Top-Down Genomic Surveillance Approach To Investigate the Genomic Epidemiology and Antibiotic Resistance Patterns of Enterococcus faecium Detected in Cancer Patients in Arkansas.

Control of hospital-associated Enterococcus faecium infection is a strenuous task due to the difficulty of identifying transmission routes and the persistence of this nosocomial pathogen despite the implementation of infection control measures that have been successful with other important nosocomial pathogens. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from 66 cancer patients at the University of Arkansas for Medical Sciences (UAMS) between June 2018 and May 2019. In the top-down approach used in this study, we employed, in addition to the 106 E. faecium UAMS isolates, a filtered set of 2,167 E. faecium strains from the GenBank database to assess the current population structure of E. faecium species and, consequently, to identify the lineages associated with our clinical isolates. We then evaluated the antibiotic resistance and virulence profiles of hospital-associated strains from the species pool, focusing on antibiotics of last resort, to establish an updated classification of high-risk and multidrug-resistant nosocomial clones. Further investigation of the clinical isolates collected from UAMS patients using whole-genome sequencing analytical methodologies (core genome multilocus sequence typing [cgMLST], core single nucleotide polymorphism [coreSNP] analysis, and phylogenomics), with the addition of patient epidemiological data, revealed a polyclonal outbreak of three sequence types occurring simultaneously in different patient wards. The integration of genomic and epidemiological data collected from the patients increased our understanding of the relationships and transmission dynamics of the E. faecium isolates. Our study provides new insights into genomic surveillance of E. faecium to assist in monitoring and further limiting the spread of multidrug-resistant E. faecium. IMPORTANCE Enterococcus faecium is a member of the gastrointestinal microbiota. Although its virulence is low in healthy, immunocompetent individuals, E. faecium has become the third leading cause of health care-associated infections in the United States. This study provides a comprehensive analysis of over 100 E. faecium isolates collected from cancer patients at the University of Arkansas for Medical Sciences (UAMS). We employed a top-down analytical approach (from population genomics to molecular biology) to classify our clinical isolates into their genetic lineages and thoroughly evaluate their antibiotic resistance and virulence profiles. The addition of patient epidemiological data to the whole-genome sequencing analytical methodologies performed in the study allowed us to increase our understanding of the relationships and transmission dynamics of the E. faecium isolates. This study provides new insights into genomic surveillance of E. faecium to help monitor and further limit the spread of multidrug-resistant E. faecium.

Pseudomonas aeruginosa Pangenome: Core and Accessory Genes of a Highly Resourceful Opportunistic Pathogen.

In this chapter, we leverage a novel approach to assess the seamless population structure of Pseudomonas aeruginosa, using the full repertoire of genomes sequenced to date (GenBank, April 6, 2020). In order to assess the set of core functions that represents the species as well as the differences in these core functions among the phylogroups observed in the population structure analysis, we performed pangenome analyses at the species level and at the phylogroup level. The existence of the phylogroups described in the population structure analyses was supported by their different profiles of antibiotic-resistant determinants. Finally, we utilized a presence/absence matrix of protein families from the entire species to evaluate if P. aeruginosa phylogroups can be differentiated according to their accessory genomic content. Our analysis shows that the core genome of P. aeruginosa is approximately 62% of the average gene content for the species, and it is highly enriched with pathways related to the metabolism of carbohydrates and amino acids as well as cellular processes and cell maintenance. The analysis of the accessory genome of P. aeruginosa performed in this chapter confirmed not only the existence of the three phylogroups previously described in the population structure analysis, but also of 29 genetic substructures (subgroups) within the main phylogroups. Our work illustrates the utility of populations genomics pipelines to better understand highly complex bacterial species such as P. aeruginosa.

Insertion sequences and other mobile elements associated with antibiotic resistance genes in Enterococcus isolates from an inpatient with prolonged bacteraemia.

Insertion sequences (ISs) and other transposable elements are associated with the mobilization of antibiotic resistance determinants and the modulation of pathogenic characteristics. In this work, we aimed to investigate the association between ISs and antibiotic resistance genes, and their role in the dissemination and modification of the antibiotic-resistant phenotype. To that end, we leveraged fully resolved Enterococcus faecium and Enterococcus faecalis genomes of isolates collected over 5 days from an inpatient with prolonged bacteraemia. Isolates from both species harboured similar IS family content but showed significant species-dependent differences in copy number and arrangements of ISs throughout their replicons. Here, we describe two inter-specific IS-mediated recombination events and IS-mediated excision events in plasmids of E. faecium isolates. We also characterize a novel arrangement of the ISs in a Tn1546-like transposon in E. faecalis isolates likely implicated in a vancomycin genotype-phenotype discrepancy. Furthermore, an extended analysis revealed a novel association between daptomycin resistance mutations in liaSR genes and a putative composite transposon in E. faecium, offering a new paradigm for the study of daptomycin resistance and novel insights into its dissemination. In conclusion, our study highlights the role ISs and other transposable elements play in the rapid adaptation and response to clinically relevant stresses such as aggressive antibiotic treatment in enterococci.

