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Planet, P. J.

Publications and source records attributed to Planet, P. J..

At least 19 recordsLinked to original sources

Multi-lineage emergence of USA300 MRSA in in Chile and a potential role for environmental copper resistance

Methicillin-resistant S. aureus (MRSA) is prevalent worldwide, but individual strains of MRSA tend to spread and expand in relatively confined geographic areas. The USA300 lineage of MRSA is most well-known for its rapid epidemic spread in North America during the first decade of the 2000's leading to an overall increase in serious skin infections and the emergence of methicillin resistance in the majority of community acquired S. aureus infections. A distinct but closely related South American epidemic clade of MRSA USA300 (USA300-SAE), first reported in 2006, proliferated rapidly in Northern South America but was rarely found in other Latin American countries. In a new survey of 274 invasive MRSA isolates from 2022 collected in Chile, we saw a marked increase in USA300-SAE in Southern South America. We found that Chilean USA300-SAE is phylogenetically diverse and was likely introduced multiple times from multiple different South American countries over the course of more than a decade, but only recently became a highly prevalent clone. Using novel molecular diagnostic methods for isolates collected in 2023, we estimate that USA300-SAE made up more than 22% of invasive MRSA strains in Chile in that year and is likely still increasing. We show that copper-associated genes in the signature copper and mercury resistance mobile element (COMER) of USA300-SAE are strongly conserved, and that isogenic mutants of the copper resistance genes in COMER are highly susceptible to copper killing at concentrations seen in tap water. These findings suggest that environmental exposure to copper may provide a fitness advantage that enhances spread of this lineage.

microbiology↗

Prevalence, Production and the Role of Staphylococcus aureus Superantigens in Cystic Fibrosis Lung Disease.

Staphylococcus aureus (SA), the most common cystic fibrosis (CF) lung pathogen, is uniquely capable of producing superantigen (SAg) exotoxins, which are the most potent activators of the immune system. Although the proinflammatory roles of SA-SAgs is well-established, their role in the immunopathogenesis of CF lung disease is unexplored. Herein, we demonstrate that 60-80% of pediatric and adult CF SA isolates carried at least one SA-SAg gene, with the former harboring potent SA-SAgs (Staphylococcal enterotoxin A and B) more frequently (30-60%). Biofilms of clinical SA isolates readily produced biologically active SA-SAgs in artificial sputum medium and purified SA-SAgs retained their bioactivity in human CF sputum in vitro. Repeated intratracheal challenge with purified SA-SAgs induced a robust pulmonary inflammatory response in CF mouse models ({beta}ENAC and CFGC transgenic mice) expressing HLA-DR3 in a dose-dependent manner, with the low dose favoring a type 2 eosinophilic lung inflammatory response, and a high dose eliciting a type 1 inflammatory response with neutrophilic lung inflammation and higher mortality. In vivo neutralization of IFN-{gamma} also promoted SA-SAg-driven type 2 inflammation. Intratracheal infection with sub-lethal dose of a clinical SA isolate producing SEB, but not the SEB-deficient mutant isogenic SA strain, also elicited an eosinophilic inflammatory response.

immunology↗

Hidden Diversity in Enterococcus faecalis Revealed by CRISPR2 Screening: Eco-evolutionary Insights into a Novel Subspecies

