Wadsworth Center

Glyphosate, the active ingredient in herbicide Roundup, is the most widely used environmental contaminant that has been extensively studied for its potential carcinogenic effects. In the US alone, a staggering 81% of the US population ≥6 years of age is exposed to glyphosate. Notably, this coincides with the alarming rise in the incidence of hypertension, the single largest risk factor for global mortality through cardiovascular diseases. Here we asked if there is a link between glyphosate exposure and hypertension, the premise being that glyphosate targets the shikimate pathway present in gut microbiota coupled with more recent knowledge that gut microbiota causally regulate blood pressure. We hypothesized that glyphosate elevates hypertension through microbiota-mediated mechanisms and document a highly concerning detrimental effect of glyphosate by demonstrating a causal link between oral glyphosate exposure and significant elevation in blood pressure. Gut microbiota was identified as the central mediator of this effect. Mechanistically, glyphosate-mediated elevation in blood pressure was through disruption of both gut-liver and gut-vascular homeostasis via FXR-signaling and accumulation of the microbial metabolite shikimic acid, respectively. Together, these findings underscore the need to reconsider the unabated use of this herbicide, which adversely affects a cardinal sign of health.

BackgroundGroup A Streptococcus (GAS) is highly contagious and can cause invasive infections including bacteremia, sepsis, and necrotizing fasciitis. Thus, it is important to limit transmission and treat infections promptly and appropriately. In 2019, the Centers for Disease Control and Prevention (CDC) listed resistant GAS as a concerning threat due to increasing rates of clindamycin (CLI) and erythromycin (ERY) resistance. The Wadsworth Center (WC) is one of 10 laboratories in the country funded to perform GAS surveillance through the Emerging Infections Program (EIP) and receives isolates of GAS from hospitals, laboratories, and local health departments. WC also receives isolates from healthcare-associated investigations. In the past 6 years, over 2,200 GAS isolates have been received, including isolates from over 100 outbreaks.MethodsThese isolates are analyzed using multiple methods for identification and characterization, including antimicrobial resistance (AR) and whole genome sequencing (WGS). Antimicrobial susceptibility testing (AST) is performed using the bioMérieux E-test gradient strip to assess increased resistance to CLI and ERY.ResultsTo date, WC has tested over 1,400 GAS isolates and identified CLI resistance in 130 isolates and ERY resistance in 230 isolates. Inducible clindamycin resistance (ICR) in GAS can develop when the pathogen is exposed to other antibiotics, such as ERY, and can result in treatment failure. To address this concern, the ICR test is currently under validation and will be used alongside the E-test when applicable. In addition to culture AST, isolates are sent for WGS and analyzed using an AR bioinformatic gene detection pipeline to screen for genes potentially leading to AR in strains circulating in New York State (NYS). To date, this method has identified over 400 samples containing one or more AR genes. Data analysis is underway to assess the relationship between the presence of AR genes and the correlation with phenotypic AST results.ConclusionThe surveillance testing performed at WC represents an extensive testing algorithm that can be used for the identification of trends in antimicrobial resistance in GAS. Data analysis may lead to the development of new assays to improve testing algorithms at WC and other public health laboratories.DisclosuresAll Authors: No reported disclosures

CRISPR-Cas systems are prokaryotic adaptive immune systems that use RNA-guided protein complexes to target invading nucleic acid. A surveillance complex consisting of protein and a CRISPR-RNA (crRNA) binds target nucleic acid via base-pairing interactions, typically leading to processing of the target nucleic acid by a nuclease. CRISPR-Cas systems are classified based on their mechanism of action, with type I systems being the most prevalent in nature. Type I CRISPR-Cas systems target DNA, and require extensive complementarity between the crRNA and the target DNA. Moreover, type I systems require the presence of a “Protospacer Adjacent Motif” (PAM) sequence in the target DNA immediately adjacent to the expected region of base-pairing with the crRNA. Classical biotypes of the bacterial pathogen Vibrio cholerae have active type I-E CRISPR-Cas systems. While the optimal PAM sequence for this CRISPR-Cas system is known to be AAY, the activity of other sequences as possible PAMs has not been determined. Here, we quantify the effectiveness of all possible trinucleotide sequences in the PAM position for the V. cholerae type I-E CRISPR-Cas system. Our data indicate a hierarchy of PAM efficacy, with 15 of the 64 trinucleotide sequences functioning as a PAM.