Complete Genome Sequence of a Non-Carbapenemase-Producing Carbapenem-Resistant Providencia rettgeri Strain Isolated from a Clinical Urine Sample in Arkansas.

Here, we report the complete genome sequence of Providencia rettgeri isolate PROV_UAMS_01, which was recovered in 2021 from a urine sample from a hospitalized patient in Arkansas, USA. The genome sequence of P. rettgeri isolate PROV_UAMS_01 comprises a single chromosomal replicon with a G+C content of 40.51% and a total of 3,887 genes.

Isolation of AmpC- and extended spectrum ß-lactamase-producing Enterobacterales from fresh vegetables in the United States.

Vegetables may serve as a reservoir for antibiotic resistant bacteria and resistance genes. AmpC ß-lactamases and extended spectrum beta-lactamases (ESBL) inactivate commonly used ß-lactam antibiotics, including penicillins and cephalosporins. In this study, we determined the prevalence of AmpC and ESBL-producing Enterobacterales in retail vegetables in the United States. A total of 88 vegetable samples were collected for the screening of AmpC and ESBL-producing Enterobacterales using CHROMagar ESBL agar. These vegetables included washed ready-to-eat salad (23), microgreens/sprouts (13), lettuce (11), herbs (11), spinach (5), mushrooms (5), brussels sprouts (4), kale (3), and other vegetable samples (13). AmpC and ESBL activity in these isolates were determined using double disk combination tests. Two vegetable samples (2.27%), organic basil and brussels sprouts, were positive for AmpC-producing Enterobacterales and eight samples (9.09%), including bean sprouts, organic parsley, organic baby spinach, and several mixed salads, were positive for ESBL-producing Enterobacterales. Whole genome sequencing was used to identify the bacterial species and resistance genes in these isolates. Genes encoding AmpC ß-lactamases were found in Enterobacter hormaechei strains S43-1 and 74-2, which were consistent with AmpC production phenotypes. Multidrug-resistant E. hormaechei strains S11-1, S17-1, and S45-4 possess an ESBL gene, blaSHV66 , whereas five Serratia fonticola isolates contain genes encoding a minor ESBL, FONA-5. In addition, we used shotgun metagenomic sequencing approach to examine the microbiome and resistome profiles of three spinach samples. We found that Pseudomonas was the most prevalent bacteria genus in the spinach samples. Within the Enterobacteriaceae family, Enterobacter was the most abundant genus in the spinach samples. Moreover, antibiotic resistance genes encoding 12 major classes of antibiotics, including ß-lactam antibiotics, aminoglycoside, macrolide, fluoroquinolone, and others, were found in these spinach samples. Therefore, vegetables can serve as an important vehicle for transmitting antibiotic resistance. The study highlights the need for antibiotic resistance surveillance in vegetable products.

Mash-based analyses of Escherichia coli genomes reveal 14 distinct phylogroups.

In this study, more than one hundred thousand Escherichia coli and Shigella genomes were examined and classified. This is, to our knowledge, the largest E. coli genome dataset analyzed to date. A Mash-based analysis of a cleaned set of 10,667 E. coli genomes from GenBank revealed 14 distinct phylogroups. A representative genome or medoid identified for each phylogroup was used as a proxy to classify 95,525 unassembled genomes from the Sequence Read Archive (SRA). We find that most of the sequenced E. coli genomes belong to four phylogroups (A, C, B1 and E2(O157)). Authenticity of the 14 phylogroups is supported by several different lines of evidence: phylogroup-specific core genes, a phylogenetic tree constructed with 2613 single copy core genes, and differences in the rates of gene gain/loss/duplication. The methodology used in this work is able to reproduce known phylogroups, as well as to identify previously uncharacterized phylogroups in E. coli species.

Towards a better metabolic engineering reference: the microbial chassis.

Highlight of the work of Calero and Nikel published in Microbial Biotechnology journal a few months ago.