Identification and Safety Assessment of Enterococcus casseliflavus KB1733 Isolated from Traditional Japanese Pickle Based on Whole-Genome Sequencing Analysis and Preclinical Toxicity Studies.

The present study involves the precise identification and safety evaluation of Enterococcus casseliflavus KB1733, previously identified using 16S rRNA analysis, through whole-genome sequencing, phenotypic analysis, and preclinical toxicity studies. Analyses based on the genome sequencing data confirm the identity of KB1733 as E. casseliflavus and show that the genes related to vancomycin resistance are only present on the chromosome, while no virulence factor genes are present on the chromosome or plasmid. Phenotypic analyses of antibiotic resistance and hemolytic activity also indicated no safety concerns. A bacterial reverse mutation test showed there was no increase in revertant colonies of heat-killed KB1733. An acute toxicity test employing heat-killed KB1733 at a dose of 2000 mg/kg body weight in rats resulted in no deaths and no weight gain or other abnormalities in the general condition of the animals, with renal depression foci and renal cysts only occurring at the same frequency as in the control. Taking the background data into consideration, the effects on the kidneys observed in the current study were not caused by KB1733. Our findings suggest that KB1733 is non-pathogenic to humans/animals, although further studies involving repeated oral toxicity tests and/or clinical tests are required.

Identification and biological characteristics of Enterococcus casseliflavus TN-47 isolated from Monochamus alternatus.

With the widespread use of antibiotics, the incidence of antibiotic resistance in microorganisms has increased. Monochamus alternatus is a trunk borer of pine trees. This study aimed to investigate the in vitro antimicrobial and biological characteristics of Enterococcus casseliflavus TN-47 (PP411196), isolated from the gastrointestinal tract of M. alternatus in Jilin Province, PR China. Among 13 isolates obtained from the insects, five were preliminarily screened for antimicrobial activity. E. casseliflavus TN-47, which exhibited the strongest antimicrobial activity, was identified. E. casseliflavus TN-47 possessed antimicrobial activity against Staphylococcus aureus USA300 and Salmonella enterica serovar Pullorum ATCC 19945. Furthermore, E. casseliflavus TN-47 was sensitive to tetracyclines, penicillins (ampicillin, carbenicillin, and piperacillin), quinolones and nitrofuran antibiotics, and resistant to certain beta-lactam antibiotics (oxacillin, cefradine and cephalexin), macrolide antibiotics, sulfonamides and aminoglycosides. E. casseliflavus TN-47 could tolerate low pH and pepsin-rich conditions in the stomach and grow in the presence of bile acids. E. casseliflavus TN-47 retained its strong auto-aggregating ability and hydrophobicity. This strain did not exhibit any haemolytic activity. These results indicate that E. casseliflavus TN-47 has potential as a probiotic. This study provides a theoretical foundation for the future applications of E. casseliflavus TN-47 and its secondary metabolites in animal nutrition and feed.

Polycystic intrahepatic infection caused by Enterococcus casseliflavus: a case report and literature review.

BACKGROUND: Enterococcus casseliflavus is a rare pathogenic bacterium that is characterized by vancomycin resistance and can lead to multiple infections in the human body. This report describes a rare case of polycystic intrahepatic infection with E. casseliflavus which necessitated antibiotic treatment and surgical intervention involving cystic drainage. CASE PRESENTATION: A 59-year-old woman, a long-term hemodialysis patient, was hospitalized due to a 5-day history of fever, abdominal pain, and diarrhea, which were possibly caused by the ingestion of contaminated food. Her blood culture yielded a positive result for E. casseliflavus, and she was initially treated with piperacillin/tazobactam and linezolid. Later, the antibiotic regimen was adjusted to include meropenem and linezolid. Despite treatment, her body temperature remained elevated. However, subsequent blood cultures were negative for E.casseliflavus.Conventional CT scans and ultrasound examinations did not identify the source of infection. However, a PET-CT examination indicated an intrahepatic cyst infection. Following MRI and ultrasound localization, percutaneous intrahepatic puncture and drainage were performed on the 20th day. Fluoroquinolones were administered for 48 days. On the 32nd day, MRI revealed a separation within the infected cyst, leading to a repeat percutaneous drainage at a different site. Subsequently, the patient's temperature returned to normal. The infection was considered resolved, and she was discharged on the 62nd day. Follow-up results have been favorable thus far. CONCLUSIONS: Based on the findings from this case, it is recommended to promptly conduct PET-CT examination to exclude the possibility of intracystic infection in cases of polycystic liver infection that are challenging to control. Furthermore, timely consideration should be given to puncture drainage in difficult cases.

