Emerging enterococcus pore-forming toxins with MHC/HLA-I as receptors.

Enterococci are a part of human microbiota and a leading cause of multidrug resistant infections. Here, we identify a family of Enterococcus pore-forming toxins (Epxs) in E. faecalis, E. faecium, and E. hirae strains isolated across the globe. Structural studies reveal that Epxs form a branch of ß-barrel pore-forming toxins with a ß-barrel protrusion (designated the top domain) sitting atop the cap domain. Through a genome-wide CRISPR-Cas9 screen, we identify human leukocyte antigen class I (HLA-I) complex as a receptor for two members (Epx2 and Epx3), which preferentially recognize human HLA-I and homologous MHC-I of equine, bovine, and porcine, but not murine, origin. Interferon exposure, which stimulates MHC-I expression, sensitizes human cells and intestinal organoids to Epx2 and Epx3 toxicity. Co-culture with Epx2-harboring E. faecium damages human peripheral blood mononuclear cells and intestinal organoids, and this toxicity is neutralized by an Epx2 antibody, demonstrating the toxin-mediated virulence of Epx-carrying Enterococcus.

Tracing the Enterococci from Paleozoic Origins to the Hospital.

We examined the evolutionary history of leading multidrug resistant hospital pathogens, the enterococci, to their origin hundreds of millions of years ago. Our goal was to understand why, among the vast diversity of gut flora, enterococci are so well adapted to the modern hospital environment. Molecular clock estimation, together with analysis of their environmental distribution, phenotypic diversity, and concordance with host fossil records, place the origins of the enterococci around the time of animal terrestrialization, 425-500 mya. Speciation appears to parallel the diversification of hosts, including the rapid emergence of new enterococcal species following the End Permian Extinction. Major drivers of speciation include changing carbohydrate availability in the host gut. Life on land would have selected for the precise traits that now allow pathogenic enterococci to survive desiccation, starvation, and disinfection in the modern hospital, foreordaining their emergence as leading hospital pathogens.

Global diversity of enterococci and description of 18 previously unknown species.

Enterococci are gut microbes of most land animals. Likely appearing first in the guts of arthropods as they moved onto land, they diversified over hundreds of millions of years adapting to evolving hosts and host diets. Over 60 enterococcal species are now known. Two species, Enterococcus faecalis and Enterococcus faecium, are common constituents of the human microbiome. They are also now leading causes of multidrug-resistant hospital-associated infection. The basis for host association of enterococcal species is unknown. To begin identifying traits that drive host association, we collected 886 enterococcal strains from widely diverse hosts, ecologies, and geographies. This identified 18 previously undescribed species expanding genus diversity by >25%. These species harbor diverse genes including toxins and systems for detoxification and resource acquisition. Enterococcus faecalis and E. faecium were isolated from diverse hosts highlighting their generalist properties. Most other species showed a more restricted distribution indicative of specialized host association. The expanded species diversity permitted the Enterococcus genus phylogeny to be viewed with unprecedented resolution, allowing features to be identified that distinguish its four deeply rooted clades, and the entry of genes associated with range expansion such as B-vitamin biosynthesis and flagellar motility to be mapped to the phylogeny. This work provides an unprecedentedly broad and deep view of the genus Enterococcus, including insights into its evolution, potential new threats to human health, and where substantial additional enterococcal diversity is likely to be found.

Detection of cefiderocol and aztreonam/avibactam resistance in epidemic Escherichia coli ST-361 carrying blaNDM-5 and blaKPC-3 from foreign fighters evacuated from Ukraine.

Genomic surveillance detected clonal Escherichia coli sequence type-361 isolates carrying blaNDM-5, blaKPC-3, blaCTX-M-15, and rmtB1 from a patient in Ukraine and four wounded foreign soldiers evacuated to Germany. Isolates were non-susceptible to carbapenems, aminoglycosides, and cefiderocol and aztreonam/avibactam due to a PBP3 YRIN insertion and the blaCMY-145 AmpC ß-lactamase. Coordinated surveillance efforts across civilian, military, and veteran healthcare systems are essential to prevent further spread as international volunteers return home after medical evacuation from Ukraine.

Pseudomonas aeruginosa ventricular assist device infections: findings from ineffective phage therapies in five cases.

