. Functional genetic analysis and structural modeling of gyrB suggest that M330I, V340L, R485C, D500A, D533A, A543T, A543V and T546M mutations are not sufficient to confer resistance as determined by agar proportion. Only three mutations, N538D, E540V and R485C+T539N, conferred resistance to all four fluoroquinolones in at least one genetic background. The D500H and D500N mutations conferred resistance only to levofloxacin and ofloxacin while N538K and E540D consistently conferred resistance to moxifloxacin only. Transductants and clinical isolates harboring T539N, T539P or N538T+T546M mutations exhibited low-level resistance to moxifloxacin only but not consistently. These findings indicate that certain mutations in gyrB confer fluoroquinolone resistance, but the level and pattern of resistance varies among the different mutations. The results from this study provide support for the inclusion of the QRDR of gyrB in molecular assays used to detect fluoroquinolone resistance in M. tuberculosis. |
Human coronavirus in young children hospitalized for acute respiratory illness and asymptomatic controls
Prill MM , Iwane MK , Edwards KM , Williams JV , Weinberg GA , Staat MA , Willby MJ , Talbot HK , Hall CB , Szilagyi PG , Griffin MR , Curns AT , Erdman DD . Pediatr Infect Dis J 2011 31 (3) 235-40 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 with asymptomatic controls and to assess the severity of outcomes among hospitalized children with HCoV compared with other respiratory viruses. METHODS: From December 2003-April 2004 and October 2004-April 2005, we conducted prospective, population-based surveillance of children <5 years of age hospitalized for ARI/fever in three US counties. Asymptomatic outpatient controls were enrolled concurrently. Nasal/throat swabs were tested for HCoV species HKU1, NL63, 229E, and OC43 by RT-PCR. 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. CONCLUSION: In this study of children hospitalized for acute respiratory illness and/or fever, HCoV infection was not associated with hospitalization or with increased severity of illness. |
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