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For a One-Health investigation of antimicrobial resistance (AMR) in Enterococcus spp., isolates from humans and beef cattle along with abattoirs, manured fields, natural streams, and wastewater from both urban and cattle feedlot sources were collected over two years. Species identification of Enterococcus revealed distinct associations across the continuum. Of the 8430 isolates collected, Enterococcus faecium and Enterococcus faecalis were the main species in urban wastewater (90%) and clinical human isolates (99%); Enterococcus hirae predominated in cattle (92%) and feedlot catch-basins (60%), whereas natural streams harbored environmental Enterococcus spp. Whole-genome sequencing of E. faecalis (n = 366 isolates) and E. faecium (n = 342 isolates), revealed source clustering of isolates, indicative of distinct adaptation to their respective environments. Phenotypic resistance to tetracyclines and macrolides encoded by tet(M) and erm(B) respectively, was prevalent among Enterococcus spp. regardless of source. For E. faecium from cattle, resistance to β-lactams and quinolones was observed among 3% and 8% of isolates respectively, compared to 76% and 70% of human clinical isolates. Clinical vancomycin-resistant E. faecium exhibited high rates of multi-drug resistance, with resistance to all β-lactam, macrolides, and quinolones tested. Differences in the AMR profiles among isolates reflected antimicrobial use practices in each sector of the One-Health continuum.

Background Enterococcus is ubiquitous in nature and is a commensal of both the bovine and human gastrointestinal (GI) tract. It is also associated with clinical infections in humans. Subtherapeutic administration of antibiotics to cattle selects for antibiotic resistant enterococci in the bovine GI tract. Antibiotic resistance genes (ARGs) may be present in enterococci following antibiotic use in cattle. If located on mobile genetic elements (MGEs) their dissemination between Enterococcus species and to pathogenic bacteria may be promoted, reducing the efficacy of antibiotics. Results We present a comparative genomic analysis of twenty-one Enterococcus spp. isolated from bovine feces including Enterococcus hirae ( n  = 10), Enterococcus faecium ( n  = 3), Enterococcus villorum ( n  = 2), Enterococcus casseliflavus ( n  = 2), Enterococcus faecalis ( n  = 1), Enterococcus durans ( n  = 1), Enterococcus gallinarum ( n  = 1) and Enterococcus thailandicus ( n  = 1). The analysis revealed E. faecium and E. faecalis from bovine feces share features with human clinical isolates, including virulence factors. The Tn 917 transposon conferring macrolide-lincosamide-streptogramin B resistance was identified in both E. faecium and E. hirae , suggesting dissemination of ARGs on MGEs may occur in the bovine GI tract. An E. faecium isolate was also identified with two integrative conjugative elements (ICEs) belonging to the Tn 916 family of ICE, Tn 916 and Tn 5801 , both conferring tetracycline resistance. Conclusions This study confirms the presence of enterococci in the bovine GI tract possessing ARGs on MGEs, but the predominant species in cattle, E. hirae is not commonly associated with infections in humans. Analysis using additional complete genomes of E. faecium from the NCBI database demonstrated differential clustering of commensal and clinical isolates, suggesting that these strains may be specifically adapted to their respective environments.

Enterococci, common hospital-acquired infections in immunocompromised patients, have garnered attention in clinical microbiology. To determine the clinical relevance of enterococci as food-borne pathogens, 116 fish, 90 vegetables, and 120 human diarrheal samples were tested for E. faecalis and E. faecium pathogenicity. Conventionally, 69 of 326 (21.17%) samples were positive for Enterococcus species, 52 (15.95%) of which were molecularly classified as E. faecalis and 13 (3.99%) as E. faecium . The E. faecalis contamination percentage of fresh fish (19.70%) was higher than frozen fish (4%). Cauliflower had the highest E. faecalis percentage (16.67%) when fish and vegetable samples didn’t harbor the E. faecium atpA gene. 23.33% and 10.83% of participants’ samples were molecularly confirmed as E. faecalis and E. faecium positive, respectively. E. faecalis isolates had all virulence genes, with gel s being the most common (65.38%), while cylA and asa1 genes couldn’t be detected in E. faecium isolates. E. faecalis showed the highest resistance against vancomycin and tetracycline (69.23%), whereas E. faecium extremely resisted tetracycline (76.92%) and erythromycin (69.23%) with the recognition of MDR among 44.2% of E. faecalis and 38.5% of E. faecium isolates. The great similarity of our isolates showed the clinical importance of food-borne antibiotic-resistant enterococci.