Enterococcus faecalis is a commensal bacterium that colonizes the gut of humans and animals, and a major opportunistic pathogen, known for causing multidrug-resistant healthcare-associated infections (HAIs). Its ability to thrive in diverse environments and disseminate antimicrobial resistance genes (ARGs) across ecological niches highlights the importance of understanding its ecological, evolutionary, and epidemiological dynamics. The CRISPR2 locus has been used as a valuable marker for assessing clonality and phylogenetic relationships in E. faecalis. In this study, we identified a group of E. faecalis strains lacking CRISPR2, forming a distinct, well-supported clade. We demonstrate that this clade meets the genomic criteria for classification as a novel subspecies, here referred to as "subspecies B". Through a comprehensive pangenome analysis and comparative genomics, we explored the adaptive ecological traits underlying this diversification process, identifying clade-specific features and their predicted functional roles. Our findings suggest that the frequent isolation of subspecies B from meat products and processing facilities may reflect dissemination routes involving environmental contamination (e.g., water, plants, soil) from avian species. The absence of key virulence traits required for pathogenicity in mammals, particularly in humans, and the lack of clinically relevant resistance determinants indicate that subspecies B may currently pose minimal threat to public health compared to the broadly disseminated "subspecies A". Nevertheless, the unclear potential for genetic exchange between these subspecies, and the frequent association of subspecies B with food sources, calls for continued genomic surveillance of E. faecalis from a One Health perspective to detect and mitigate the emergence of high-risk variants in advance.

microbiology↗

Regulation of airway fumarate by host and pathogen promotes S. aureus pneumonia

Staphylococcus aureus is a leading cause of healthcare-associated pneumonia, contributing significantly to morbidity and mortality worldwide. As a ubiquitous colonizer of the upper respiratory tract, S. aureus must undergo substantial metabolic adaptation to achieve persistent infection in the distinctive microenvironment of the lung. We observed that fumC, which encodes the enzyme that converts fumarate to malate, is highly conserved with low mutation rates in S. aureus isolates from chronic lung infections. Fumarate, a pro-inflammatory metabolite produced by macrophages during infection, is regulated by the host fumarate hydratase (FH) to limit inflammation. Here, we demonstrate that fumarate, which accumulates in the chronically infected lung, is detrimental to S. aureus, blocking primary metabolic pathways such as glycolysis and oxidative phosphorylation (OXPHOS). This creates a metabolic bottleneck that drives staphylococcal FH (FumC) activity for airway adaptation. FumC not only degrades fumarate but also directs its utilization into critical pathways including the tricarboxylic acid (TCA) cycle, gluconeogenesis and hexosamine synthesis to maintain metabolic fitness and form a protective biofilm. Itaconate, another abundant immunometabolite in the infected airway enhances FumC activity, in synergy with fumarate. In a mouse model of pneumonia, a {Delta}fumC mutant displays significant attenuation compared to its parent and complemented strains, particularly in fumarate- and itaconate-replete conditions. Our findings underscore the pivotal role of immunometabolites in promoting S. aureus pulmonary adaptation.

microbiology↗

Carbohydrate consumption drives adaptive mutations in Escherichia coli associated with increased risk for systemic infection

Dissemination of organisms from the gut microbiota is a major contributor to sepsis and critical illness. Patients with cirrhosis are prone to systemic infections and are commonly prescribed the carbohydrate lactulose to manage hepatic encephalopathy (HE) 1. Commensal metabolism of lactulose is believed to reduce pathobiont colonization through short-chain fatty acid production, but its direct effects on gut pathobionts remain unexplored 2. Here, we show that lactulose consumption unexpectedly selects for mutations in Escherichia coli lactose (lac) operon regulation, enhancing its metabolic fitness and colonization capacity. This is mediated by selection for constitutive expression of the lac operon through mutations in its regulatory components. Using in vitro systems, murine models, and clinical samples, we demonstrate that these mutations enable E. coli to exploit lactulose as a carbon source, bypassing host carbohydrate metabolism and increasing its intestinal colonization. Despite its long-standing use in HE treatment, we find that lactulose has a paradoxical association with risk of infection hospitalization in patients with cirrhosis in a large epidemiologic study. The emergence of lactulose-adapted E. coli strains could be suppressed by a dietary oligosaccharide that competitively inhibits lactulose uptake. These findings reveal a mechanism by which dietary substrates exert selective pressure on the microbiome, with implications for diet-based strategies to modulate microbial evolution and infection risk.

microbiology↗

Microbial succession at weaning is guided by microbial metabolism of host glycans.

