Public health officials have raised concerns that plasmid transfer between Enterobacteriaceae species may spread resistance to carbapenems, an antibiotic class of last resort, thereby rendering common healthcare-associated infections nearly impossible to treat. We performed comprehensive surveillance and genomic sequencing to identify carbapenem-resistant Enterobacteriaceae in the NIH Clinical Center patient population and hospital environment in order to to articulate the diversity of carbapenemase-encoding plasmids and survey the mobility of and assess the mobility of these plasmids between bacterial species. We isolated a repertoire of carbapenemase-encoding Enterobacteriaceae, including multiple strains of Klebsiella pneumoniae, Klebsiella oxytoca, Escherichia coli, Enterobacter cloacae, Citrobacter freundii, and Pantoea species. Long-read genome sequencing with full end-to-end assembly revealed that these organisms carry the carbapenem-resistance genes on a wide array of plasmids. Klebsiella pneumoniae and Enterobacter cloacae isolated simultaneously from a single patient harbored two different carbapenemase-encoding plasmids, overriding the epidemiological scenario of plasmid transfer between organisms within this patient. We did, however, find evidence supporting horizontal transfer of carbapenemase-encoding plasmids between Klebsiella pneumoniae, Enterobacter cloacae and Citrobacter freundii in the hospital environment. Our comprehensive sequence data, with full plasmid identification, challenges assumptions about horizontal gene transfer events within patients and identified wider possible connections between patients and the hospital environment. In addition, we identified a new carbapenemase-encoding plasmid of potentially high clinical impact carried by Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Pantoea species, from unrelated patients and the hospital environment.

Nonsusceptibility to first-line antibiotics is associated with decreased survival in GNBSIs. DTR is a simple bedside prognostic measure of treatment-limiting coresistance.

Voltage-gated potassium channels such as Shaker help to control electrical signalling in neurons by regulating the passage of K+ across cell membranes. Ion flow is controlled by a voltage-dependent gate at the intracellular side of the pore, formed by the crossing of four alpha-helices--the inner-pore helices. The prevailing model of gating is based on a comparison of the crystal structures of two bacterial channels--KcsA in a closed state and MthK in an open state--and proposes a hinge motion at a conserved glycine that splays the inner-pore helices wide open. We show here that two types of intersubunit metal bridge, involving cysteines placed near the bundle crossing, can occur simultaneously in the open state. These bridges provide constraints on the open Shaker channel structure, and on the degree of movement upon opening. We conclude that, unlike predictions from the structure of MthK, the inner-pore helices of Shaker probably maintain the KcsA-like bundle-crossing motif in the open state, with a bend in this region at the conserved proline motif (Pro-X-Pro) not found in the bacterial channels. A narrower opening of the bundle crossing in Shaker K+ channels may help to explain why Shaker has an approximately tenfold lower conductance than its bacterial relatives.

Carbapenemase-producing Enterobacteriaceae (CPE), which are resistant to most or all known antibiotics, constitute a global threat to public health. Transposable elements are often associated with antibiotic resistance determinants, suggesting a role in the emergence of resistance. One insertion sequence, IS26, is frequently associated with resistance determinants, but its role remains unclear. We have analyzed the genomic contexts of 70 IS26 copies in several clinical and surveillance CPE isolates from the National Institutes of Health Clinical Center. We used target site duplications and their patterns as guides and found that a large fraction of plasmid reorganizations result from IS26 replicative transpositions, including replicon fusions, DNA inversions, and deletions. Replicative transposition could also be inferred for transposon Tn4401, which harbors the carbapenemase blaKPC gene. Thus, replicative transposition is important in the ongoing reorganization of plasmids carrying multidrug-resistant determinants, an observation that carries substantial clinical and epidemiological implications for understanding how such extreme drug resistance phenotypes evolve.

Carbapenem-resistant Enterobacteriaceae (CRE) are among the most severe threats to the antibiotic era. Multiple different species can exhibit resistance due to many different mechanisms, and many different mobile elements are capable of transferring resistance between lineages. We prospectively sampled CRE from hospitalized patients from three Boston-area hospitals, together with a collection of CRE from a single California hospital, to define the frequency and characteristics of outbreaks and determine whether there is evidence for transfer of strains within and between hospitals and the frequency with which resistance is transferred between lineages or species. We found eight species exhibiting resistance, with the majority of our sample being the sequence type 258 (ST258) lineage of Klebsiella pneumoniae. There was very little evidence of extensive hospital outbreaks, but a great deal of variation in resistance mechanisms and the genomic backgrounds carrying these mechanisms. Local transmission was evident in clear phylogeographic structure between the samples from the two coasts. The most common resistance mechanisms were KPC (K. pneumoniae carbapenemases) beta-lactamases encoded by bla KPC2 , bla KPC3 , and bla KPC4 , which were transferred between strains and species by seven distinct subgroups of the Tn4401 element. We also found evidence for previously unrecognized resistance mechanisms that produced resistance when transformed into a susceptible genomic background. The extensive variation, together with evidence of transmission beyond limited clonal outbreaks, points to multiple unsampled transmission chains throughout the continuum of care, including asymptomatic carriage and transmission of CRE. This finding suggests that to control this threat, we need an aggressive approach to surveillance and isolation.carbapenem resistance | Enterobacteriaceae | comparative genomics | whole-genome sequencing | molecular evolution