Metagenome-assembled bacterial genomes recovered from the datasets of Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae).

Spodoptera frugiperda (Smith) (Lepidoptera: Noctuidae), also known as the fall armyworm, is an economically important and widespread polyphagous pest. Microorganisms associated to this insect during life cycle play important ecological roles. We report 3 metagenome-assembled bacterial genomes reconstructed from a metagenome dataset obtained from S. frugiperda larvae F3 3rd-instar reared using artificial diet under laboratory conditions. Genome data for Enterococcus casseliflavus indicated a genome length of 3,659,8333 bp and GC content of 42.54%. Genome data for E. mundtii indicated a genome length of 2,921,701 bp and GC content of 38.37%. Finally, genome data for Lactiplantibacillus plantarum indicated a genome length of 3,298,601 bp, GC content of 44.31%. Genome analysis allowed us to identify genus-specific protein families (PLFams), transporters and antibiotic resistance-related genes among others. DNA sequences were deposited in National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) as Bioproject accession PRJNA899064.

Enterococcus casseliflavus infection in a captive Southern Cassowary (Casuarius casuarius): clinical and post-mortem findings.

An adult 22-year-old, male southern cassowary (Casuarius casuarius) presented for lethargy, dysorexia and soft feces. Clinical findings under chemical immobilization were unremarkable except for heterophilic toxic changes on blood smear, reduced albumin/globulin ratio and potentially elevated liver parameters. The animal was treated for suspected hepatobiliary insufficiency and received supportive treatment with daily subcutaneous fluids and medication and was monitored with a quality-of-life assessment. It was eventually euthanized after further deterioration. Post-mortem examination revealed vegetative valvular endocarditis, bile duct obstruction, cholangiohepatitis and splenitis with intralesional Enterococcus casseliflavus isolated. This is a first documented case of E. casseliflavus infection in a bird with multiorgan involvement.

Endophthalmitis caused by Enterococcus casseliflavus: a systematic review of literature.

Introduction: Enterococcus casseliflavus is a rare pathogen in human infections, despite being widely distributed in natural environments. This systematic review aims to evaluate the evidence related to endophthalmitis caused by E. casseliflavus. Methods: A thorough search of PubMed, PubMed Central, and Scopus databases was conducted, covering the period up to October 2022. Results: A total of 53 records were identified, with 8 studies reporting a total of 21 cases meeting the inclusion criteria. Among these studies, 7 described isolated case reports, while 1 study described 14 cases. The overall quality of the reports was good, as all articles were determined to have low risk of bias. Vancomycin susceptibility was reported in only one case of isolated case reports, while the remaining cases were all vancomycin resistant. With regard to management, in most cases intravenous ampicillin and linezolid were administered, while only one study reported administration of vancomycin. Conclusions: Ophthalmologists should be aware of the potential for E. casseliflavus to cause endophthalmitis infections and the challenges associated with its intrinsic resistance to vancomycin.

Isolation, Identification, and Fermentation Medium Optimization of a Caproic Acid­Producing Enterococcus casseliflavus Strain from Pit Mud of Chinese Strong Flavor Baijiu Ecosystem.

Caproic acid is the precursor material of ethyl hexanoate, a representative flavor substance in strong flavor baijiu (SFB). Increasing the content of caproic acid in SFB helps to improve its quality. In the present study, caproic acid-producing bacteria from the pit mud of an SFB ecosystem were isolated, purified, and characterized. Strain BF-1 with the highest caproic acid yield (0.88 g/l) was selected. The morphological and molecular identification analysis showed that strain BF-1 was Enterococcus casseliflavus. The genome of E. casseliflavus BF-1 was sequenced and was found to be 2,968,377 bp in length with 3,270 open reading frames (ORFs). The caproic acid biosynthesis pathway in E. casseliflavus BF-1 was predicted based on the KAAS annotation. The virulence factors in the genome of strain BF-1 were annotated, which showed that E. casseliflavus BF-1 is safe at the genetic level. After adding essential nutrients based on the KAAS annotation, the optimum medium conditions for acid production by strain BF-1 were obtained by performing orthogonal experiments. The caproic acid yield of strain BF-1 reached 3.03 g/l, which was 3.44-fold higher than the initial yield. The optimized fer- mentation of caproic acid production by BF-1 was reported for the first time. The strain could be further used to regulate the ecosystem in baijiu production to improve its quality.