The weaning transition from a milk-based to a solid-food diet supports critical developmental changes to the intestinal microbiome and immune system. However, the specific microbial and host features that influence microbial succession at weaning are not well understood. Here, we developed a simple approach to investigate the complex dynamics of microbial succession during weaning by co-housing gnotobiotic mice colonized with the defined pre-weaning community PedsCom and the adult-derived consortium OMM12. As expected, co-housing PedsCom mice with OMM12 recapitulated microbial succession at weaning and induced immune system maturation in PedsCom mice. Unexpectedly, we found that the OMM12 microbes with the highest host glycan utilization profiles were the most adept colonizers of PedsCom mice. Genomic analysis confirmed that PedsCom is deficient in the carbohydrate-active enzymes responsible for degrading host-derived glycans, including mucins, compared to adult-derived consortia. We validated a role for glycan utilization in vivo by demonstrating that the mucus-degrading commensal microbe Akkermansia muciniphila critically depends on the metabolism of mucin glycans for colonization of PedsCom mice. These findings highlight the importance of host-derived glycans in shaping microbial communities during the weaning transition and suggest host glycans as novel targets to modulate intestinal microbial populations, introduce beneficial probiotics, and enhance immune system development during weaning.

microbiology↗

Comparison of gnotobiotic communities reveals unexpected amino acid metabolism by the pre-weaning microbiome.

The intestinal microbiome during infancy and childhood has distinct compositions and metabolic functions to that of adults. We recently published a gnotobiotic mouse model of the pre-weaning microbiome (PedsCom), which retains a pre-weaning configuration during the transition from a milk-based diet to solid foods and leads to a stunted immune system and susceptibility to enteric infection. Here we compared the phylogenetic and metabolic relationship of the PedsCom consortium to the adult-derived gnotobiotic communities, Altered Schaedler Flora and Oligo-MM12. We find that PedsCom contains several unique functions relative to adult-derived mouse consortia. In particular, amino acid degradation metabolic modules are more prevalent among PedsCom isolates, which is in line with the ready availability of these nutrients in milk. Indeed, metabolomic analysis showed significantly lower levels of free amino acids in the intestinal contents of adult PedsCom colonized mice versus Oligo-MM12 controls. Thus, enhanced amino acid metabolism is a prominent feature of the pre-weaning microbiome that may facilitate design of early life microbiome interventions.

microbiology↗

Early-life exposures to specific commensal microbes prevent type 1 diabetes.

Early-life disruptions of the gut microbiome have long-lasting impacts on the risk of developing autoimmune diseases. How the composition of the early-life microbiota contributes to autoimmunity and whether manipulating it can prove therapeutically beneficial remains largely unexplored. Here we demonstrate that a simple consortium of nine early-life commensal bacteria (PedsCom) prevents type 1 diabetes (T1D) in diabetes-susceptible NOD mice. Remarkably, we find that this protection is completely dependent upon early-life colonization. During this critical time window of early-life colonization and immune development, specific microbes unexpectedly translocate from the gut to peripheral tissues and induce the tolerogenic responses required for T1D protection. These findings highlight how the timing and localization of microbial interactions during a pivotal stage of immune development contribute to protection from T1D. Altogether, these findings suggest an opportunity to develop microbial therapies for human infants to prevent autoimmune diseases. One sentence summaryA defined consortium of early-life microbes shapes immune development and prevents type 1 diabetes.

immunology↗

Heavy Metal Pollution From a Major Earthquake and Tsunami in Chile Is Associated With Geographic Divergence of Clinical Isolates of Methicillin-Resistant Staphylococcus aureus in Latin America

