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Novelty, diversity, and genetic dark matter in enterococci of invertebrates.

Enterococci appear to have originated in the guts of early terrestrializing arthropods and invertebrates over 425 million years ago-hosts that are now highly diverse and widespread in nature today. Yet most knowledge of the genus comes from human infection-associated lineages with genomes swollen by the recent accretion of foreign DNA conveyed by mobile elements. Because invertebrates dominate terrestrial animal diversity and biomass, they would be predicted to constitute a major but little-explored reservoir of enterococcal diversity. We therefore systematically examined Enterococcus association and species diversification in invertebrate hosts of the comparatively natural, isolated, but well-characterized environment of the Azorean island of Terceira. Over 100 invertebrate specimens were examined for associated enterococci, which were taxonomically classified by whole-genome sequencing. Supporting the existence of a large pool of uncharacterized enterococci and Enterococcus-adapted genes, 40% (eight of 20) of the Enterococcus species identified were either undescribed, including four candidate new species described here, or very recently discovered. In contrast, control isolates from vertebrates were exclusively of known species typical of sampling elsewhere, discounting geographic isolation as a main driver of the novelty observed. Further, because of the abundance of E. casseliflavus and E. flavescens in this collection, we obtained the resolution necessary to quantify the divergence and decipher the drivers of speciation in the controversial division between these naturally vancomycin-resistant species. These findings provide robust support for the existence of a large pool of new species and unexplored adaptive traits in invertebrate-associated enterococci-diverse environmental survival traits optimized for expression in an enterococcal background, and well positioned for transmission into human-associated enterococcal strains.IMPORTANCEEnterococci are auxotrophic gut-associated bacteria that co-evolved with their terrestrial hosts over many eons. In the last 75 years-the "antibiotic era"-E. faecalis and E. faecium gained genes for antibiotic resistance and enhanced virulence, emerging as leading causes of multidrug-resistant infection. Little is known about the source of those genes or the pathway by which they entered human-associated strains. A recent global survey suggested a potentially large repository of uncharacterized genetic diversity in the enterococci of invertebrates. We directly tested this prospect by examining enterococci of invertebrate hosts in a largely natural and pastoral environment. Our findings provide clear evidence that invertebrates naturally harbor vast unexplored enterococcal diversity. Moreover, associations are likely driven by intrinsic host selection factors rather than geographic isolation. This expands our knowledge of Enterococcus biodiversity, including the identification of four novel species, identifying a vast reservoir of enterococcal genes available to species that colonize and infect humans.

Animals

[Differential diagnosis of enterococci].

The most stable differential signs of enterococci are: growth in the medium at pH 10.2, growth in broth containing 40% bile, citrate utilization, resistance to 0,05% potassium tellurite, 2, 3, 5-triphenyltetrazolium chloride (TTC) reduction, the staining of colonies (plaques) on a medium with manganese, iron and zinc salts, glycerine fermentation under anaerobic conditions, mannite fermentation, the presence of hemolysin, of the proteolytic enzyme, and mobility. Combined differential-diagnostic nutrient medium permits to determine simultaneously 5 enterococci signs--resistance to nalidixic acid and to crystal violet, TTC reduction, hemolytic and proteolytic activity. The suggested scheme of enterococci laboratory diagnosis including a set of hard nutrient media poured into multisection Petri dish is simple reliable and accessible for any bacteriological laboratory.

Culture Media

Emergence of an optrA-positive Enterococcus faecalis ST699 lineage in animal-derived foods in Beijing, China.

Enterococci from animal-derived foods are key reservoirs for antimicrobial resistance (AMR) in the food chain. However, comparative genomic studies investigating the distribution of the oxazolidinone resistance gene optrA among food- and human-derived Enterococci remain limited. This study assessed linezolid-resistant Enterococci from retail meat and healthy humans in Beijing, China (2023-2024). Among 87 isolates, E. faecalis and E. faecium predominated. Food-derived isolates showed broader resistance profiles than human isolates. Fourteen optrA-positive strains were identified, accounting for 92.9% of food isolates. optrA frequently co-localized with erm(A), ant(9)-Ia, and fexA on Tn554-family transposons, suggesting a potentially transferable multidrug resistance module. Notably, an optrA-positive E. faecalis ST699 clone was identified for the first time in Chinese retail meat. This clone formed a distinct lineage and carried a complete Tn554-optrA island. A representative ST699 isolate exhibited enhanced fitness and virulence potential in the Galleria mellonella model. These findings highlight animal-derived foods as important reservoirs of linezolid-resistant Enterococci and provide genomic evidence consistent with their role as potential sources of optrA-mediated resistance. The emergence of a multidrug-resistant E. faecalis ST699 clone with enhanced fitness characteristics underscores the need for continued surveillance of foodborne antimicrobial resistance within the One Health framework.

