Study on Biofilm Formation Among Enterococcus Isolates and Association With Their Antibiotic Resistance Patterns.

Background Enterococci are a part of the normal intestinal flora of humans. They have emerged as one of the leading causes of nosocomial infection. The evolved antibiotic resistance mechanisms coupled with the virulence properties of enterococci have made it a successful pathogen. Aim This study aimed to determine the ability of biofilm formation among the clinical enterococci isolates and the antimicrobial resistance pattern of the strains. Materials and methods Clinical samples of patients who attended Saveetha Medical College and Hospital, Chennai, India, over six months. Identification and characterization of Enterococcus species were done using various biochemical tests. Antibiotic susceptibility patterns for each isolate were performed using the Kirby- Bauer disc diffusion method. Results The formation of biofilm formation was detected using the microtiter plate method. In total, 90 Enterococcus species were isolated; Enterococcus faecalis were 63 (70%), Enterococcus faecium were 25 (28%) and Enterococcus gallinarum were 2 (2%)independently. E. faecalis displayed advanced resistance rates compared to other Enterococcus species. Resistance against penicillin was found in 42 strains (47%) and resistance to ampicillin was observed in 39 strains (43%). This was followed by resistance to high-level gentamicin in 35 strains (39%) and resistance to ciprofloxacin in 32 strains (36%). Resistance to vancomycin and linezolid also were noted in some strains. Conclusion Our results indicate that E. faecalis exhibits an increasing rate of antimicrobial resistance but lower biofilm conformation. The unique traits of E. faecalis raise concerns for the associated infections, especially hospital-acquired infections.

Exploring the antimicrobial potential of 4,5,7-trihydroxyflavanone (THF) against vancomycin-resistant Enterococcus gallinarum infections: in vitro and in silico investigations.

Enterococcus gallinarum and other Enterococcus species commonly inhabit the human gastrointestinal tract. While the pathogenicity of Enterococcus gallinarum remains incompletely understood, its infections are alarmingly severein humans, as evidenced by numerous cases. Formerly, Vancomycin was the preferred drug, but recent findings indicate that clinical isolates of Enterococcus gallinarum are resistant, leading to the emergence of vancomycin-resistant enterococci (VRE) strains. The escalation of drug resistance is often linked to overexpressed virulence factors, some of which are implicated in biofilm formation in Enterococcus infections. Henceforth, this research investigates the potential of phytocompounds to combat E. gallinarum infection, employing both in vitro and in silico methodologies. In vitro techniques were employed to assess the efficacy of various phytocompounds, ultimately identifying 4,5,7-trihydroxyflavanone (THF) as particularly effective in inhibiting microbial growth. THF displayed over 80% antibacterial activity at 200 µg/ml against E. gallinarum. Subsequent qualitative and quantitative hemolysin assays implicated hemolysin as a target of THF. Molecular docking analysis of THF and Hemolysin A revealed a strong binding affinity. Notably, residues Asn18, Asp85, and His199 formed hydrogen bonds, while His22 and His86 were involved in robust π-π stacking and π-cation interactions with THF. Overall, this study highlights THF's potential in combating E. gallinarum infections.Communicated by Ramaswamy H. Sarma.

Refractory Peritonitis Related to Peritoneal Dialysis Caused by Enterococcus gallinarum: A Case Report.

A 65-year-old woman with a history of peritoneal dialysis undergoing hemodialysis at our hospital presented with a fever after experiencing gastroenteritis symptoms. She had an implanted peritoneal dialysis catheter for draining chylous ascites. After commencing empirical treatment with meropenem, peritoneal effluent samples revealed an increased white blood cell count, and peritonitis was diagnosed. Enterococcus gallinarum was detected in blood and effluent cultures. Meropenem was changed to vancomycin based on susceptibility testing but subsequently restarted and thereafter changed to ampicillin following exacerbation of peritonitis. Finally, catheter removal led to complete recovery. E. gallinarum is vancomycin-resistant and a rare cause of peritonitis.

Genome sequence of Enterococcus gallinarum AH4, a milk oligosaccharide-degrading strain isolated from suckling rats.