Left ventricular assist devices (LVAD) are increasingly used for management of heart failure; infection remains a frequent complication. Phage therapy has been successful in a variety of antibiotic refractory infections and is of interest in treating LVAD infections. We performed a retrospective review of four patients that underwent five separate courses of intravenous (IV) phage therapy with concomitant antibiotic for treatment of endovascular Pseudomonas aeruginosa LVAD infection. We assessed phage susceptibility, bacterial strain sequencing, serum neutralization, biofilm activity, and shelf-life of phage preparations. Five treatments of one to four wild-type virulent phage(s) were administered for 14-51 days after informed consent and regulatory approval. There was no successful outcome. Breakthrough bacteremia occurred in four of five treatments. Two patients died from the underlying infection. We noted a variable decline in phage susceptibility following three of five treatments, four of four tested developed serum neutralization, and prophage presence was confirmed in isolates of two tested patients. Two phage preparations showed an initial titer drop. Phage biofilm activity was confirmed in two. Phage susceptibility alone was not predictive of clinical efficacy in P. aeruginosa endovascular LVAD infection. IV phage was associated with serum neutralization in most cases though lack of clinical effect may be multifactorial including presence of multiple bacterial isolates with varying phage susceptibility, presence of prophages, decline in phage titers, and possible lack of biofilm activity. Breakthrough bacteremia occurred frequently (while the organism remained susceptible to administered phage) and is an important safety consideration.

Distribution and expression of the aac(6')-Im (aacA16) aminoglycoside resistance gene.

BACKGROUND: The aac(6')-Im (aacA16) amikacin, netilmicin and tobramycin resistance gene cassette had been circulating globally undetected for many years in a sublineage of Acinetobacter baumannii global clone 2. OBJECTIVES: To identify sources for the aac(6')-Im fragment found in A. baumannii. METHODS: MinION long-read sequencing and Unicycler hybrid assemblies were used to determine the genetic context of the aac(6')-Im gene. Quantitative reverse transcriptase PCR was used to measure expression. RESULTS: Among >60 000 non-Acinetobacter draft genomes in the MRSN collection, the aac(6')-Im gene was detected in Pseudomonas putida and Enterobacter hormaechei isolates recovered from patients in Thailand between 2016 and 2019. Genomes of multiply resistant P. putida MRSN365855 and E. hormaechei MRSN791417 were completed. The class 1 integron containing the aac(6')-Im cassette was in the chromosome in MRSN365855, and in an HI2 plasmid in MRSN791417. However, MRSN791417 was amikacin susceptible and the gene was not expressed due to loss of the Pc promoter of the integron. Further examples of aac(6')-Im in plasmids from or the chromosome of various Gram-negative species were found in the GenBank nucleotide database. The aac(6')-Im context in integrons in pMRSN791417-8 and a Klebsiella plasmid pAMR200031 shared similarities with the aac(6')-Im region of AbGRI2-Im islands in A. baumannii. In other cases, the cassette array including the aac(6')-Im cassette was different. CONCLUSIONS: The aac(6')-Im gene is widespread, being found so far in several different species and in several different gene cassette arrays. The lack of amikacin resistance in E. hormaechei highlights the importance of correlating resistance gene content and antibiotic resistance phenotype.

Anatomy of an extensively drug-resistant Klebsiella pneumoniae outbreak in Tuscany, Italy.

A protracted outbreak of New Delhi metallo-ß-lactamase (NDM)-producing carbapenem-resistant Klebsiella pneumoniae started in Tuscany, Italy, in November 2018 and continued in 2020 and through 2021. To understand the regional emergence and transmission dynamics over time, we collected and sequenced the genomes of 117 extensively drug-resistant, NDM-producing K. pneumoniae isolates cultured over a 20-mo period from 76 patients at several healthcare facilities in southeast Tuscany. All isolates belonged to high-risk clone ST-147 and were typically nonsusceptible to all first-line antibiotics. Albeit sporadic, resistances to colistin, tigecycline, and fosfomycin were also observed as a result of repeated, independent mutations. Genomic analysis revealed that ST-147 isolates circulating in Tuscany were monophyletic and highly genetically related (including a network of 42 patients from the same hospital and sharing nearly identical isolates), and shared a recent ancestor with clinical isolates from the Middle East. While the blaNDM-1 gene was carried by an IncFIB-type plasmid, our investigations revealed that the ST-147 lineage from Italy also acquired a hybrid IncFIB/IncHIB-type plasmid carrying the 16S methyltransferase armA gene as well as key virulence biomarkers often found in hypervirulent isolates. This plasmid shared extensive homologies with mosaic plasmids circulating globally including from ST-11 and ST-307 convergent lineages. Phenotypically, the carriage of this hybrid plasmid resulted in increased siderophore production but did not confer virulence to the level of an archetypical, hypervirulent K. pneumoniae in a subcutaneous model of infection with immunocompetent CD1 mice. Our findings highlight the importance of performing genomic surveillance to identify emerging threats.

