Putnamkryger4384

An analysis of the genome sequence of Yersinia mollaretii ATCC 43969 identified the blaYEM gene encoding YEM-1, a putative subclass B2 metallo-β-lactamase. The objectives of our work were to produce, purify and complete the kinetic characterization of YEM-1. YEM-1 displayed the narrowest substrate range among known subclass B2 metallo-β-lactamases since it can hydrolyze imipenem but not other carbapenems, such as biapenem, meropenem, doripenem and ertapenem, with a high catalytic efficiency. A possible explanation of this activity profile is the presence of tyrosine at residue 67 (loop L1), threonine at residue 156 (loop L2) and serine at residue 236 (loop L3). We showed that the substitution of Y67 broadened the activity profile of the enzyme for all carbapenems but still resulted in poor activity toward the other β-lactam classes.Most microbes live in spatially confined sub-populations. Under spatial structure, the efficacy of natural selection is often reduced (relative to homogeneous conditions), due to the increased importance of genetic drift and local competition. Additionally, under spatial structure, the fittest genotype may not always be the one with better access to the heterogeneous distribution of nutrients. The effect of radial expansion may be particularly relevant for the elimination of antibiotic resistance mutations, as their dynamics within bacterial populations are strongly dependent on their growth rate. Here, we use Escherichia coli to systematically compare the allele frequency of streptomycin, rifampicin and fluoroquinolone single and double resistance mutants after 24h of coexistence with a susceptible strain under radial expansion (local competition) and homogeneous (global competition) conditions. We show that there is a significant effect of structure on the maintenance of double resistances which is not observed for single resistances. Radial expansion also facilitates the persistence of double resistances when competing against their single counterparts. Importantly, we found that spatial structure reduces the rate of compensation of the double mutant RpsLK43TRpoBH526Y and that a strongly compensatory mutation in homogeneous conditions becomes deleterious under spatial structure. Overall, our results unravel the importance of spatial structure for facilitating the maintenance and accumulation of multiple resistances over time and for determining the identity of compensatory mutations.The treatment of infections caused by carbapenem-resistant Enterobacterales, especially New Delhi metallo-β-lactamase (NDM)-producing bacteria, is challenging. Although less common in the United States than some other carbapenemase-producers, NDM-producing bacteria are a public health threat due to the limited treatment options available. Here we report on the antibiotic susceptibility of 275 contemporary NDM-producing Enterobacterales collected from 30 U.S. HL 362 clinical trial states through the Centers for Disease Control and Prevention's Antibiotic Resistance Laboratory Network. The aim of the study was to determine the susceptibility of these isolates against 32 currently available antibiotics using reference broth microdilution and explore the in vitro activity of 3 combination agents that are not yet available. Categorical interpretations were determined using Clinical and Laboratory Standards Institute (CLSI) interpretative criteria. For agents without CLSI criteria, Food and Drug Administration (FDA) interpretative criteria were used. The percentage of susceptible isolates did not exceed 90% for any of the FDA-approved antibiotics tested. The antibiotics with breakpoints that had the highest in vitro activity were tigecycline (86.5% susceptible), eravacycline (66.2% susceptible), and omadacycline (59.6% susceptible) 18.2% of isolates were susceptible to aztreonam. All NDM-producing isolates tested were multidrug-resistant, and 116 isolates were extensively drug-resistant (42.2%) 207 (75.3%) isolates displayed difficult-to-treat resistance. The difficulty in treating infections caused by NDM-producing Enterobacterales highlights the need for containment and prevention efforts to keep these infections from becoming more common.In 2019, the WHO tuberculosis (TB) treatment guidelines were updated to recommend only limited use of streptomycin, in favor of newer agents or amikacin as the preferred aminoglycoside for drug-resistant Mycobacterium tuberculosis However, the emergence of resistance to newer drugs, such as bedaquiline, has prompted a reanalysis of antitubercular drugs in search of untapped potential. Using 211 clinical isolates of M. tuberculosis from South Africa, we performed phenotypic drug susceptibility testing (DST) to aminoglycosides by both critical concentration and MIC determination in parallel with whole-genome sequencing to identify known genotypic resistance elements. Isolates with low-level streptomycin resistance mediated by gidB were frequently misclassified with respect to streptomycin resistance when using the WHO-recommended critical concentration of 2 μg/mL. We identified 29 M. tuberculosis isolates from South Africa with low-level streptomycin resistance concomitant with high-level amikacin resistance, conferred by gidB and rrs 1400, respectively. Using a large global dataset of M. tuberculosis genomes, we observed 95 examples of this corresponding resistance genotype (gidB-rrs 1400), including identification in 81/257 (31.5%) of XDR isolates. In a phylogenetic analysis, we observed repeated evolution of low-level streptomycin and high-level amikacin resistance in multiple countries. Our findings suggest that current critical concentration methods and the design of molecular diagnostics need to be revisited to provide more accurate assessments of streptomycin resistance for gidB containing isolates. For patients harbouring isolates of M. tuberculosis with high-level amikacin resistance conferred by rrs 1400, and for whom newer agents are not available, treatment with streptomycin may still prove useful, even in the face of low-level resistance conferred by gidB.The Src homology 2 (SH2) domain has a highly conserved architecture that recognizes linear phosphotyrosine motifs and is present in a wide range of signaling pathways across different evolutionary taxa. A hallmark of SH2 domains is the arginine residue in the conserved "FLVR" motif that forms a direct salt bridge with bound phosphotyrosine. Here, we solve the X-ray crystal structures of the C-terminal SH2 domain of p120RasGAP (RASA1) in its apo and peptide-bound form. We find that the arginine residue in the FLVR motif does not directly contact pTyr-1087 of a bound phosphopeptide derived from p190RhoGAP; rather, it makes an intramolecular salt bridge to an aspartic acid. Unexpectedly, coordination of phosphotyrosine is achieved by a modified binding pocket which appears early in evolution. Using isothermal titration calorimetry, we find that substitution of the FLVR arginine R377A does not cause a significant loss of phosphopeptide binding, but rather a tandem substitution of R398A (SH2 position βD4) and K400A (SH2 position βD6) is required to disrupt the binding.