Acinetobacter radioresistens and Enterococcus casseliflavus co-infection with endocarditis, bacteremia, and pneumonia.

Acinetobacter species are Gram-negative coccobacilli found to cause a multitude of infections. However, they are a rare cause of bacteremia with Acinetobacter radioresistens accounting for less than 10 % of Acinetobacter infections. In this report, we describe a patient presenting with acute encephalopathy, fever, and hypoxia who was initially found to have bilateral perihilar and lower lobar peribronchial thickening on chest x-ray. Two sets of blood cultures obtained on admission were positive for Acinetobacter radioresistens and Enterococcus casseliflavus and one set of blood cultures returned positive for Leclercia adecarboxylata although believed to be a skin contaminant. Susceptibilities confirmed all bacteria were pan-sensitive. The patient was also found to have an aortic valve vegetation which was not amenable to surgical intervention. He was treated with 42 days of daptomycin and cefepime. At present, co-infection with Acinetobacter radioresistens and Enterococcus casseliflavus with manifestations of polymicrobial endocarditis has never been reported. Though this co-infection was pan-sensitive, there is an increasing rate of resistance to commonly used, broad-spectrum antibiotics such as ß-lactams, which will continue to pose a challenge between balancing treatment and antibiotic stewardship.

Probiotic potentials of the silkworm gut symbiont Enterococcus casseliflavus ECB140, a promising L-tryptophan producer living inside the host.

AIMS: L-tryptophan is an essential aromatic amino acid for the growth and development of animals. Studies about enteric L-tryptophan-producing bacteria are scarce. In this report, we characterized the probiotic potential of Enterococcus casseliflavus ECB140, focusing on its L-tryptophan production abilities. METHODS AND RESULTS: ECB140 strain was isolated from the silkworm gut and can survive under strong alkaline environmental conditions. Bacterial colonization traits (motility and biofilm) were examined and showed that only ECB140 produced flagellum and strong biofilms compared with other Enterococcus strains. Comparative genome sequence analyses showed that only ECB140 possessed a complete route for L-tryptophan synthesis among all 15 strains. High-performance liquid chromatography and qRT-PCR confirmed the capability of ECB140 to produce L-tryptophan. Besides, the genome also contains the biosynthesis pathways of several other essential amino acids, such as phenylalanine, threonine, valine, leucine, isoleucine and lysine. These results indicate that ECB140 has the ability to survive passage through the gut and could act as a candidate probiotic. CONCLUSIONS: The study describes a novel, natural silkworm gut symbiont capable of producing L-tryptophan. Enterococcus casseliflavus ECB140 physical and genomic attributes offer possibilities for its colonization and provide L-tryptophan for lepidopteran insects.

Enterococcus casseliflavus KB1733 Isolated from a Traditional Japanese Pickle Induces Interferon-Lambda Production in Human Intestinal Epithelial Cells.

The association between lactic acid bacteria (LAB) and their immunostimulatory effects has attracted considerable attention; however, it remains unclear whether LAB can induce interferon-lambdas (IFN-λs) in human epithelial cells under conditions that do not mimic infection. In this study, we first employed a reporter assay to screen for a potential strain capable of inducing IFN-λ3 among 135 LAB strains derived from traditional Japanese pickles. Next, we assessed the strain's ability to induce the expression of IFN-λ genes and interferon-stimulated genes (ISGs), and to produce IFN-λs. As a result, we screened and isolated Enterococcus casseliflavus KB1733 (KB1733) as a potential strain capable of inducing IFN-λ3 expression. Furthermore, we clarified that KB1733 induced the expression of IFN-λ genes and ISGs related to antiviral functions, and that KB1733 induced IFN-λ1 and -λ3 expression in a dose-dependent manner up to 10 µg/mL. In addition, KB1733 significantly increased IFN-λ1 production compared to Enterococcus casseliflavus JCM8723T, which belongs to the same genera and species as KB1733. In conclusion, we isolated a unique LAB strain from traditional Japanese pickles that is capable of stimulating IFN-λ production, although further study is needed to investigate how KB1733 protects against viruses in mice and humans.

