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Abstract Retrons are bacterial genetic elements that encode a reverse transcriptase and, in combination with toxic effector proteins, can serve as antiphage defense systems. However, the mechanisms of action of most retron effectors, and how phages evade retrons, are not well understood. Here, we show that some phages can evade retrons and other defense systems by producing specific tRNAs. We find that expression of retron-Eco7 effector proteins (PtuA and PtuB) leads to degradation of tRNATyr and abortive infection. The genomes of T5 phages that evade retron-Eco7 include a tRNA-rich region, including a highly expressed tRNATyr gene, which confers protection against retron-Eco7. Furthermore, we show that other phages (T1, T7) can use a similar strategy, expressing a tRNALys, to counteract a tRNA anticodon defense system (PrrC170).

Abstract Antimicrobial resistance is a global health concern; Enterobacterales resistant to third-generation cephalosporins (3GCs) and carbapenems are of the highest priority. Here, we conducted genome sequencing and standardized quantitative antimicrobial susceptibility testing of 4,195 isolates of Escherichia coli and Klebsiella pneumoniae resistant to 3GCs and Enterobacterales with reduced meropenem susceptibility collected across Japan. Our analyses provided a complete classification of 3GC resistance mechanisms. Analyses with complete reference plasmids revealed that among the bla CTX-M extended-spectrum β-lactamase genes, bla CTX-M-8 was typically encoded in highly similar plasmids. The two major AmpC β-lactamase genes were bla CMY-2 and bla DHA-1. Long-read sequencing of representative plasmids revealed that approximately 60% and 40% of bla CMY-2 and bla DHA-1 were encoded by such plasmids, respectively. Our analyses identified strains positive for carbapenemase genes but phenotypically susceptible to carbapenems and undetectable by standard antimicrobial susceptibility testing. Systematic long-read sequencing enabled reconstruction of 183 complete plasmid sequences encoding three major carbapenemase genes and elucidation of their geographical distribution stratified by replicon types and species carrying the plasmids and potential plasmid transfer events. Overall, we provide a blueprint for a national genomic surveillance study that integrates standardized quantitative antimicrobial susceptibility testing and characterizes resistance determinants.

Ottilie et al. employ an experimental evolution approach to investigate the role of transcription factors in yeast chemical resistance. Most emergent mutations in resistant strains were enriched in transcription factor coding genes, highlighting their importance in drug resistance.

Abstract The spread of vancomycin-resistant enterococci (VRE) is a major threat in nosocomial settings. A large-scale multiclonal VRE outbreak has rarely been reported in Japan due to low VRE prevalence. We evaluated the transmission of vancomycin resistance in a multiclonal VRE outbreak, conducted biological and genomic analyses of VRE isolates, and assessed the implemented infection control measures. In total, 149 patients harboring VanA-type VRE were identified from April 2017 to October 2019, with 153 vancomycin-resistant Enterococcus faecium isolated being grouped into 31 pulsotypes using pulsed-field gel electrophoresis, wherein six sequence types belonged to clonal complex 17. Epidemic clones varied throughout the outbreak; however, they all carried vanA-plasmids (pIHVA). pIHVA is a linear plasmid, carrying a unique structural Tn1546 containing vanA; it moves between different Enterococcus spp. by genetic rearrangements. VRE infection incidence among patients in the “hot spot” ward correlated with the local VRE colonization prevalence. Local prevalence also correlated with vancomycin usage in the ward. Transmission of a novel transferrable vanA-plasmid among Enterococcus spp. resulted in genomic diversity in VRE in a non-endemic setting. The prevalence of VRE colonization and vancomycin usage at the ward level may serve as VRE cross-transmission indicators in non-intensive care units for outbreak control.

Abstract The undifferentiated state of human induced pluripotent stem cells (hiPSCs) depends on their cell–cell and cell–substrate adhesions. In this study, we report that exposure to botulinum hemagglutinin (HA), an E-cadherin function-blocking agent, selectively removed cells that deviated from the undifferentiated state in hiPSC colonies. After HA treatment, cell–cell adhesion was disrupted, deviated cells detached from colony centers, and dividing cells filled these spaces. Because E-cadherin-mediated adhesion was disrupted in undifferentiated cells, stress-fiber formation and focal adhesions were diminished; however, these were subsequently restored, and the cells retained expression of undifferentiated stem cell markers and their differentiation potential. In contrast, actin structures and focal adhesions were lost from deviated cells, and they subsequently died. In undifferentiated and deviated cells, the cadherin/integrin-regulator Rap1 was localized at cell–cell adhesions and in the cytoplasm, respectively. Concurrent HA and Rap1-inhibitor treatment accelerated the deviated-cell detachment and delayed the recovery of hiPSC morphology, but this effect was significantly attenuated by co-treatment with Rap1 activator. Thus, Rap1 regulated E-cadherin–integrin interplay in hiPSC colonies exhibiting deviation, while HA-mediated selective removal of these deviated cells helped maintain the undifferentiated state in the remaining hiPSCs.

Abstract The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.