Infections caused by drug-resistant bacteria are a major problem worldwide. Carbapenem-resistant Klebsiella pneumoniae, most notably isolates classified as multilocus sequence type (ST) 258, have emerged as an important cause of hospital deaths. ST258 isolates are predominantly multidrug resistant, and therefore infections caused by them are difficult to treat. It is not known why the ST258 lineage is the most prevalent cause of multidrug-resistant K. pneumoniae infections in the United States and other countries. Here we tested the hypothesis that carbapenem-resistant ST258 K. pneumoniae is a single genetic clone that has disseminated worldwide. We sequenced to closure the genomes of two ST258 clinical isolates and used these genomes as references for comparative genome sequencing of 83 additional clinical isolates recovered from patients at diverse geographic locations worldwide. Phylogenetic analysis of the SNPs in the core genome of these isolates revealed that ST258 K. pneumoniae organisms are two distinct genetic clades. This unexpected finding disproves the single-clone hypothesis. Notably, genetic differentiation between the two clades results from an ∼215-kb region of divergence that includes genes involved in capsule polysaccharide biosynthesis. The region of divergence appears to be a hotspot for DNA recombination events, and we suggest that this region has contributed to the success of ST258 K. pneumoniae. Our findings will accelerate research on novel diagnostic, therapeutic, and vaccine strategies designed to prevent and/or treat infections caused by multidrug resistant K. pneumoniae.antibiotic resistance | carbapenemase | Enterobacteriaceae | plasmid

We sequenced the genomes of 3,615 strains of serotype Emm protein 1 (M1) group A Streptococcus to unravel the nature and timing of molecular events contributing to the emergence, dissemination, and genetic diversification of an unusually virulent clone that now causes epidemic human infections worldwide. We discovered that the contemporary epidemic clone emerged in stepwise fashion from a precursor cell that first contained the phage encoding an extracellular DNase virulence factor (streptococcal DNase D2, SdaD2) and subsequently acquired the phage encoding the SpeA1 variant of the streptococcal pyrogenic exotoxin A superantigen. The SpeA2 toxin variant evolved from SpeA1 by a single-nucleotide change in the M1 progenitor strain before acquisition by horizontal gene transfer of a large chromosomal region encoding secreted toxins NAD + -glycohydrolase and streptolysin O. Acquisition of this 36-kb region in the early 1980s into just one cell containing the phage-encoded sdaD2 and speA2 genes was the final major molecular event preceding the emergence and rapid intercontinental spread of the contemporary epidemic clone. Thus, we resolve a decades-old controversy about the type and sequence of genomic alterations that produced this explosive epidemic. Analysis of comprehensive, population-based contemporary invasive strains from seven countries identified strong patterns of temporal population structure. Compared with a preepidemic reference strain, the contemporary clone is significantly more virulent in nonhuman primate models of pharyngitis and necrotizing fasciitis. A key finding is that the molecular evolutionary events transpiring in just one bacterial cell ultimately have produced millions of human infections worldwide.pathogenesis | phylogeography | mobile genetic element | flesh-eating disease | molecular clock

Integration of rapid identification and susceptibility techniques with antimicrobial stewardship significantly improved time to optimal therapy, and it decreased hospital length of stay and total costs. This innovative strategy has ramifications for other areas of patient care.

Coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and no proven treatments are available. Convalescent plasma therapy has been used with varying degrees of success to treat severe microbial infections for >100 years. Patients (n Z 25) with severe and/or life-threatening COVID-19 disease were enrolled at the Houston Methodist hospitals from March 28, 2020, to April 14, 2020. Patients were transfused with convalescent plasma, obtained from donors with confirmed severe acute respiratory syndrome coronavirus 2 infection who had recovered. The primary study outcome was safety, and the secondary outcome was clinical status at day 14 after transfusion. Clinical improvement was assessed on the basis of a modified World Health Organization six-point ordinal scale and laboratory parameters. Viral genome sequencing was performed on donor and recipient strains. At day 7 after transfusion with convalescent plasma, nine patients had at least a one-point improvement in clinical scale, and seven of those were discharged. By day 14 after transfusion, 19 (76%) patients had at least a one-point improvement in clinical status, and 11 were discharged. No adverse events as a result of plasma transfusion were observed. Whole genome sequencing data did not identify a strain genotype-disease severity correlation. The data indicate that administration of convalescent plasma is a safe treatment option for those with severe COVID-19 disease.

Coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2, has spread globally, and proven treatments are limited. Transfusion of convalescent plasma collected from donors who have recovered from COVID-19 is among many approaches being studied as potentially efficacious therapy. We are conducting a prospective, propensity score–matched study assessing the efficacy of COVID-19 convalescent plasma transfusion versus standard of care as treatment for severe and/or critical COVID-19. We present herein the results of an interim analysis of 316 patients enrolled at Houston Methodist hospitals from March 28 to July 6, 2020. Of the 316 transfused patients, 136 met a 28-day outcome and were matched to 251 non-transfused control COVID-19 patients. Matching criteria included age, sex, body mass index, comorbidities, and baseline ventilation requirement 48 hours from admission, and in a second matching analysis, ventilation status at day 0. Variability in the timing of transfusion relative to admission and titer of antibodies of plasma transfused allowed for analysis in specific matched cohorts. The analysis showed a significant reduction ( P = 0.047) in mortality within 28 days, specifically in patients transfused within 72 hours of admission with plasma with an anti-spike protein receptor binding domain titer of ≥1:1350. These data suggest that treatment of COVID-19 with high anti–receptor binding domain IgG titer convalescent plasma is efficacious in early-disease patients.

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R e s e a R c h a R t i c l e 3 5 4 6jci.org Volume 125 Number 9 September 2015 ogous recombinational molecular genetic event resulted in upregulation of production of extracellular NADase and SLO toxins and recently stimulated an epidemic of serotype M89 GAS infections caused by a recently emerged clone. ResultsPre-epidemic and epidemic serotype M1 strains. Mouse invasive infection and nonhuman primate pharyngitis and invasive infection studies show that the MGAS2221 strain, genetically representative of the epidemic ("new") clone, is more virulent than reference strain SF370, genetically representative of pre-epidemic ("old") serotype M1 strains (3). Strain MGAS2221 lacks polymorphisms in regulatory genes known to influence virulence, such as covRS, ropB, and mga. A previous expression microarray analysis of the transcriptomes of pre-epidemic and epidemic M1 strains found that only 8 core chromosomal genes were differentially transcribed between the two strain groups in early logarithmic growth phase (12). Given the clear evidence that pre-epidemic and epidemic M1 strains differ significantly in virulence, we tested the hypothesis that the expression microarray analysis had failed to identify critical virulence genes differentially regulated between the two strain groups. We reassessed global transcriptome differences in a panel of pre-epidemic and epidemic M1 strains using RNA sequencing (RNA-Seq) analysis, a more sensitive and accurate method of transcript analysis. RNA-Seq analysis detected 5 differentially expressed genes, confirming our previous expression microarray findings of very limited gene transcript differences between pre-epidemic and epidemic strains. Of particular interest, all 5 differentially regulated genes were located in the 36-kb region of recombinational replacement. These genes include nga, ifs (encoding Ifs, an endogenous inhibitor of SPN; refs. 15, 16), slo, and two small genes of uncertain function (Spy0142 and Spy0144; SPN enhances GAS survival by inhibiting pathogen internalization by host cells and also augments SLO cytotoxicity (7-10). SLO is a potent oxygen-sensitive cytolytic toxin that forms pores in hostcell membranes (10). The coordinated activities of SPN and SLO prevent maturation of phagolysosomes and thereby decrease phagocytic killing of GAS (9, 11). Several lines of evidence suggest that the nga and slo genes may play a critical role in M1 epidemicity. First, the transcript levels of nga and slo are significantly higher in epidemic strains than in preepidemic M1 GAS strains (12). Second, epidemic M1 strains produce more SPN and SLO activity than pre-epidemic strains (12,13). By comparing the genome sequences of a genetically representative epidemic (MGAS2221) strain and a pre-epidemic (SF370) strain, we discovered that the two strains differ by 59 SNPs and 2 indels in the nga-slo region of recombination (3). Among these SNPs, 3 were of particular interest because they are located in regions that, in principle, might influence nga and slo transcript level and strain phe...

Many animal species are susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and could act as reservoirs; however, transmission in free-living animals has not been documented. White-tailed deer, the predominant cervid in North America, are susceptible to SARS-CoV-2 infection, and experimentally infected fawns can transmit the virus. To test the hypothesis that SARS-CoV-2 is circulating in deer, 283 retropharyngeal lymph node (RPLN) samples collected from 151 free-living and 132 captive deer in Iowa from April 2020 through January of 2021 were assayed for the presence of SARS-CoV-2 RNA. Ninety-four of the 283 (33.2%) deer samples were positive for SARS-CoV-2 RNA as assessed by RT-PCR. Notably, following the November 2020 peak of human cases in Iowa, and coinciding with the onset of winter and the peak deer hunting season, SARS-CoV-2 RNA was detected in 80 of 97 (82.5%) RPLN samples collected over a 7-wk period. Whole genome sequencing of all 94 positive RPLN samples identified 12 SARS-CoV-2 lineages, with B.1.2 (n = 51; 54.5%) and B.1.311 (n = 19; 20%) accounting for ∼75% of all samples. The geographic distribution and nesting of clusters of deer and human lineages strongly suggest multiple human-to-deer transmission events followed by subsequent deer-to-deer spread. These discoveries have important implications for the long-term persistence of the SARS-CoV-2 pandemic. Our findings highlight an urgent need for a robust and proactive “One Health” approach to obtain enhanced understanding of the ecology, molecular evolution, and dissemination of SARS-CoV-2.