Analysis of mcr-1-containing sequences identified a common ϳ2,607-bp DNA segment that in many cases is flanked on one or both ends by ISApl1. We present evidence that mcr-1 is mobilized by an ISApl1 composite transposon which has, in some cases, subsequently lost one or both copies of ISApl1. We also show that mcr-1 can be mobilized in some circumstances by a single upstream copy of ISApl1 in conjunction with the remnants of a downstream ISApl1. Colistin is increasingly relied upon to treat infections caused by multidrug-resistant, carbapenemase-producing Enterobacteriaceae (1). Consequently, there is great concern surrounding plasmid-borne colistin resistance mediated by mcr-1 (2). mcr-1 has now been reported in Enterobacteriaceae from five continents: Asia, Europe, Africa, South America, and North America (reviewed in reference 3). A recent study detected mcr-1 from Escherichia coli collected in the early 1980s, suggesting that the emergence of mcr-1 occurred much earlier than previously thought (4). Given the global dissemination of mcr-1 and the potential clinical consequences, understanding the molecular mechanisms underlying its mobility is critical. Here we provide a detailed examination of the genetic context surrounding mcr-1 and present evidence that mcr-1 is primarily mobilized by an ISApl1 composite transposon.There is increasing evidence that ISApl1 plays a pivotal role in the mobilization of mcr-1 (5, 6). ISApl1 was first detected in Actinobacillus pleuropneumoniae, an etiological agent of disease in swine (7). It belongs to the IS30 family and possesses features and activities similar to those of IS30 members. It is flanked by 27-bp inverted repeats (designated IRL and IRR, for inverted repeat left and inverted repeat right, respectively) and contains a 927-bp open reading frame (ORF) that encodes a DD(E/D) superfamily transposase protein. Like IS30 and other family members, ISApl1 moves through a covalently closed double-stranded DNA (dsDNA) circular intermediate containing 2 bp of host flanking DNA between the abutted transposon ends and, upon integration, generates 2-bp target site duplications (TSDs) (7-9). Such circular intermediates are characteristic of a large number of IS families (10) and appear to be produced by a replicative mechanism known as copy out-paste in (11). Furthermore, the ends of these IS elements are often preferred target sites for insertion by the same IS (12).We employed the Geneious R9.1 software suite (Geneious, Auckland, New Zealand) to analyze 77 mcr-1-containing sequences, 19 of which were too fragmented to be useful (see Table S1 in the supplemental material). This represents every unique sequence present in NCBI as of this writing (August 2016). A common feature in all sequences is an ϳ2,607-bp DNA segment containing mcr-1 and a putative 765-bp ORF encoding a protein similar to a PAP2 superfamily protein (Fig. 1). In 7 sequences, this segment is flanked by two copies of ISApl1 in the same orientation, reminiscent of a composite transposon (Fig. 2A). In a further...

IMPORTANCE Broad-spectrum antibiotics are recommended for all patients with suspected sepsis to minimize the risk of undertreatment. However, little is known regarding the net prevalence of antibiotic-resistant pathogens across all patients with community-onset sepsis or the outcomes associated with unnecessarily broad empiric treatment. OBJECTIVE To elucidate the epidemiology of antibiotic-resistant pathogens and the outcomes associated with both undertreatment and overtreatment in patients with culture-positive communityonset sepsis. DESIGN, SETTING, AND PARTICIPANTS This cohort study included 17 430 adults admitted to 104 US hospitals between January 2009 and December 2015 with sepsis and positive clinical cultures within 2 days of admission. Data analysis took place from January 2018 to December 2019. EXPOSURES Inadequate empiric antibiotic therapy (ie, Ն1 pathogen nonsusceptible to all antibiotics administered on the first or second day of treatment) and unnecessarily broad empiric therapy (ie, active against methicillin-resistant Staphylococcus aureus [MRSA]; vancomycin-resistant Enterococcus [VRE]; ceftriaxone-resistant gram-negative [CTX-RO] organisms, including Pseudomonas aeruginosa; or extended-spectrum β-lactamase [ESBL] gram-negative organisms when none of these were isolated). MAIN OUTCOMES AND MEASURES Prevalence and empiric treatment rates for antibiotic-resistant organisms and associations of inadequate and unnecessarily broad empiric therapy with in-hospital mortality were assessed, adjusting for baseline characteristics and severity of illness. RESULTS Of 17 430 patients with culture-positive community-onset sepsis (median [interquartile range] age, 69 [57-81] years; 9737 [55.9%] women), 2865 (16.4%) died in the hospital. The most common culture-positive sites were urine (9077 [52.1%]), blood (6968 [40.0%]), and the respiratory tract (2912 [16.7%]). The most common pathogens were Escherichia coli (5873 [33.7%]), S aureus (3706 [21.3%]), and Streptococcus species (2361 [13.5%]). Among 15 183 cases in which all antibiotic-pathogen susceptibility combinations could be calculated, most (12 398 [81.6%]) received adequate empiric antibiotics. Empiric therapy targeted resistant organisms in 11 683 of 17 430 cases (67.0%; primarily vancomycin and anti-Pseudomonal β-lactams), but resistant organisms were uncommon (MRSA, 2045 [11.7%]; CTX-RO, 2278 [13.1%]; VRE, 360 [2.1%]; ESBLs, 133 [0.8%]). The net prevalence for at least 1 resistant gram-positive organism (ie, MRSA or VRE) was 13.6% (2376 patients), and for at least 1 resistant gram-negative organism (ie, CTX-RO, ESBL, or CRE), it was 13.2% (2297 patients). Both inadequate and unnecessarily broad empiric antibiotics were associated with (continued) Key Points Question What is the prevalence of antibiotic resistance in communityonset sepsis, and is there risk associated with the receipt of empiric broadspectrum antibiotics? Findings In this cohort study of 17 430 adults with culture-positive sepsis admitted to 104 US hospitals, 67.0% received empiric b...

The effect of surges in COVID-19 caseload on COVID-19 survival rates is unclear, especially independent of temporal changes in survival. This retrospective cohort study used data from a large U.S. hospital database to study the association between caseload surges and risk-adjusted mortality in patients with COVID-19.