Enterococcus faecalis

In vitro activity of josamycin against aerobic gram-positive cocci and anaerobes.

Josamycin, a new macrolide antibiotic, was compared with ampicillin, erythromycin, and clindamycin in vitro against 25 isolates each of pneumococci, enterococci, Staphylococcus aureus, S. epidermidis, and nonenterococcal hemolytic streptococci and against 25 anaerobes including 10 Bacteroides fragilis. Minimal inhibitory concentration and minimal bactericidal concentration data were obtained for the aerobic organisms, using serial twofold tube dilutions in Mueller-Hinton broth. Minimal inhibitory concentrations were determined for the anaerobes by the agar dilution technique. Josamycin was comparable to erythromycin and clindamycin in activity against the pneumococci, streptococci, and staphylococci and was more active than clindamycin against enterococci. It was somewhat less active than ampicillin against enterococci and S. epidermidis and showed its greatest in vitro activity against anaerobes, being comparable to clindamycin.

Ampicillin

Clinical Characteristics and Genomic Analysis of Vancomycin-Resistant Enterococcus faecium in a Tertiary Hospital in Huizhou.

OBJECTIVE: To characterize the clinical and genomic features of vancomycin-resistant enterococci (VRE) in a tertiary hospital in Huizhou and identify risk factors to inform local infection control. METHODS: A retrospective study included 58 VRE and 25 vancomycin-susceptible Enterococci (VSE) strains (August 2023-May 2025). Clinical data and antimicrobial susceptibility were analyzed; whole-genome sequencing (WGS) was performed on 54 VRE strains. RESULTS: Midstream urine was the primary VRE-positive specimen. ICU admission, polyantibiotic use (≥3 agents), and urinary catheterization were key risk factors for VRE. All VRE isolates were Enterococcus faecium and showed a predominantly clonal population structure, dominated by CC17/ST80 (68.8%) and CC2/ST106 (64.6%) under the two multilocus sequence typing schemes; five novel STs were ultimately identified in the latter scheme. VRE was universally resistant to ampicillin, with high resistance to penicillin, levofloxacin, and teicoplanin, while linezolid and tigecycline remained effective. Genotypically, 94.8% carried vanA, 100% carried virulence gene esp, and aminoglycoside and macrolide resistance genes were prevalent. A unique VRE strain (VRE48) showed resistance without canonical van genes, harboring a Ddl Ser210Tyr mutation.

Humans

Genome-wide analysis of Enterococcus faecalis genes that facilitate interspecies competition with Lactobacillus crispatus.

Enterococci are opportunistic pathogens notorious for causing a variety of infections. While both Enterococcus faecalis and Lactobacillus crispatus are commensal residents of the vaginal tract, the molecular mechanisms that enable E. faecalis to take advantage of a vaginal biome with lower counts of lactobacilli to colonize the vaginal tract and induce aerobic vaginitis remain unknown. Here, we show that L. crispatus eradicates E. faecalis in a contact-independent manner. Using transposon sequencing to identify E. faecalis OG1RF transposon (Tn) mutants that are either under-represented or over-represented when co-cultured with L. crispatus, we found that Tn mutants with disruption in the dltABCD operon, that encodes the proteins responsible for the D-alanylation of teichoic acids, and OG1RF_11697 encoding for an uncharacterized hypothetical protein are more susceptible to killing by L. crispatus. Inversely, Tn mutants with disruption in ldh1, which encodes for L-lactate dehydrogenase, are more resistant to L. crispatus killing. Using the Galleria mellonella infection model, we show that co-injection of L. crispatus with E. faecalis OG1RF enhances larvae survival while this L. crispatus-mediated protection was lost in larvae co-infected with either L. crispatus and E. faecalisΔldh1 or Δldh1Δldh2 strains. Last, using RNA sequencing to identify E. faecalis genes that are differently expressed in the presence of L. crispatus, we found major changes in the expression of genes associated with glycerophospholipid metabolism, central metabolism, and general stress responses. The findings in this study provide insights into how E. faecalis mitigate assaults by L. crispatus.IMPORTANCEEnterococcus faecalis is an opportunistic pathogen notorious for causing a multitude of infections. As vaginal commensals, E. faecalis must interact with Lactobacillus crispatus, but how E. faecalis overcomes or mitigate assaults by L. crispatus killing remains unknown. We show that L. crispatus eradicates E. faecalis temporally in a contact-independent manner. Using high-throughput molecular approaches, we identified genetic determinants that enable E. faecalis to compete with L. crispatus. This study represents an important first step for the identification of adaptive genetic traits required for enterococci to tolerate assaults by lactobacilli.