Detection of carbapenemase producing Acinetobacter baumannii ST19 from Georgia and Ukraine carrying bla OXA-23, bla OXA-72, and/or bla NDM-5, December 2019 to June 2023.

In 2003-2023, amid 5,436 Acinetobacter baumannii isolates collected globally through the Multidrug-Resistant Organism Repository and Surveillance Network, 97 were ST19PAS, 34 of which carbapenem-resistant. Strains (n = 32) sampled after 2019 harboured either bla OXA-23, bla OXA-72, and/or bla NDM-5. Phylogenetic analysis of the 97 isolates and 11 publicly available ST19 genomes revealed three sub-lineages of carbapenemase-producing isolates from mainly Ukraine and Georgia, including an epidemic clone carrying all three carbapenemase genes. Infection control and global surveillance of carbapenem-resistant A. baumannii remain important.

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).

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.

Complete genome of the extensively antibiotic-resistant GC1 Acinetobacter baumannii isolate MRSN 56 reveals a novel route to fluoroquinolone resistance.

OBJECTIVES: To examine the causes of antibiotic resistance in the extensively resistant global clone 1 (GC1) Acinetobacter baumannii isolate MRSN 56 recovered at a US military treatment facility. METHODS: MRSN 56 was sequenced using MinION (Oxford Nanopore) and the reads combined with available Illumina MiSeq data using Unicycler. Acquired resistance genes were identified using ABRicate and their environment examined. ISAba1 and ISAba125 copies were located. RESULTS: MRSN 56 is ST1IP:ST231Ox:KL1:OCL1 and the complete genome includes four small plasmids, none of which carry resistance genes. The acquired resistance genes were found at four locations in the chromosome in addition to AbaR28 (aphA1, aacC1, aadA1, sul1) in comM. Tn2006 (oxa23, carbapenem resistance) was both in AbaR4 and alone elsewhere. Two copies of Tn7 (dfrA1, sat, aadA1) were identified. One was associated with a 22 852 bp adjacent segment [tetA(B), sul2] derived from the AbGRI1 island, and this novel configuration was designated Tn7+. Tn7+ was incorporated in the position preferred by Tn7, downstream of glmS, by transposition using a sequence in AbGRI1 resembling the Tn7 terminal inverted repeats. Tn7 was found at a secondary site. Fluoroquinolone resistance appears to involve a mutation in gyrA combined with inactivation by ISAba1 of the marR gene in the mar operon and constitutive expression of marA from the promoter internal to ISAba1. CONCLUSIONS: MRSN 56 represents a new sublineage of GC1 lineage 1 with novel features that had not been detected previously. The involvement of the mar operon in fluoroquinolone resistance has not been noted previously.

Characterization of KPC-82, a KPC-2 Variant Conferring Resistance to Ceftazidime-Avibactam in a Carbapenem-Nonsusceptible Clinical Isolate of Citrobacter koseri.

KPC-82 is a KPC-2 variant identified in a carbapenem-nonsusceptible Citrobacter koseri that confers high-level resistance to ceftazidime-avibactam. Genomic analysis revealed that blaKPC-82 is carried by a chromosomally integrated Tn4401 transposon (disrupting porin gene phoE) and evolved by a 6-nucleotide tandem repeat duplication causing a two-amino-acid insertion (Ser-Asp) within the Ala267-Ser275 loop. Similar to related KPC variants, KPC-82 showed decreased carbapenemase activity when expressed in a heterologous background and remained susceptible to carbapenem/ß-lactamase inhibitor combinations.

Transferable Resistance Gene optrA in Enterococcus faecalis from Swine in Brazil.

OptrA is an ATP-binding cassette (ABC)-F protein that confers resistance to oxazolidinones and phenicols and can be either plasmid-encoded or chromosomally encoded. Here, we isolated 13 Enterococcus faecalis strains possessing a linezolid MIC of ≥4 mg/liter from nursery pigs in swine herds located across Brazil. Genome sequence comparison showed that these strains possess optrA in different genetic contexts occurring in 5 different E. faecalis sequence type backgrounds. The optrA gene invariably occurred in association with an araC regulator and a gene encoding a hypothetical protein. In some contexts, this genetic island was able to excise and form a covalently closed circle within the cell; this circle appeared to occur in high abundance and to be transmissible by coresident plasmids.

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.

A mutation in the glycosyltransferase gene lafB causes daptomycin hypersusceptibility in Enterococcus faecium.