Key Points Enterococci are some of the most versatile organisms found to infect hospitalized patients. The epidemiology of enterococcal infections has evolved since the emergence of these pathogens and has seen the rise of Enterococcus faecium as a nosocomial pathogen with serious clinical implications. The effect of antibiotics on the microbiota of the gastrointestinal tract and subsequent alterations in the regulation of the gut immune system can favour colonization by multidrug-resistant enterococci. Enterococcal genomes are extremely malleable, with the ability to exchange large fragments of chromosomal DNA. In addition, the lack of CRISPR (clustered regularly interspaced short palindromic repeats) elements has a potential role in the adaptation of hospital-associated enterococci. Specific pathogenicity factors contribute to the ability of enterococci to produce disease and/or survive in the gastrointestinal tract of mammals. The major factors include secreted and cell surface-associated determinants. Antibiotic resistance is widespread for the anti-enterococcal antibiotics that are most commonly used in clinical practice, and the mechanisms of resistance for many of these antibiotics are known. These antibiotics include ampicillin, linezolid, daptomycin and quinupristin–dalfopristin, and there is also high-level resistance to aminoglycosides. Such resistances have important therapeutic implications. Arias and Murray discuss the factors that may have contributed to the rise of enterococci as nosocomial pathogens, with an emphasis on the epidemiology and pathogenesis of these species and their mechanisms of resistance to the most relevant anti-enterococcal agents used in clinical practice. The genus Enterococcus includes some of the most important nosocomial multidrug-resistant organisms, and these pathogens usually affect patients who are debilitated by other, concurrent illnesses and undergoing prolonged hospitalization. This Review discusses the factors involved in the changing epidemiology of enterococcal infections, with an emphasis on Enterococcus faecium as an emergent and challenging nosocomial problem. The effects of antibiotics on the gut microbiota and on colonization with vancomycin-resistant enterococci are highlighted, including how enterococci benefit from the antibiotic-mediated eradication of Gram-negative members of the gut microbiota. Analyses of enterococcal genomes indicate that there are certain genetic lineages, including an E. faecium clade of ancient origin, with the ability to succeed in the hospital environment, and the possible virulence determinants that are found in these genetic lineages are discussed. Finally, we review the most important mechanisms of resistance to the antibiotics that are used to treat vancomycin-resistant enterococci.

Background This study investigates the safety evaluation of enterocin-producing 11 E. mundtii and two E. faecium strains previously isolated from small livestock colostrums. Enterococcus species do not possess Generally Recognized as Safe (GRAS) status. Hence, it is critical to scrutinize enterococci’s antibiotic resistance, virulence characteristics, and biogenic amine production capabilities in order to assess their safety before using them as starter or adjunct cultures. Results Enterococcus strains showed susceptibility to medically significant antibiotics. Multiple-drug resistance (MDR) was found in only E. faecium HC121.4, and its multiple antibiotic resistance (MAR) index was detected to be 0.22. The tetL and aph(3')-IIIa were the most commonly found antibiotic resistance genes in the strains. However, E. mundtii strains HC56.3, HC73.1, HC147.1, and E. faecium strain HC121.4 were detected to lack any of the antibiotic resistance genes examined in this study. Only E. mundtii HC166.3 showed hemolytic activity, while none of the strains engage in gelatinase activity. The strains were identified to have virulence factor genes with a low rate. None of the virulence factor genes could be detected in E. mundtii HC26.1, HC56.3, HC73.1, HC165.3, HC166.8, and E. faecium HC121.4. The E. mundtii HC73.2 strain displayed the highest presence of virulence factor genes, namely gelE , efaA fs , cpd , and ccf . Similarly, the E. mundtii HC112.1 strain showed a significant presence of genes efaA fm , ccf , and acm . There was no decarboxylation of histidine, ornithine, or lysine seen in any of the strains. Nevertheless, E. faecium HC121.4 and HC161.1 strains could decarboxylate tyrosine, but E. mundtii HC26.1, HC56.3, HC73.1, HC73.2, HC112.1, HC147.1, HC155.2, HC165.3, HC166.3, HC166.5, and HC166.8 strains only showed a limited capacity for tyrosine decarboxylation. None of the strains possessed the hdc , odc , or ldc genes, but all of them had the tdc gene. Conclusion The E. mundtii HC56.3 and HC73.1 strains were deemed appropriate for utilization in food production. Using the remaining 11 strains as live cultures in food production activities could pose a possible risk to consumer health.