Methicillin-resistant Staphylococcus aureus (MRSA) is a priority pathogen listed by the World Health Organization. The global spread of MRSA is characterized by successive waves of epidemic clones that predominate in specific geographical regions. The acquisition of genes encoding resistance to heavy-metals is thought to be a key feature in the divergence and geographical spread of MRSA. Increasing evidence suggests that extreme natural events, such as earthquakes and tsunamis, could release heavy-metals into the environment. However, the impact of environmental exposition to heavy-metals on the divergence and spread of MRSA clones has been insufficiently explored. We assess the association between a major earthquake and tsunami in an industrialized port in southern Chile and MRSA clone divergence in Latin America. We performed a phylogenomic reconstruction of 113 MRSA clinical isolates from seven Latin American healthcare centers, including 25 isolates collected in a geographic area affected by an earthquake and tsunami that led to high levels of heavy-metal environmental contamination. We found a divergence event strongly associated with the presence of a plasmid harboring heavy-metal resistance genes in the isolates obtained in the area where the earthquake and tsunami occurred. Moreover, clinical isolates carrying this plasmid showed increased tolerance to mercury, arsenic, and cadmium. We also observed a physiological burden in the plasmid-carrying isolates in absence of heavy-metals. Our results are the first evidence that suggests that heavy-metal contamination, in the aftermath of an environmental disaster, appears to be a key evolutionary event for the spread and dissemination of MRSA in Latin America.

microbiology↗

Parallel Evolution of Linezolid Resistant Staphylococcus aureus in Patients with Cystic Fibrosis

BackgroundLinezolid is an antibiotic used to treat serious Staphylococcus aureus infections. Resistance to linezolid is considered rare but could emerge with repeated dosing. We recently reported widespread prescription of linezolid for a cohort of patients with cystic fibrosis (CF). ObjectivesThe goals of this study were to determine the incidence of linezolid resistance in CF and identify molecular mechanisms for linezolid resistance. MethodsWe identified patients with S. aureus resistant to linezolid (MIC > 4) at the University of Iowa CF Center between 2008 and 2018. We obtained isolates from these patients and retested susceptibility to linezolid using broth microdilution. We used whole genome sequencing to perform phylogenetic analysis of linezolid resistant isolates and examine sequences for mutations or accessory genes that confer linezolid resistance. Main ResultsBetween 2008 and 2018, 111 patients received linezolid and 4 of these patients cultured linezolid resistant S. aureus. We sequenced 11 resistant and 21 susceptible isolates from these 4 subjects. Phylogenetic analysis indicated that linezolid resistance developed in ST5 or ST105 backgrounds. Three individuals had linezolid resistant S. aureus with a G2576T mutation in 23S rRNA. One of these subjects additionally had a mutS- mutL- hypermutating S. aureus that produced 5 resistant isolates with multiple ribosomal subunit mutations. In one subject, the genetic basis for linezolid resistance was unclear. ConclusionsLinezolid resistance evolved in 4 of 111 patients in this study. Linezolid resistance occurred by multiple genetic mechanisms. All resistant strains developed in ST5 or ST105 MRSA backgrounds. Key PointLinezolid resistance arises through multiple genetic mechanisms and could be facilitated by mutator phenotypes. Linezolid resistance was transient, possibly due to growth disadvantage.

microbiology↗

Dynamics of the MRSA Population in A Chilean Hospital: A Phylogenomic Analysis (2000-2016)