A Diagnostic Survey of Aborted Equine Fetuses and Stillborn Premature Foals in Denmark.

Background: Loss of pregnancy in mares can have many different causes, including both infectious and non-infectious conditions. Extrapolation of findings from other studies is often uncertain as the significance of each cause varies across regions. Causes of pregnancy loss in mares have never been thoroughly studied in Denmark, so a prospective cross-sectional cohort study targeting the entire Danish population of pregnant mares was performed over a period of 13 months to obtain knowledge of the significance of individual causes. Fifty aborted or prematurely delivered stillborn fetuses were submitted for necropsy and examined by a panel of diagnostic laboratory methods. Results: Overall, a cause of fetal loss was established for 72% of the examined cases. Most cases (62%) were lost due to a non-infectious cause, of which obstruction of the feto-placental blood circulation due to severe torsion of the umbilical cord was most prevalent. Pregnancy loss due to a variety of opportunistic bacteria, including bacteria not previously associated with abortion in mares, accounted for 12%, while equid alphaherpesvirus (EHV) type 1 was the cause of pregnancy loss in 8% of the cases. EHV type 4 and Chlamydiaceae species were identified in some cases, but not regarded as the cause of fetal loss. Conclusion: Umbilical cord torsion was found to be the most prevalent cause of fetal loss in Danish mares, while infectious causes such as EHV type 1 and streptococci only accounted for a minor proportion of the losses. The study highlights the need for defined criteria for establishing an abortion diagnosis in mares, particularly in relation to EHV types 1 and 4.

Successful treatment of aortic valve endocarditis caused by Enterococcus casseliflavus: a case report.

BACKGROUND: Enterococcus casseliflavus is rarely isolated from human specimens. To the best of our knowledge, there are no reports on its detailed treatment course and prognosis. Here, we present the first known case of E. casseliflavus endocarditis with a detailed treatment course. CASE PRESENTATION: An 86-year-old Japanese woman was transferred to the emergency department with dyspnoea, wheezing, and lumbago. Her medical history included hypertension, chronic kidney disease, idiopathic interstitial pneumonia, and rectal carcinoma. Physical examination revealed expiratory wheezes and a diastolic murmur (Levine 2/6) at the 4th right sternal border. Chest radiography revealed bilateral interstitial opacities and slight cardiac dilatation. Transthoracic echocardiography demonstrated the presence of mobile vegetation with perforation, prolapse, and regurgitation of the aortic valve. With a suspicion of infective endocarditis, we started administering intravenous ampicillin/sulbactam. Thereafter, blood cultures identified E. casseliflavus through matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. The antimicrobial treatment was then switched to ampicillin plus gentamicin. The patient underwent aortic valve replacement on the thirteenth hospital day. She was administered intravenous ampicillin and gentamicin for 6 weeks. The patient was discharged 8 weeks after admission. CONCLUSIONS: Our case demonstrated that E. casseliflavus could cause infective endocarditis, which can be successfully treated with a 6-week regimen of ampicillin and gentamicin in combination with proper surgical treatment.

Effects of Potential Probiotic Enterococcus casseliflavus (EC-001) on Growth Performance, Immunity, and Resistance to Aeromonas hydrophila Infection in Common Carp (Cyprinus carpio).