OBJECTIVES: To verify dissemination of daptomycin-non-susceptible Enterococcus faecium in a hospital where daptomycin was not in use and to understand the evolutionary pathways connecting daptomycin hypersusceptibility to non-susceptibility. METHODS: Clonality of 26 E. faecium was assessed by PFGE and the STs of these isolates were determined. The most daptomycin-susceptible isolate was evolved in vitro by stepwise daptomycin selection, generating isolates for genome comparisons. RESULTS: The spread of a high-risk daptomycin-non-susceptible VRE clone was detected, as was the occurrence of an unusual daptomycin-hypersusceptible strain (HBSJRP18). To determine the basis for daptomycin hypersusceptibility, we evolved HBSJRP18 in vitro and identified candidate genetic alterations potentially related to daptomycin susceptibility. Both lafB, encoding glycosyltransferase, which is putatively involved in lipoteichoic acid (LTA) biosynthesis, and dak, encoding a dihydroxyacetone kinase likely involved in fatty acid metabolism, were mutated in multiple independent experiments. Trans-complementation showed that the lafB polymorphism naturally occurring in HBSJRP18 caused its daptomycin hypersusceptibility. Fourier-transform infrared spectroscopy identified differences between the extracted LTA spectra from the hypersusceptible isolate and its revertant, as well as other non-susceptible variants, supporting a role for LafB in E. faecium LTA biosynthesis. Zeta potential difference was detected in one evolved dak mutant derivative. While much more susceptible to daptomycin, HBSJRP18 showed enhanced growth in the presence of piperacillin, suggesting that this, or another cell wall-targeting antibiotic, may have selected for the daptomycin-hypersusceptible phenotype. CONCLUSIONS: Our findings provide new information on the basis for daptomycin susceptibility in E. faecium, with implications for limiting the development and spread of daptomycin resistance.

Enterococci from Wild Magellanic Penguins (Spheniscus magellanicus) as an Indicator of Marine Ecosystem Health and Human Impact.

Enterococci are commensals that proliferated as animals crawled ashore hundreds of millions of years ago. They are also leading causes of multidrug-resistant hospital-acquired infections. While most studies are driven by clinical interest, comparatively little is known about enterococci in the wild or the effect of human activity on them. Pharmaceutical pollution and runoff from other human activities are encroaching widely into natural habitats. To assess their reach into remote habitats, we investigated the identity, genetic relatedness, and presence of specific traits among 172 enterococcal isolates from wild Magellanic penguins. Four enterococcal species, 18 lineage groups, and different colonization patterns were identified. One Enterococcus faecalis lineage, sequence type 475 (ST475), was isolated from three different penguins, making it of special interest. Its genome was compared to those of other E. faecalis sequence types (ST116 and ST242) recovered from Magellanic penguins, as well as to an existing phylogeny of E. faecalis isolated from diverse origins over the past 100 years. No penguin-derived E. faecalis strains were closely related to dominant clinical lineages. Most possessed intact CRISPR defenses, few mobile elements, and antibiotic resistances limited to those intrinsic to the species and lacked pathogenic features conveyed by mobile elements. Interestingly, plasmids were identified in penguin isolates that also had been reported for other marine mammals. Enterococci isolated from penguins showed limited anthropogenic impact, indicating that they are likely representative of those naturally circulating in the ecosystem inhabited by the penguins. These findings establish an important baseline for detecting the encroachment of human activity into remote planetary environments.IMPORTANCE Enterococci are host-associated microbes that have an unusually broad range, from the built hospital environment to the guts of insects and other animals in remote locations. Despite their occurrence in the guts of animals for hundreds of millions of years, we know little about the properties that confer this range or how anthropogenic activities may be introducing new selective forces. Magellanic penguins live at the periphery of human habitation. It was of interest to examine enterococci from these animals for the presence of antibiotic resistance and other markers reflective of anthropogenic selection. Diverse enterococcal lineages found discount the existence of a single well-adapted intrinsic penguin-specific species. Instead, they appear to be influenced by a carnivorous lifestyle and enterococci present in the coastal sea life consumed. These results indicate that currently, the penguin habitat remains relatively free of pollutants that select for adaptation to human-derived stressors.

Daptomycin Resistance and Tolerance Due to Loss of Function in Staphylococcus aureus dsp1 and asp23.