The global dissemination of methicillin-resistant Staphylococcus aureus (MRSA) is associated with the emergence and establishment of clones in specific geographic areas. The Chilean-Cordobes clone (ChC) (ST5-SCCmecI) has been the predominant MRSA clone in Chile since its first description in 1998, despite the report of other emerging MRSA clones in the last years. Here, we characterize the evolutionary history of MRSA from 2000 to 2016 in a Chilean tertiary healthcare center using phylogenomic analyses. We sequenced 469 MRSA isolates collected between 2000-2016 in a tertiary healthcare center in Chile. We evaluated the temporal trends of the circulating clones and performed a phylogenomic reconstruction to characterize the clonal dynamics. We found a significant increase in the diversity and richness of sequence types (STs; Spearman r=0.8748, p<0.0001) with a Shannon diversity index increasing from 0.221 in the year 2000 to 1.33 in 2016. The temporal trend analysis revealed that in the period 2000-2003 most of the isolates (94.2%; n=98) belonged to the ChC clone. However, since then, the frequency of the ChC clone has decreased over time, accounting for 52% of the collection in the 2013-2016 period. This decline was accompanied by the rise of two emerging MRSA lineages, ST105-SCCmecII and ST72-SCCmecVI. In conclusion, the ChC clone remains the most frequent MRSA lineage in Chile. However, this lineage is gradually being replaced by several emerging clones, the most important of which is clone ST105-SCCmecII. To the best of our knowledge, this is the largest study of MRSA clonal dynamics performed in South America. ImportanceMethicillin-resistant Staphylococcus aureus (MRSA) is a major public health pathogen that disseminates through the emergence of successful dominant clones in specific geographic regions. Knowledge of the dissemination and molecular epidemiology of MRSA in Latin America is scarce and is largely based on small studies or classical typing techniques with several limitations to depict an accurate description of their genomic landscape. We used whole-genome sequencing to study 469 MRSA isolates collected between 2000-2016 in Chile to provide the largest and most detailed study of clonal dynamics of MRSA carried out in South America to date. We found a significant increase in the diversity of MRSA clones circulating over the 17-year study period. Additionally, we describe the emergence of two novel clones (ST105-SCCmecII and ST72-SCCmecVI), which have been gradually increasing their frequency over time. Our results drastically improve our understanding of the dissemination and update our knowledge about MRSA in Latin America.

microbiology↗

Acquisition, co-option, and duplication of the rtx toxin system and the emergence of virulence in Kingella

The Kingella genus includes two pathogenic species, namely K. kingae and K. negevensis, as well as strictly commensal species. Both K. kingae and K. negevensis secrete a toxin called RtxA that is absent in the commensal species. Phylogenetic analysis demonstrates that the toxin-encoding operon rtxCrtxAtolC was acquired by a common ancestor of the pathogenic Kingella species and that a preexisting type I secretion system was co-opted for toxin export. Subsequent genomic reorganization distributed the toxin machinery across two loci, with 30-35% of K. kingae strains containing two copies of the rtxA toxin gene. The rtxA duplication is largely clonal and strongly associated with invasive disease. In assays with isogenic strains, a single copy of rtxA was associated with reduced virulence in vitro. This study establishes the critical steps in the evolutionary transition from commensal to pathogen, including horizontal gene transfer, co-option of an existing secretion system, and gene duplication.

microbiology↗

Phylogenomic analyses of understudied Neisseriaceae species support the reclassification of the polyphyletic genera Kingella, Simonsiella, and Alysiella.

Taxonomic classification and phylogenetic analysis of the Neisseriaceae family has focused on the pathogens Neisseria meningitidis and Neisseria gonorrhoeae. Less is known about the relationships of commensal Neisseria species and other Neisseriaceae genera, raising the possibility that the phylogeny of this family may not agree with taxonomy. In this study we used available nucleotide sequences and a phylogenetic approach to assess the Kingella genus and its relatives. We found that this genus is both paraphyletic and polyphyletic. Kingella potus is more closely related to Neisseria bacilliformis than other Kingella species. The Alysiella and Simonsiella genera form a distinct clade within the Kingella genus that is closely related to the pathogens K. kingae and K. negevensis. We find a phylogenetic relationship between Conchiformibius, Alysiella, Simonsiella, and Kingella, which we name the CASK clade. Finally, we define the gene sets that differentiate each genus of the CASK clade from one another and from the rest of the Neisseriaceae family. ImportanceUnderstanding the evolutionary relationships between the species in the Neisseriaceae has been a persistent challenge in bacterial systematics due to high recombination rates in these species. Previous studies of this family have focused on N. meningitidis and N. gonorrhoeae. However, previously understudied Neisseriaceae species are gaining new attention, with K. kingae now recognized as a common human pathogen and with Alysiella and Simonsiella being unique in the bacterial world as multicellular organisms. A better understanding of the genomic evolution of the Neisseriaceae can lead to identification of the specific genes and traits that characterize the remarkable diversity of this family.

evolutionary biology↗

Modulation of adaptive immune responses by Akkermansia muciniphila is restricted to an early life window in NOD mice