The effects of different levels of dietary Enterococcus casseliflavus (EC-001), as a potential probiotic, were investigated on the growth performance, hemato-biochemical parameters, immune responses, and resistance to Aeromonas hydrophila infection in common carp (Cyprinus carpio) fingerlings. Accordingly, fish (N = 720; 12.0 ± 0.5 g) were distributed into four treatments receiving different dietary levels of E. casseliflavus, EC-001 (0 [control], 1 × 107, 108, and 109 CFU g-1 feed), for 8 weeks. The fish fed with a diet containing 109 CFU g-1 showed the highest weight gain and specific growth rate, along with the lowest feed conversion ratio, compared with the control group (P < 0.05). Red and white blood cells, hemoglobin, hematocrit, neutrophils, and monocytes significantly increased in the fish fed with 1 × 108 and 109 CFU g-1 (P < 0.05). Dietary inclusion of 1 × 108 and 109 CFU g-1 significantly increased serum total protein, albumin, and immunoglobulin content (P < 0.05). Feeding the fish with 1 × 109 CFU g-1 resulted in a significant increase in serum and skin mucus lysozyme activity compared with the other groups (P < 0.05). Complement component 3 and skin mucus protease activity were also significantly higher in all the fish treated with dietary E. casseliflavus (EC-001) compared with the control group (P < 0.05). The cumulative mortality in the treated fish was lower (ranging from 10 to 22%) than the control group (31%) after challenging the fish with A. hydrophila infection, while the fish fed with E. casseliflavus (EC-001) at 1 × 109 CFU g-1 exhibited the lowest mortality rate (P < 0.05). In conclusion, our results revealed the potential probiotic effects of E. casseliflavus (EC-001) for enhancing growth performance, immunity, and disease resistance of common carp.

Biodegradation of aflatoxin by bacterial species isolated from poultry farms.

Aflatoxins are carcinogenic compounds produced by certain Aspergillus spp and naturally contaminate poultry rations. Exposure to low levels of Aflatoxin B1 (AFB1) in poultry feeds is the second most threatening issue facing the poultry industry in Egypt; it can cause a reduction in growth, egg production, and compromised immune functions, resulting in significant economic loss. Hence, a safe, effective and eco-friendly detoxification method is strongly required. Biological decontamination is a promising approach to reduce aflatoxin levels within threshold limits. This study explores the biodegradation capacity of bacteria isolated from the moldy feed, soil and poultry feces in various poultry farms against AFB1 (100 ppb), G1 (100 ppb), B2 (30 ppb), G2 (30 ppb). Sixty-five bacterial isolates were initially screened using coumarin media with a concentration of (0.01%-0.5%) coumarin. Only one soil isolate (SZ1) grew at the highest concentration (0.5%). Coumarin and Aflatoxin degradation rates of ten promising isolates were measured using spectrophotometry and HPLC. Six isolates reduced AFG1 by more than 90% in the liquid medium, five reduced AFB2 while only four did the same with AFB1& AFG2. Impressively, isolate SZ1 (identified as Pseudomonas fluorescens) exhibited the best degradation capacity to both coumarin and aflatoxin with 100% degradation of AFG1 and 99% degradation of AFB1, AFB2 and AFG2. Biochemical and molecular identification of the ten isolates revealed that they belong to four genera; Bacillus (6), Pseudomonas (2), Enterococcus (1) and Stenotrophomonas (1). Factors affecting Pseudomonas fluorescens SZ1 degradation activity was further investigated. Optimum temperature, time and pH for maximum aflatoxin degradation were at 37 °C, 72 h and 7, respectively. Treatment with proteinase K reduced the degradation activity of G1 (31% ± 1.438), B1 (42% ± 1.438), G2 (19% ± 1.097), and B2 (25% ± 1.732), suggesting that the effective component in aflatoxin degradation may be protein in nature. Our study suggests the biocontrol potential of several different species isolated from poultry farms; B. haynesii, B. licheniformis, B. tequilensis, B. subtilis, B. amyloliquefaciens, Pseudomonas fluorescens, Enterococcus casseliflavus, and Stenotrophomonas maltophilia. The results proposed Pseudomonas fluorescens SZ1 as an excellent candidate for bioremediation and decontamination of aflatoxin in feed matrices. To the best of our knowledge, this is the first report identifying B. haynesii, Enterococcus casseliflavus, B. tequilensis and B. amyloliquefaciens with aflatoxin degradation activity.

Enterococcus casseliflavus Bacteraemia in a Patient with Chronic Renal Disease.

Enterococcus casseliflavus is a rare pathogen that usually causes urinary tract and abdominal infections. Its main characteristics are positive motility, yellow colonies and constitutive low-level resistance to vancomycin. We present a case of E. casseliflavus bacteraemia due to thrombophlebitis at the site of the central venous catheter used for hemodialysis in a renal patient. The biochemical identification of the microorganism was further corroborated by molecular detection of the vanC gene. The patient received antibiotic therapy initially with daptomycin and gentamicin, and then with ampicillin and ceftriaxone. The outcome was cure, and he was released from the hospital after seven weeks afebrile with negative blood cultures.

