Emergence of the E484K Mutation in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) Lineage B.1.1.345 in Upstate New York

A severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) B.1.1.345 variant carrying the E484K mutation was detected in 4 patients with no apparent epidemiological association from a hospital network in upstate New York. Subsequent analysis identified an additional 11 B.1.1.345 variants from this region between December 2020 and February 2021

Detection of Bacterial <em>16S</em> rRNA and Identification of Four Clinically Important Bacteria by Real-Time PCR

<div><p>Within the paradigm of clinical infectious disease research, <em>Acinetobacter baumannii</em>, <em>Escherichia coli</em>, <em>Klebsiella pneumoniae</em>, and <em>Pseudomonas aeruginosa</em> represent the four most clinically relevant, and hence most extensively studied bacteria. Current culture-based methods for identifying these organisms are slow and cumbersome, and there is increasing need for more rapid and accurate molecular detection methods. Using bioinformatic tools, 962,279 bacterial <em>16S</em> rRNA gene sequences were aligned, and regions of homology were selected to generate a set of real-time PCR primers that target 93.6% of all bacterial 16S rRNA sequences published to date. A set of four species-specific real-time PCR primer pairs were also designed, capable of detecting less than 100 genome copies of <em>A. baumannii</em>, <em>E. coli</em>, <em>K. pneumoniae</em>, and <em>P. aeruginosa</em>. All primers were tested for specificity <em>in vitro</em> against 50 species of Gram-positive and –negative bacteria. Additionally, the species-specific primers were tested against a panel of 200 clinical isolates of each species, randomly selected from a large repository of clinical isolates from diverse areas and sources. A comparison of culture and real-time PCR demonstrated 100% concordance. The primers were incorporated into a rapid assay capable of positive identification from plate or broth cultures in less than 90 minutes. Furthermore, our data demonstrate that current targets, such as the <em>uidA</em> gene in <em>E.coli</em>, are not suitable as species-specific genes due to sequence variation. The assay described herein is rapid, cost-effective and accurate, and can be easily incorporated into any research laboratory capable of real-time PCR.</p> </div

A diverse panel of clinical acinetobacter baumannii for research and development

Over the past two decades, Acinetobacter baumannii has emerged as a leading cause of nosocomial infections worldwide. Of particular concern are panresistant strains, leading the World Health Organization (WHO) to designate carbapenem-resistant A. baumannii as a priority 1 (critical) pathogen for research and development of new antibiotics. A key component in supporting this effort is accessibility to diverse and clinically relevant strains for testing. Here, we describe a panel of 100 diverse A. baumannii strains for use in this endeavor. Whole-genome sequencing was performed on 3,505 A. baumannii isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. Isolates were cultured from clinical samples at health care facilities around the world between 2001 and 2017. Core-genome multilocus sequence typing and high-resolution single nucleotide polymorphism (SNP)-based phylogenetic analyses were used to select a final panel of 100 strains that captured the genetic diversity of the collection. Comprehensive antibiotic susceptibility testing was also performed on all 100 isolates using 14 clinically relevant antibiotics. The final 100-strain diversity panel contained representative strains from 70 different traditional Pasteur scheme multilocus sequence types, including major epidemic clones. This diversity was also reflected in antibiotic susceptibility and antimicrobial resistance (AMR) gene content, with phenotypes ranging from pansensitive to panresistant, and over 100 distinct AMR gene alleles identified from 32 gene families. This panel provides the most diverse and comprehensive set of A. baumannii strains for use in developing solutions for combating antibiotic resistance. The panel and all available metadata, including genome sequences, will be available to industry and academic institutions and federal and other laboratories free of charge

Primer characteristics.

1<p>Probe sequences were generated for the <i>secE</i> and <i>yccT</i> real-time PCR primers, but no <i>in vitro</i> testing was performed. The probe sequences are provided here for the benefit of researchers wishing to perform multiplex real-time PCR reactions using these primers. All primers have an optimal annealing temperature of 56°C.</p>2<p>Primer efficiency was calculated from the slope and intercept of the trendline produced following amplification of serial dilutions of genomic DNA from the ATCC strains of each species, as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0048558#pone.0048558-McGann1" target="_blank">[14]</a>.</p>3<p>Amplicon size in base pairs.</p

Amplification and melting curves for selected isolates.