Early life microbiota drive immune system development and influence risk for immune dysfunction later in life, including the development of type 1 diabetes (T1D). Which specific early-life microbes modulate diabetes risk and the timing of these critical interactions are not well understood. To address this gap in knowledge, we screened for microbes that induce systemic IgG1 responses in young NOD mice. We isolated a strain of Akkermansia muciniphila that potently induces systemic IgG1 antibodies and peripheral regulatory T cells (pTregs). Since this mucus-degrading commensal protects NOD mice from T1D and is associated with lower risk of developing T1D in children, we investigated how A. muciniphila impacts early-life host-commensal interactions using gnotobiotic NOD mice colonized with a defined 9-member bacterial consortium that models the early life microbiome. We find that A. muciniphila potently induces pTregs and enhances antibody responses to other commensal microbes. Remarkably, these effects only occur when A. muciniphila colonizes NOD mice prior to weaning, establishing that the specific window of exposure to A. muciniphila shapes adaptive immune system development in diabetes-susceptible NOD mice. This time dependence provides important evidence that early-life exposure may enhance microbiota-based therapies to prevent T1D. One Sentence SummaryAkkermansia muciniphila induces peripheral Tregs and enhances antibody responses to itself and other commensals during an early life window.

immunology↗

Arresting microbiome development limits immune system maturation and resistance to infection.

Disruptions to the intestinal microbiome during weaning lead to long-term negative effects on host immune function. However, the critical host-microbe interactions occurring during weaning required for healthy immune system development remain poorly understood. We find that restricting microbiome maturation during weaning leads to stunted immune system development and increased susceptibility to enteric infection. We developed a gnotobiotic mouse model of the early-life microbiome designated as Pediatric Community (PedsCom). This nine-member consortium of microbes derived from intestinal microbiomes of preweaning mice stably colonized germfree adult mice and was efficiently transmitted to offspring for multiple generations. Unexpectedly, the relative abundance of PedsCom microbes were largely unaffected by the transition from a milk-based to a fiber rich solid food diet. PedsCom mice developed less peripheral regulatory T cells and Immunoglobulin A, hallmarks of microbiota-driven immune system development. Consistent with defects in maturation, adult PedsCom mice retain high susceptibility to salmonella infection characteristic of young mice and humans. Altogether, our work illustrates how the post-weaning transition in intestinal microbiome composition contributes to normal immune maturation and protection from enteric infection. Accurate modelling of the pre-weaning microbiome provides a window into the microbial requirements of healthy immune development and suggests an opportunity to design microbial interventions at weaning to improve immune system development in human infants. One Sentence SummaryArresting microbiome development stunts immune ontogeny

immunology↗

What do we gain when tolerating loss? The information bottleneck, lossy compression, and detecting horizontal gene transfer

Most microbes have the capacity to acquire genetic material from their environment. Recombination of foreign DNA yields genomes that are, at least in part, incongruent with the vertical history of their species. Dominant approaches for detecting these transfers are phylogenetic, requiring a painstaking series of analyses including alignment and phylogenetic tree reconstruction. These traditional pan-genomic methods do not scale. Here we propose an unsupervised, alignment-free and tree-free technique based on the sequential information bottleneck (SIB), an optimization procedure designed to extract some portion of relevant information from one random variable conditioned on another. In our case, this joint probability distribution tabulates occurrence counts of k-mers against their genomes of origin with the expectation that recombination will create a strong signal that unifies certain sets of co-occuring k-mers. We conceptualize the technique as a rate-distortion problem, measuring distortion in the relevance information as k-mers are compressed into clusters based on their co-occurrence in the source genomes. The result is fast, model-free, lossy compression of k-mers into groups that learns tracts of shared genome sequence differentiating recombined elements from the vertically inherited core. We show that the technique yields a new recombination measure based purely on information, divorced from any biases and limitations inherent to alignment and phylogeny. SignificanceThe information bottleneck, a lossy compression technique borrowed from the information theoretic and Natural Langauge Processing literature, is well suited to detecting evolutionary patterns in sets of co-occuring k-mers. Here we show that we can detect simulated and real recombination events while highlighting a core set of k-mers that comprise the vertically inherited portion of any set of genomes. Moreover, the compressibility of any given set of genomes offers a new way to compare the pangenomes of clades across the microbial tree of life. In our application, the bottleneck is informed by genome origin, our relevance variable, but the technique is general. The information bottleneck can be used for any biological contingency matrix where the goal is to learn groups from unstructured data.