Enterococcus faecalis

[Enterococcal survival in forcemeat preserved in polymer films and in cutlets made from it].

The available data suggest that at 2 degrees and 5 degrees with a 12-day long storage in unpacked and in polymeric film packed forcemeat there occurs no propagation of the enterococci. At 22-24 degrees the multiplication of Str. faecalis var. liquefaciens proceeded similarly both in packed and unpacked forcemeat. When the temperature in the central part of the cutlets prepared from the enterococci-contaminated forcemeat reaches 7-80 degrees the bulk of the Str. faecalis var liquefaciens cells perishes, but even at 80 degrees there survive individual heat-resistant cells.

Animals

[Current resistence situation in a surgical and urological department].

Spectrum and sensitivity of bacteria were studied at the Surgical (534 positive wound smears) and the Urological Clinics (7879 urine specimens). Krankenhaus Nordwest, Frankfurt/M., during the period of 1969-1971 and in 1973. The most common organisms identified in wound smears were E. coli, followed by Staph. areus, Aerobacter and Proteus species. E. coli were also predominant in urine, but followed by Enterococci, Proteus and Pseudomonas. E. coli, Proteus species and especially Pseudomonas increased in number whereas Enterococci decreased. There was no pronounced increase in resistance to 9 current antibiotics as well as to chemotherapeutics during the observation period which was particularly striking in the case of Ampicillin used on a large scale. The results of our study support the presently employed therapeutic method using bactericidal antibiotics of the penicillin group in strict indications.

Anti-Bacterial Agents

Development of linezolid and daptomycin resistance in vancomycin resistant Enterococcus faecium during antibiotic treatment.

The increasing incidence of vancomycin-resistant enterococci (VRE) over the past decade has reduced treatment options largely to linezolid and daptomycin. However, the emergence of resistance to both agents further complicates the management of VRE infections. While the mechanisms of linezolid resistance are relatively well understood, those underlying daptomycin resistance remain less clearly defined. In this study, we analyzed genomic changes associated with the development of linezolid and daptomycin resistance in initially susceptible isolates following treatment at a Danish university hospital. Phenotypic susceptibility testing and whole-genome sequencing were performed on eight isolates obtained from the same patient. We identified two distinct Enterococcus faecium clones with different mechanisms of linezolid resistance. Linezolid resistance was associated with a G2576T mutation in the 23S rRNA gene (ST80 clone) and the presence of the poxtA gene (ST3082 clone). The ST80 clone also developed daptomycin resistance during therapy. We found that daptomycin resistance might result from either a G173R substitution in a gene annotated as an "ABC transporter ATP-binding protein (LolD)" or a nonsense mutation (Q58*) in phosphoketolase, with both alterations potentially acting synergistically, but further studies are warranted to confirm if these mutations can confer resistance. Together with these findings, the study demonstrates that a single patient may harbor multiple E. faecium clones simultaneously, highlighting the risk of treatment failure if all clones are not accurately identified.

Daptomycin

Clinical sequelae of gut microbiome development and disruption in hospitalized preterm infants.