Subclinical mastitis poses a hidden threat to dairy productivity and animal health, often harbouring antimicrobial-resistant pathogens. It is becoming increasingly recognized that Enterococcus species cause mastitis in dairy cows. Accurately characterizing the regional epidemiology of enterococcal mastitis, determining its correlations with management variables, and comprehending its effects on udder health all depend on accurate species information. This study investigated the occurrence, antibiotic resistance and virulence factors of Enterococcus faecalis and Enterococcus faecium in cow dung and milk samples from cows with subclinical mastitis. Subclinical mastitis was identified in 39.0% (68/174) of cows and 27.8% (194/696) of quarters, based on results from the California Mastitis Test (CMT) and somatic cell counts (SCC), respectively. Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) and Polymerase Chain Reaction (PCR) targeting the ddl gene confirmed the predominance of E. faecalis (93%) and E. faecium (6.4%) in milk samples, while cow dung samples yielded only E. faecalis (100%). Notably, among the E. faecalis isolates from milk samples, 17.2% exhibited vancomycin resistance, whereas streptomycin resistance was found in a smaller proportion of isolates (6.8%). All (100%) E. faecium isolates from the same milk samples showed resistance to vancomycin. The findings also revealed that 11 (32.3%) of E. faecium isolates from cow dung were resistant to vancomycin. Multidrug resistance (MDR) was observed in 20.6% of milk and 6.8% of cow dung isolates. The vanA gene was the most prevalent antibiotic resistance gene (ARG), detected in 96% of E. faecalis isolates. Virulence profiling of Enterococcus spp. isolates showed varying gene prevalence in milk ( asa1 : 33.3%, ace : 12.7%, esp : 10%) and cow dung samples ( gelE : 53.2%, hyl : 38.2%). This study has indicated a significant occurrence of antimicrobial-resistant E. faecalis and E. faecium strains obtained from subclinical cattle mastitis. These findings emphasize the role of Enterococcus spp., especially vancomycin-resistant strains, as emerging threats in bovine subclinical mastitis, with possible implications for zoonotic transmission and antimicrobial stewardship in dairy systems.

Patients with hematological malignancies or undergoing hematopoietic stem cell transplantation are vulnerable to colonization and infection with multidrug-resistant organisms, including vancomycin-resistant Enterococcus faecium (VREfm). Over a 10-y period, we collected and sequenced the genomes of 110 VREfm isolates from gastrointestinal and blood cultures of 24 pediatric patients undergoing chemotherapy or hematopoietic stem cell transplantation for hematological malignancy at St. Jude Children’s Research Hospital. We used patient-specific reference genomes to identify variants that arose over time in subsequent gastrointestinal and blood isolates from each patient and analyzed these variants for insight into how VREfm adapted during colonization and bloodstream infection within each patient. Variants were enriched in genes involved in carbohydrate metabolism, and phenotypic analysis identified associated differences in carbohydrate utilization among isolates. In particular, a Y585C mutation in the sorbitol operon transcriptional regulator gutR was associated with increased bacterial growth in the presence of sorbitol. We also found differences in biofilm-formation capability between isolates and observed that increased biofilm formation correlated with mutations in the putative E. faecium capsular polysaccharide (cps) biosynthetic locus, with different mutations arising independently in distinct genetic backgrounds. Isolates with cps mutations showed improved survival following exposure to lysozyme, suggesting a possible reason for the selection of capsule-lacking bacteria. Finally, we observed mutations conferring increased tolerance of linezolid and daptomycin in patients who were treated with these antibiotics. Overall, this study documents known and previously undescribed ways that VREfm evolve during intestinal colonization and subsequent bloodstream infection in immunocompromised pediatric patients.