<p>(<b>A</b>) Amplification curves of three of the eight <i>E. coli</i> isolates that were <i>uidA</i>-negative, but <i>yccT</i>-positive; MRSN 1628 (Red line), MRSN 1681 (Blue Line), and MRSN 7544 (Green Line). MRSN 7851 (Yellow Line) was both <i>uidA</i> and <i>yccT</i>-positive. <i>E.coli</i> ATCC 35218 (Black line) was used as a positive control and <i>K. pneumoniae</i> ATCC 1706 (Grey Line) was used a negative control. Each amplification curve is one representative sample from quintuplicate experiments. (<b>B</b>) Melting curve analysis of the amplicons produced by the <i>yccT</i> primer pair from 30 clinical isolates of <i>E. coli</i> demonstrating a highly conserved sequence in all strains. Melting curve of the <i>yccT</i> amplicon from <i>E. coli</i> ATCC 35218 is shown in red. All 30 isolates represented diverse pulse-types as determined by PFGE. RFU - Relative Fluorescent Units; -d(RFU)dt – Relative change in RFU over time (in seconds).</p

Six Extensively Drug-Resistant Bacteria in an Injured Soldier, Ukraine

Blood and surveillance cultures from an injured service member from Ukraine grew Acinetobacter baumannii, Klebsiella pneumoniae, Enterococcus faecium, and 3 distinct Pseudomonas aeruginosa strains. Isolates were nonsusceptible to most antibiotics and carried an array of antibiotic resistant genes, including carbapenemases (blaIMP-1, blaNDM-1, blaOXA-23, blaOXA-48, blaOXA-72) and 16S methyltransferases (armA and rmtB4)

A one-year genomic investigation of Escherichia coli epidemiology and nosocomial spread at a large US healthcare network

Abstract Background Extra-intestinal pathogenic Escherichia coli (ExPEC) are a leading cause of bloodstream and urinary tract infections worldwide. Over the last two decades, increased rates of antibiotic resistance in E. coli have been reported, further complicating treatment. Worryingly, specific lineages expressing extended-spectrum β-lactamases (ESBLs) and fluoroquinolone resistance have proliferated and are now considered a serious threat. Obtaining contemporary information on the epidemiology and prevalence of these circulating lineages is critical for containing their spread globally and within the clinic. Methods Whole-genome sequencing (WGS), phylogenetic analysis, and antibiotic susceptibility testing were performed for a complete set of 2075 E. coli clinical isolates collected from 1776 patients at a large tertiary healthcare network in the USA between October 2019 and September 2020. Results The isolates represented two main phylogenetic groups, B2 and D, with six lineages accounting for 53% of strains: ST-69, ST-73, ST-95, ST-131, ST-127, and ST-1193. Twenty-seven percent of the primary isolates were multidrug resistant (MDR) and 5% carried an ESBL gene. Importantly, 74% of the ESBL-E.coli were co-resistant to fluoroquinolones and mostly belonged to pandemic ST-131 and emerging ST-1193. SNP-based detection of possible outbreaks identified 95 potential transmission clusters totaling 258 isolates (12% of the whole population) from ≥ 2 patients. While the proportion of MDR isolates was enriched in the set of putative transmission isolates compared to sporadic infections (35 vs 27%, p = 0.007), a large fraction (61%) of the predicted outbreaks (including the largest cluster grouping isolates from 12 patients) were caused by the transmission of non-MDR clones. Conclusion By coupling in-depth genomic characterization with a complete sampling of clinical isolates for a full year, this study provides a rare and contemporary survey on the epidemiology and spread of E. coli in a large US healthcare network. While surveillance and infection control efforts often focus on ESBL and MDR lineages, our findings reveal that non-MDR isolates represent a large burden of infections, including those of predicted nosocomial origins. This increased awareness is key for implementing effective WGS-based surveillance as a routine technology for infection control

A panel of diverse Klebsiella pneumoniae clinical isolates for research and development

Klebsiella pneumoniae are a leading cause of healthcare-associated infections worldwide. In particular, strains expressing extended-spectrum β-lactamases (ESBLs) and carbapenemases pose serious treatment challenges, leading the World Health Organization (WHO) to designate ESBL and carbapenem-resistant Enterobacteriaceae as \u27critical\u27 threats to human health. Research efforts to combat these pathogens can be supported by accessibility to diverse and clinically relevant isolates for testing novel therapeutics. Here, we describe a panel of 100 diverse K. pneumoniae isolates that are publicly available to assist the research community in this endeavour. Whole-genome sequencing (WGS) was performed on 3878 K. pneumoniae clinical isolates housed at the Multidrug-Resistant Organism Repository and Surveillance Network. The isolates were cultured from 63 facilities in 19 countries between 2001 and 2020. Core-genome multilocus sequence typing and high-resolution single-nucleotide polymorphism-based phylogenetic analyses captured the genetic diversity of the collection and were used to select the final panel of 100 isolates. In addition to known multidrug-resistant (MDR) pandemic lineages, the final panel includes hypervirulent lineages and isolates with specific and diverse resistance genes and virulence biomarkers. A broad range of antibiotic susceptibilities, ranging from pan-sensitive to extensively drug-resistant isolates, are described. The panel collection, and all associated metadata and genome sequences, are available at no additional cost and will be an important resource for the research community and for the design and development of novel antimicrobial agents and diagnostics against this important pathogen