Aberrant preterm infant gut microbiota assembly predisposes to early-life disorders and persistent health problems. Here, we characterize gut microbiome dynamics over the first 3 months of life in 236 preterm infants hospitalized in three neonatal intensive care units using shotgun metagenomics of 2,512 stools and metatranscriptomics of 1,381 stools. Strain tracking, taxonomic and functional profiling, and comprehensive clinical metadata identify Enterobacteriaceae, enterococci, and staphylococci as primarily exploiting available niches to populate the gut microbiome. Clostridioides difficile lineages persist between individuals in single centers, and Staphylococcus epidermidis lineages persist within and, unexpectedly, between centers. Collectively, antibiotic and non-antibiotic medications influence gut microbiome composition to greater extents than maternal or baseline variables. Finally, we identify a persistent low-diversity gut microbiome in neonates who develop necrotizing enterocolitis after day of life 40. Overall, we comprehensively describe gut microbiome dynamics in response to medical interventions in preterm, hospitalized neonates.

Humans

Emergence of antibiotic resistance in hospitals, 1935-1975.

A limited review of the changes in susceptibility of common bacterial pathogens to available antibacterial agents is presented. Significant developments in recent years include the following: (1) the emergence of Streptococcus pneumoniae with decreased resistance to penicillin and of some strains resistant to several antibiotics; (2) a decline in prevalence of multi-drug-resistant Staphylococcus aureus after 1960 following their increasing prevalence in the preceding years (these changes were methicillin-resistant (and multi-drug-resistant) S. aureus and the marked differences in their prevalence in different areas (these changes also were related to appearance of new phages in those organisms); (4) an increasing resistance to multiple drugs among enterococci but not among viridans streptococci or among nonenterococcal group D streptococci; (5) the emergence of beta-lactamase-producing Neisseria gonorrhoeae; (6) the emergence and spread of sulfonamide-resistant Neisseria meningitidis; (7) the occurrence of beta-lactamase-producing strains of Haemophilus influenzae and occasional strains resistant to chloramphenicol; (8) the focal occurrence of chloramphenicol-resistant Salmonella typhi in Vietnam and in epidemic form in Mexico; (9) the demonstration of marked differences in prevalence of resistance to multiple drugs in common pathogens to the most widely used antibiotics in different geographic areas. The dominant factor in the emergence and spread of antibiotic-resistant bacterial pathogens, whether in hospital wards or in the community, is clearly the intensive use of the antibiotic agents to which resistance emerges and then spreads.

Bacteria

Changing ecology of acute bacterial empyema: occurrence and mortality at Boston City Hospital during 12 selected years from 1935 to 1972.

The occurrence, etiology, and demography of acute bacterial empyema are presented to reflect the widespread use of sulfonamides, penicillin, and other active antibiotics. In community-acquired (C-A) cases Streptococcus pneumoniae, hemolytic streptococci, and Staphylococcus aureus were the most frequent single organisms identified in initial positive cultures of pleural fluid during 1935. S. pneumoniae declined steadily until 1953 but continued to occur frequently in C-A cases. Hemolytic streptococci became infrequent. S. aureus increased and became the most frequent organism in 1955 and declined to original levels after 1965 while gram-negative rods increased. S. aureus, aerobic gram-negative rods, and enterococci were most frequent in originally mixed infections, hospital-acquired cases, and superinfections. Anaerobes with or without aerobes were mostly seen in C-A cases and did not vary in incidence. Mortality increased with age. Overall mortality was greater during the 10 antibiotic years, associated with a marked decrease in the proportion of patients younger than 50 years and increase in those over 60 years old, and was greater in patients with serious underlying diseases subjected to more complicated surgical procedures.

Age Factors

In vitro study of netilmicin compared with other aminoglycosides.