Culturomics Discloses Anti-Tubercular Enterococci Exclusive of Pulmonary Tuberculosis: A Preliminary Report.

Mycobacterium tuberculosis causes pulmonary tuberculosis, a deadly infection of which the clinical expression and prognosis are not fully understood at the individual level, apart from genetic susceptibility traits. We investigated whether individual gut microbiota may correlate with pulmonary tuberculosis status. Culturomics investigations of gut microbiota in two pulmonary tuberculosis patients and two controls in Burkina Faso found 60 different bacterial species in patients and 97 in controls, including 45 in common. Further analysis of the results at the individual level indicated seven bacteria, including Enterococcus mundtii and Enterococcus casseliflavus, which were exclusively cultured in controls. Blind quantitative PCR-based exploration of faeces samples in two cohorts in Burkina Faso and in France confirmed a nonsignificant association of E. mundtii and E. casseliflavus with controls. Further in vitro explorations found four E. mundtii and E. casseliflavus strains inhibiting the growth of M. tuberculosis strains representative of four different lineages as well as Mycobacterium africanum, Mycobacterium canettii, and Mycobacterium bovis, in an inoculum-dependent manner. Heat-killed E. mundtii or E. casseliflavus were ineffective. These unprecedented observations of direct interactions between gut E. mundtii and E. casseliflavus with M. tuberculosis complex mycobacteria suggest that gut microbiota may modulate the expression of pulmonary tuberculosis.

Processing Wastewaters from Spanish-Style cv. Chalkidiki Green Olives: A Potential Source of Enterococcus Casseliflavus and Hydroxytyrosol.

The purpose of this study was to examine the isolation of indigenous lactic acid bacteria (LAB) with functional properties from Spanish-style cv. Chalkidiki green olive processing wastewaters (GOW). Predominant indigenous LAB could serve as bioaugmentation agents/starter culture for table olives production and protected designation of origin specification. Spontaneous fermentation of fresh GOW over different temperatures (15 °C to 50 °C) and pH values (3.5 to 11.5) for 30 d enabled the isolation/molecular identification of the lactic acid bacterium Enterococcus casseliflavus and the plant-associated bacterium Bacillus amyloliquefaciens subsp. plantarum. E. casseliflavus was found to reduce chemical oxygen demand by 72%. Its resistance to extreme pH values, salinity, and temperature was successfully modeled and the minimum inhibitory concentration of oleuropein against the bacterial growth was determined (0.9 g/L). Furthermore, hydroxytyrosol content was doubled (up to 553 mg/L) after GOW spontaneous fermentation under acidic conditions at 15 °C to 30 °C for 120 d, creating an additional source of input. These results highlight the significance and potential of E. casseliflavus in Spanish-style cv. Chalkidiki green olive processing.

Antimicrobial resistance and genetic relationships of enterococci from siblings and non-siblings Heliconius erato phyllis caterpillars.