evolutionary biology↗

Comparative Analysis of Emerging B.1.1.7+E484K SARS-CoV-2 isolates from Pennsylvania

Rapid whole genome sequencing of SARS-CoV-2 has presented the ability to detect new emerging variants of concern in near real time. Here we report the genome of a virus isolated in Pennsylvania in March 2021 that was identified as lineage B.1.1.7 (VOC-202012/01) that also harbors the E484K spike mutation, which has been shown to promote "escape" from neutralizing antibodies in vitro. We compare this sequence to the only 5 other B.1.1.7+E484K genomes from Pennsylvania, all of which were isolated in mid March. Beginning in February 2021, only a small number (n=60) of isolates with this profile have been detected in the US, and only a total of 253 have been reported globally (first in the UK in December 2020). Comparative genomics of all currently available high coverage B.1.1.7+E484K genomes (n=235) available on GISAID suggested the existence of 7 distinct groups or clonal complexes (CC; as defined by GNUVID) bearing the E484K mutation raising the possibility of 7 independent acquisitions of the E484K spike mutation in each background. Phylogenetic analysis suggested the presence of at least 3 distinct clades of B.1.1.7+E484K circulating in the US, with the Pennsylvanian isolates belonging to two distinct clades. Increased genomic surveillance will be crucial for detection of emerging variants of concern that can escape natural and vaccine induced immunity.

genomics↗

Occurrence and associated characteristics of a mutated ant(6')-Ia gene among Enterococcus faecium strains expressing phenotypic susceptibility to high levels of streptomycin

Enterococcal high-level resistance to streptomycin (HLR-S) (MIC [&ge;] 2000 {micro}g/ml), conferred by production of a variety of aminoglycoside modifying enzymes (AMES), has been reported worldwide and a nucleotidyltransferase (ANT) enzyme encoded by the ant(6)-Ia gene is frequently associated with this phenotype. However, during a study conducted by our group on whole genome sequencing (WGS) analyses of Enterococcus faecium isolates, we observed that 32 E. faecium strains identified as susceptible to high-levels of streptomycin by the disk diffusion method had the of ant(6)-Ia gene annotated in their genomes. Antimicrobial susceptibility to streptomycin was reassessed by phenotypic testing and the presence of the ant(6)-Ia gene was confirmed by PCR in all the isolates. Alignment of the ant(6)-Ia gene with a reference sequence revealed a deletion of the first 48 nucleotides and four nonsynonymous mutations, leading to the substitution of a Glutamine to Methionine and an Aspartic Acid to Asparagine in the amino acid sequence. The protein structure was modelled by using the Phyre2 platform and the results indicated alterations in the N-terminus region leading to changes in the predicted binding site. Also, by searching the NCBI database we identified the genomes of 71 strains carrying the mutated gene. MLST analysis revealed that most strains carrying the mutated gene, including those described in this study belonged to hospital-adapted lineages, suggesting the occurrence of clonal dissemination of a subset of mutated isolates. HIGHLIGHTSO_LIThe presence of a mutated ant(6)-Ia gene was identified among Enterococcus faecium isolates expressing phenotypic susceptibility to high levels of streptomycin. C_LIO_LINonsynonymous mutations and inactivating changes in the ant(6)-Ia gene led to incongruities between phenotypes and genotypes. C_LIO_LIAlterations in the amino acid sequence had impacts on protein structure, with changes in the N-terminus region and the binding site. C_LI

microbiology↗