Ribosomes translate the genetic code in mRNA to synthesize proteins in all living organisms. Decoding of mRNA occurs in the small subunit of the ribosome and is mediated by tRNA anticodons. Regions near the decoding center are a target for antibiotics, such as aminoglycosides and tetracyclines, where their presence results in errors in protein synthesis. More than two decades of high-resolution structural studies have shown how such medically important antimicrobials (MIAs) bind to the small subunit of the bacterial ribosome. Here, we comprehensively analyze these previously reported structures to help understand the variability with which MIAs bind to small subunits of bacterial ribosomes. We previously solved the hibernating 70S ribosome structure of the bacterial pathogen Borrelia burgdorferi (Bbu), the causative agent of Lyme disease, but there is no structure of any MIA bound to this ribosome reported. Our structural analysis makes it possible to use inexpensive computational methods to predict binding of these MIAs to the Bbu ribosomal 30S small subunit. For this, we used structural analogy, restrained energy minimization, and single-point binding free energy computations. We find the single-point binding free energy of the MIAs to be very sensitive to small conformational changes in the MIA and its environment. Incorporating this knowledge in structure-guided design could aid in development of narrow-spectrum MIAs targeting specific bacterial pathogens.

In the neonatal intensive care unit (NICU), outbreaks caused by methicillin-susceptible Staphylococcus aureus (MSSA) are less commonly described than outbreaks caused by methicillin-resistant Staphylococcus aureus (MRSA) despite the increased burden of MSSA infections. To investigate a NICU MSSA outbreak utilizing whole-genome sequencing (WGS) and multi-locus sequencing typing (MLST) to identify transmission events. An investigation was initiated in a level IV NICU after four patients developed MSSA skin and soft tissue infections (SSTI) within threeweeks. MLST and WGS were performed on MSSA isolates obtained from clinical and surveillance specimens. During the outbreak, 16 infants developed MSSA infections including SSTIs (n = 15) and bacteremia (n = 1). Thirteen SSTIs presented on neonates' faces, all of whom were on non-invasive respiratory support. During 7 rounds of surveillance, an additional 31 patients were found to be colonized with MSSA. MLST identified a predominant cluster (ST-121). WGS found that all ST-121 isolates were closely related (≤10 genetic variants between isolates) suggesting likely transmission events, harbored the mupA gene, exhibited mupirocin MIC values ≥1,024 µg/mL, and were associated with infection. Multiple infection control measures were implemented including the "bare below the elbows" practice. No further mupirocin-resistant isolates were recovered or ST-121 SSTIs identified after Week 26. WGS analysis furthered the MLST analysis and identified a single MLST as the outbreak-related strain. Successful control of this outbreak was achieved with a multitude of infection prevention and control methods.

Abstract Stable acquisitions of metabolism, such as the endosymbiotic incorporation of eukaryotic chloroplasts, are thought to proceed through mechanisms that increase the genetic repertoire of the host and allow for vertical integration of new metabolism. Here we test these predictions using the chloroplast-stealing marine ciliate genus Mesodinium by comparing transcriptomes from species that represent a spectrum from full heterotrophy to nearly full phototrophy. In contrast to theory, we find a striking divestment in metabolic autonomy with increased reliance on acquired photosynthesis. Indeed, the highly photosynthetic, red tide-forming Mesodinium rubrum appears to have lost the capacity to synthesize amino acids, metabolize fatty acids, and produce peroxisomes. Our results portray a metabolic parasite, masquerading as a free-living ciliate, yet incapable of satisfying most of its basic anabolic needs.

ABSTRACT Isoniazid (INH) is a critical antibiotic used worldwide for treatment and prophylaxis of tuberculosis. Drug resistance (DR) to INH is the single most common type of DR, mediated by multiple genes/loci including katG, inhA, mabA, mabA-inhA and the oxyR-ahpC intergenic region. Over the course of 6 years, we performed a 2-phase study of 3,696 Mycobacterium tuberculosis complex (MTBC) strains aiming to determine the molecular basis of INH resistance and assess whole genome sequencing (WGS) for predicting resistance. In phase 1, we performed a side-by-side study including 1,767 strains with paired phenotypic drug susceptibility testing (DST) and genotypic DST. We found WGS capable of accurately predicting INH resistance with sensitivity of 90.3%, and specificity of 99.8%. The negative predictive value of WGS for INH susceptibility was 98.8%. Based on these findings, we developed a molecular testing algorithm where phenotypic DST was reduced and applied this new testing algorithm in phase 2 to 1,929 MTBC strains, resulting in streamlined testing, reduced cost and decreasing turnaround time (TAT). The prevalence of INH resistance among MTBC strains in New York was found to be 10.2%. Of the 3,696 isolates tested, 337 were predicted INH resistant by WGS. Of 41 additional strains exhibiting phenotypic INH resistance, 38 were found to have mutations in genes known to be associated with INH resistance. This study demonstrates the utility of WGS as a molecular tool for predicting INH DR and shows that the vast majority of INH resistance in MTBC has a molecular basis in known resistance loci.