Netilmicin (Sch 20569) is an ethyl derivative of gentamicin C(1a) that is active against most Enterobacteriaceae, Pseudomonas aeruginosa, and Staphylococcus aureus isolates. Among 342 clinical isolates tested, all staphylococci; 92% of Escherichia coli, 93% of Klebsiella pneumoniae, and 92% of Enterobacter were inhibited by 0.8 mug or less of netilmicin per ml, but only 78% of P. aeruginosa were inhibited by 3.1 mug or less per ml. Most clinical isolates of enterococci, Serratia marcescens, and Providencia were not inhibited by 3.1 mug of netilmicin per ml. Like other aminoglycosides, the netilmicin in vitro activity was markedly influenced by the growth medium used, with activity decreased by sodium, calcium, and magnesium. Netilmicin was more active at alkaline pH. Addition of magnesium to Pseudomonas or Serratia pretreated with netilmicin produced inhibition of killing. Netilmicin was more active than gentamicin, sisomicin, tobramycin, or amikacin against E. coli and K. pneumoniae. Netilmicin inhibited growth of all gentamicin-resistant isolates of Klebsiella and Citrobacter tested, but only 73% of E. coli; Pseudomonas and Providencia were resistant to netilmicin. Most Serratia (95%) and indole-positive Proteus (83%) isolates were resistant to netilmicin but were inhibited by amikacin.

Amikacin

Role of lime treatment in the removal of bacteria, enteric viruses, and coliphages in a wastewater reclamation plant.

Lime flocculation/sedimentation in the first process unit of a 4,500-m3/day wastewater reclamation plant reduced numbers of microorganisms extensively when operated at pH 11.2. The efficiency was much less at lower pH values, and some bacteria even multiplied at pH 9.6. Data on reduction in the number of microorganisms in the lime treatment and subsequent units indicate that inactivation by hydroxide alkalinity plays an important role in the efficiency of lime treatment. Reductions in the numbers of enteric viruses were higher than those of coliphages, enterococci, and total plate and coliform bacteria, which indicate that lime treatment can be monitored by means of coliphage and conventional bacteriological tests. This paper illustrates the valuable role of high-pH lime treatment in reducing the load of pathogenic microorganisms on subsequent units, including ultimate disinfection processes, which is important in the multiple safety barrier concept of wastewater reclamation processes.

Bacteria

Comparative genomic characterization and antimicrobial resistance of bacteremia-causing Enterococcus faecium and Enterococcus faecalis in a Chinese hospital.

Enterococci are common commensals of the human gut and important opportunistic pathogens, with Enterococcus faecium and Enterococcus faecalis being the most clinically prevalent species. A significant epidemiological shift has emerged with an increasing clinical burden of E. faecium. To compare genomic evolution of E. faecium and E. faecalis, we performed whole-genome sequencing on 93 E. faecium and 32 E. faecalis isolates causing bloodstream infections at a single hospital (2022-2024). Analysis of patient demographics revealed that E. faecium infections originated from fewer sources than E. faecalis, with a higher proportion deriving from intra-abdominal infections. Multilocus sequence typing identified ST78 and ST789 as the predominant sequence types for E. faecium, whereas ST16 and ST179 were most common for E. faecalis. E. faecium carried more antimicrobial resistance genes and putative virulence marker (PVM)-type virulence genes than E. faecalis, with vancomycin resistance predominantly mediated by vanHAX (33/93, 35.5%) and a single E. faecalis isolate also carrying vanHAX (1/32, 3.1%); the structurally incomplete vanHMX gene cluster was detected in 11 E. faecium isolates. Pan-genome analysis indicated a larger core genome in E. faecalis compared to E. faecium, consistent with greater plasmid replicon diversity in the latter. Intra-host comparisons showed that two E. faecalis pairs from the same patient were clonally related, with one isolate acquiring a vanHAX plasmid conferring vancomycin resistance. In contrast, E. faecium isolates exhibited marked genomic diversity even among clonally related pairs. These findings suggest that E. faecium possesses greater genomic plasticity and adaptive potential to the clinical environment.IMPORTANCEThis study provides a detailed comparison of clinical and genomic features between Enterococcus faecium and Enterococcus faecalis from the same hospital setting. We show that E. faecium isolates, mainly ST78/ST789, carry more antimicrobial resistance genes and a higher number of putative virulence marker (PVM) genes than E. faecalis, reflecting their hospital-adapted nature. E. faecium also exhibits a smaller core genome and greater diversity of plasmid replicon types, indicating higher genomic plasticity and capacity for horizontal gene transfer. By contrast, E. faecalis retains a larger core genome and a set of classical virulence factors, and its within-host isolates are clonally related. These distinct genomic profiles help to understand how the two species adapt to clinical environments and may inform more targeted infection control strategies and resistance surveillance.

Enterococcus faecium