Drs. Cesar Arias and Barbara Murray write that we have arrived at a point as frightening as the preantibiotic era: for patients infected with multidrug-resistant bacteria, there is no magic bullet. In March 1942, a 33-year-old woman lay dying of streptococcal sepsis in a New Haven, Connecticut, hospital, and despite the best efforts of contemporary medical science, her doctors could not eradicate her bloodstream infection. Then they managed to obtain a small amount of a newly discovered substance called penicillin, which they cautiously injected into her. After repeated doses, her bloodstream was cleared of streptococci, she made a full recovery, and she went on to live to the age of 90. 1 Sixty-six years after her startling recovery, a report 2 described a 70-year-old man in San Francisco with endocarditis caused by vancomycin-resistant . . .

Globally, fermented foods (FFs), which may be traditional or industrially-produced, are major sources of nutrition. In the traditional practice, the fermentation process is driven by communities of virtually uncharacterized microflora indigenous to the food substrate. Some of these flora can have virulent or antibiotic resistance properties, posing risk to consumers. Others, such as Enterococcus faecalis and Enterococcus faecium , may also be found in such foods. Enterococci that harbor antibiotic resistance or virulence factors can cycle among animals, food, humans and the environment, thereby transferring these harmful properties at the gene level to harmless commensals in the food matrix, animals and humans. In this work, several microbial isolates obtained from different FF sources were analyzed for their identity and virulence and/or antibiotic resistance properties. For identification aiming at enterococci, isolates that were Gram-positive and catalase- and oxidase-negative were subjected to multiple tests including for growth in broth containing 6.5% NaCl, growth and hydrolytic activity on medium containing bile-esculin, hemolytic activity on blood agar, and growth at 45°C and survival after incubation at 60°C for 30 min. Furthermore, the isolates were tested for susceptibility/resistance to a select group of antibiotics. Finally, the isolates were molecularly-characterized with respect to species identity and presence of virulence-encoding genes by amplification of target genes. Most sources contained enterococci, in addition to most of them also containing Gram-negative flora. Most of these also harbored virulence factors. Several isolates were also antibiotic-resistant. These results strongly suggest attention should be given to better control presence of such potentially pathogenic species.

Vancomycin-resistant enterococci (VRE) are a major public health concern, yet little is known about their circulation in fish. This study investigated the occurrence, glycopeptide resistance genotypes, virulence characteristics, and sequence types (STs) of VRE isolated from diseased fishes and humans. Isolates were identified using multiplex polymerase chain reaction (PCR) assay and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and tested for antimicrobial susceptibility. VRE isolates were screened for the presence of glycopeptide resistance genes and eight virulence genes. Multilocus sequence typing (MLST) was determined to assess the clonality of VRE isolates from fishes and humans. Among 60 human samples, 20 Enterococcus species isolates (33.33%) including Enterococcus faecalis and Enterococcus faecium (10 of each), were identified. The overall prevalence of E. faecalis was 42.86% (30/70) in Oreochromis niloticus and 48.0% (24/50) in Clarias gariepinus . E. faecium was found in 15.71% (11/70) of Oreochromis niloticus and 14.0% (7/50) of Clarias gariepinus . Over 50% of human isolates were multidrug resistant (MDR) and 30% exhibited an extensive drug resistant (XDR) phenotype. Fish isolates also displayed high MDR (70.83%) and XDR (29.17%) rates. Forty-nine (53.26%; 34 from fish and 15 from human) isolates were VRE including 30 isolates of E. faecalis (VREfs) and 19 isolates of E. faecium (VREfm). The vanA gene was the most frequent among VREfs (83.33%) and VREfm (100%) isolates. The vanB gene was found in 26.67% of VREfs and 15.79% of VREfm. Three out of 10 VREfm (30%) and 2/24 (8.33%) VREfs isolates of fish origin carried both vanA and vanB genes. vanC gene was found in 13.33% (4/30) of VREfs of human and fish origin. One VREfs isolate from human urine carried both vanA and vanC genes. High frequency of the virulence genes  gelE , sprE , asa1 , esp, and cylA were observed;  efa  and ace gene was more associated with VREfs, while hyl  gene was more frequently detected in VREfm. Different combinations of virulence genes suggesting synergistic pathogenic potential. MLST revealed both overlapping and host-specific STs among the examined Enterococcus isolates from humans and fish. Experimental infection of O. niloticus with VREfs and VREfm caused a 100% and 60% mortality rate within 6 days postinfection, respectively with characteristic disease symptoms. The emergence of VRE and the high prevalence of virulence traits could be regarded as an alarming situation. The call for increased infection control and antibiotic stewardship measures is timely and relevant to combat the spread of VRE in fish and humans.