Lipopeptide daptomycin is a last-line cell-membrane-targeting antibiotic to treat multidrug-resistant Staphylococcus aureus Alarmingly, daptomycin-resistant S. aureus isolates have emerged. The mechanisms underlying daptomycin resistance are diverse and share similarities with resistances to cationic antimicrobial peptides and other lipopeptides, but they remain to be fully elucidated. We selected mutants with increased resistance to daptomycin from a library of transposon insertions in sequent type 8 (ST8) S. aureus HG003. Insertions conferring increased daptomycin resistance were localized to two genes, one coding for a hypothetical lipoprotein (SAOUHSC_00362, Dsp1), and the other for an alkaline shock protein (SAOUHSC_02441, Asp23). Markerless loss-of-function mutants were then generated for comparison. All transposon mutants and knockout strains exhibited increased daptomycin resistance compared to those of wild-type and complemented strains. Null and transposon insertion mutants also exhibited increased resistance to cationic antimicrobial peptides. Interestingly, the Δdsp1 mutant also showed increased resistance to vancomycin, a cell-wall-targeting drug with a different mode of action. Null mutations in both dsp1 and asp23 resulted in increased tolerance as reflected by reduced killing to both daptomycin and vancomycin, as well as an increased tolerance to surfactant (Triton X-100). Neither mutant exhibited increased resistance to lysostaphin, a cell-wall-targeting endopeptidase. These findings identified two genes core to the S. aureus species that make previously uncharacterized contributions to antimicrobial resistance and tolerance in S. aureus.

Transferable vancomycin resistance in clade B commensal-type Enterococcus faecium.

Objectives: Vancomycin-resistant Enterococcus faecium is a leading cause of MDR hospital infection. Two genetically definable populations of E. faecium have been identified: hospital-adapted MDR isolates (clade A) and vancomycin-susceptible commensal strains (clade B). VanN-type vancomycin resistance was identified in two isolates of E. faecium recovered from blood and faeces of an immunocompromised patient. To understand the genomic context in which VanN occurred in the hospitalized patient, the risk it posed for transmission in the hospital and its origins, it was of interest to determine where these strains placed within the E. faecium population structure. Methods: We obtained the genome sequence of the VanN isolates and performed comparative and functional genomics of the chromosome and plasmid content. Results: We show that, in these strains, VanN occurs in a genetic background that clusters with clade B E. faecium, which is highly unusual. We characterized the chromosome and the conjugative plasmid that carries VanN resistance in these strains, pUV24. This plasmid exhibits signatures of in-host selection on the vanN operon regulatory system, which are associated with a constitutive expression of vancomycin resistance. VanN resistance in clade B strains may go undetected by current methods. Conclusions: We report a case of vancomycin resistance in a commensal lineage of E. faecium responsible for an atypical bacteraemia in an immunocompromised patient. A reservoir of transferable glycopeptide resistance in the community could pose a concern for public health.

Pheromone killing of multidrug-resistant Enterococcus faecalis V583 by native commensal strains.

Multidrug-resistant Enterococcus faecalis possess numerous mobile elements that encode virulence and antibiotic resistance traits as well as new metabolic pathways, often constituting over one-quarter of the genome. It was of interest to determine how this large accretion of mobile elements affects competitive growth in the gastrointestinal (GI) tract consortium. We unexpectedly observed that the prototype clinical isolate strain V583 was actively killed by GI tract flora, whereas commensal enterococci flourished. It was found that killing of V583 resulted from lethal cross-talk between accumulated mobile elements and that this cross-talk was induced by a heptapeptide pheromone produced by native E. faecalis present in the fecal consortium. These results highlight two important aspects of the evolution of multidrug-resistant enterococci: (i) the accretion of mobile elements in E. faecalis V583 renders it incompatible with commensal strains, and (ii) because of this incompatibility, multidrug-resistant strains sharing features found in V583 cannot coexist with commensal strains. The accumulation of mobile elements in hospital isolates of enterococci can include those that are inherently incompatible with native flora, highlighting the importance of maintaining commensal populations as means of preventing colonization and subsequent infection by multidrug-resistant strains.

Identification of a Functionally Unique Family of Penicillin-Binding Proteins.

Penicillin-binding proteins (PBPs) are enzymes involved in the assembly of the bacterial cell wall, a major target for antibiotics. These proteins are classified by mass into high-molecular-weight PBPs, which are transpeptidases that form peptidoglycan cross-links, and low-molecular-weight PBPs, which are typically hydrolases. We report a functionally unique family of low-molecular-weight PBPs that act as transpeptidases rather than hydrolases, but they do not cross-link peptidoglycan. We show that these PBPs can exchange d-amino acids bearing chemical tags or affinity handles into peptidoglycan precursors, including Lipid II, enabling biochemical studies of proteins involved in cell wall assembly. We report that, in two organisms, the PBPs incorporate lysine into cellular peptidoglycan and that, further, the PBPs have the unprecedented ability to transfer the primary ε-amine of lysine to peptidoglycan.