BACKGROUND: Studies evaluating bacteria in insects can provide information about host-microorganism-environment interactions. The gut microbial community has a profound effect on different physiological functions of insects. Enterococcus spp. are part of the gut community in humans and other animals, as well as in insects. The presence and antimicrobial resistance profile of enterococci are well studied in different animals; however, data for Heliconius erato phyllis (Lepidoptera: Nymphalidae) do not yet exist. Therefore, the aims of this study were to evaluate the distribution of enterococcal species, their antimicrobial resistance profile and virulence genes, and the genetic relationships between enterococci isolated from fecal samples from sibling and non-sibling H. erato phyllis caterpillars collected from different sites in South Brazil. METHODS: Three H. erato phyllis females were captured (two from a forest fragment and one from an urban area), and kept individually in open-air insectaries. Eggs were collected and caterpillars (siblings and non-siblings) were fed daily with Passiflora suberosa leaves. Fecal samples (n = 12) were collected from fifth-instar caterpillars, inoculated in selective medium, and 15 bacterial colonies were randomly selected from each sample. Enterococci were identified by PCR and MALDI-TOF, analyzed by disk diffusion antimicrobial susceptibility tests, and screened for resistance and virulence genes by PCR. The genetic relationships between the strains were determined using pulsed-field gel electrophoresis (PFGE). RESULTS: A total of 178 enterococci strains were identified: E. casseliflavus (74.15%; n = 132), E. mundtii (21.34%; n = 38), E. faecalis (1.12%; n = 2) and Enterococcus sp. (3.37%; n = 6). High rates of resistance to rifampicin (56%) and erythromycin (31%) were observed; 120 (67.41%) of the isolates showed resistance to at least one antibiotic and six (3.37%) were multidrug-resistant.None of the erythromycin-resistant strains was positive for the erm(B) and msrC genes. The virulence genes esp, ace, and gelE were observed in 35%, 7%, and 1% of the strains, respectively. PFGE separated the enterococci into 22 patterns, four being composed of strains from sibling caterpillars. CONCLUSION: Enterococcus casseliflavus was the dominant species in fecal samples of fifth-instar caterpillars. Resistant enterococci strains may be related to environmental pollution or the resistome. The PFGE analysis showed genetic relationships between some strains, suggesting that the enterococci isolated from fecal samples of the sibling caterpillars might have come from common sources, e.g., via diet (herbivory) and/or vertical transmission (through the egg surface). Further studies will be conducted to better understand the role of Enterococcus in the microbial community of the gastrointestinal tract of these insects, and the mechanisms involved in acquisition and maintenance of enterococci.

Exogenous endophthalmitis caused by Enterococcus casseliflavus: A case report.

BACKGROUND: Endophthalmitis caused by Enterococci is rare, and cases involving vancomycin-resistant enterococci are even rarer. We report the first case of Enterococcus casseliflavus endophthalmitis associated with injury caused by a pig. We also review reported cases of exogenous endophthalmitis caused by Enterococcus casseliflavus and discuss the clinical management and prognosis of this disease. CASE SUMMARY: A 43-year-old man with no previous visual problems complained of endophthalmitis in his left eye following injury caused by a pig. Visual acuity was light perception and B-ultrasonography revealed vitreous opacities with retinal detachment. He was treated with intravitreal vancomycin and ceftazidime after refusing vitrectomy. However, the vitreous opacities and retinal detachment deteriorated and he underwent vitrectomy 5 d post-injury. Intraoperatively, advanced rhegmatogenous and tractional retinal detachment with proliferative vitreoretinopathy were observed. On postoperative day 5, vitreous cultures grew Gram-positive cocci identified as Enterococcus casseliflavus. A 2-wk course of intravenous ampicillin and dexamethasone was commenced. On postoperative day 14, visual acuity improved to hand movement. At 6 mo post-injury, visual acuity improved to 20/667, but optic atrophy was present. CONCLUSION: Systemic administration of linezolid in the treatment of Enterococcus casseliflavus endophthalmitis can improve visual acuity. However, intravitreal amikacin should be considered despite concerns of toxicity when oral linezolid fails to prompt improvement.

Expression and characterization of a recombinant stevioside hydrolyzing ß-glycosidase from Enterococcus casseliflavus.

The demand for steviol glycosides, non-caloric sweet components of Stevia rebaudiana Bertoni (stevia) leaves, has increased considerably as a benefit to enhance human health. However, the supply has remained challenging due to limited production, with the lack of a specific steviol glycoside hydrolyzing enzyme. In this study, a novel ß-glucosidase (EcBgl) from Enterococcus casseliflavus was cloned and expressed in Escherichia coli. An EcBgl consists of 721 amino acids corresponding to a molecular mass of 79.37 kDa. The EcBgl was purified to homogeneity, followed by enzyme characterization. The enzyme showed optimum pH and temperature at 6.0 and 37 °C, and exhibited the kinetic constants kcat/Km for pNPG and kcat/Km for stevioside of 8583 mM-1s-1 and 95.41 mM-1s-1, respectively. When compared to the stevioside hydrolyzing ß-glycosidases previously reported, EcBgl was found to be the most efficient enzyme. EcBgl also rendered hydrolysis of the stevioside to produce rubusoside, a rare steviol glycoside with a pharmaceutical solubilizing property, by cleaving at the glucose moiety. In addition, the enzyme demonstrated substantial resistance against amygdalin, so it served as a potential enzyme in agricultural and pharmaceutical applications.