We had previously isolated Enterococcus gallinarum AH4, a strain capable of degrading rat milk oligosaccharides. In this study, we determined the whole-genome sequence of AH4. This whole-genome information will expand our understanding of milk oligosaccharide-mediated symbioses between bacteria and host mammals.

Identification and Characterization of a Novel α-L-Fucosidase from Enterococcus gallinarum and Its Application for Production of 2'-Fucosyllactose.

2'-fucosyllactose (2'FL) is an important nutrient in human milk that stimulates beneficial microbiota and prevents infection. α-L-fucosidase is a promising component for 2'FL synthesis. In this study, a soil-oriented α-L-fucosidase-producing strain from Enterococcus gallinarum ZS1 was isolated. Escherichia coli was employed as a host for cloning and expressing the α-L-fucosidase gene (entfuc). The EntFuc was predicted as a member of the GH29 family with a molecular mass of 58 kDa. The optimal pH and temperature for the activity of EntFuc were pH 7.0 and 30 °C, respectively. The enzyme exhibited a strictly specific activity for 4-Nitrophenyl-α-L-fucopyranoside (pNP-Fuc) and had a negligible effect on hydrolyzing 2'FL. EntFuc could catalyze the synthesis of 2'FL via transfucosylation action from pNP-Fuc and lactose. The yield of 2'FL reached 35% under optimal conditions. This study indicated that EntFuc with a high conversion rate is a promising enzyme source for the biosynthesis of 2'FL.

Detoxification of p-nitrophenol (PNP) using Enterococcus gallinarum JT-02 isolated from animal farm waste sludge.

Enterococcus gallinarum (JT-02) isolated and identified from the animal farm waste sludge was found to be capable of biodegrading p-nitrophenol (PNP), an organic compound used to manufacture drugs, fungicides, insecticides, dyes, and to darken leather. The intention of this study was to optimize the biodegradation by finding the optimal conditions for the specific strain through single-factor experiments. The bacterial strain was grown in Luria Bertani broth and various parameters were optimized to achieve the prime settings for the p-nitrophenol (PNP) biodegradation. The results indicated that the best setups for the biodegradation by the strain JT-02 was 100 mg/L of PNP; pH 7; 30 °C; 150 rpm in a shaker incubator and 3% (v/v) of inoculum dose. Once the optimal conditions were found, the bacteria were capable of degrading p-nitrophenol (98.21%) in 4 days. Intermediates produced during PNP biodegradation were identified using High Performance Liquid Chromatography (HPLC) analysis and the biodegradation pathway was elucidated. Phytotoxicity studies were carried out with Vigna radiata seeds to confirm the applicability and efficiency of PNP biodegradation.

Isolation and characterization of a novel Enterococcus phage Phi_Eg_SY1.

Enterococcus gallinarum, a gut pathobiont, is an opportunistic pathogen that carries the risk of antibiotic resistance in the clinic and has been proven to drive autoimmunity in both mice and humans. Screening for novel bacteriophages targeting Enterococcus gallinarum is expected to provide a promising strategy for controlling such infections or regulating related chronic diseases. In the present study, we isolated a novel lytic Enterococcus gallinarum phage, Phi_Eg_SY1, which presents favourable thermostability and pH stability. Further assays indicated that Phi_Eg_SY1 can efficiently adsorb and lyse the host bacteria in vitro. Genomic and phylogenetic analyses suggested that Phi_Eg_SY1 does not contain virulence or lysogeny genes and presents a novel unassigned evolutionary lineage among the related dsDNA phages. Phi_Eg_SY1 is therefore considered to be suitable for further applications.

Isolation and screening of microorganisms for high yield of succinic acid production.

This study involves the isolation of succinic acid (SA)-producing microorganisms from different samples, including the rumen, sludge, soil, and wastewater. For primary screening, 29 isolates exhibited a zone of clearance around the colony, indicating acid production. For secondary screening using thin-layer chromatography, only two isolates symbolized SA production according to their Rf values. These two isolates were further identified as Bacillus velezensis and Enterococcus gallinarum by phylogenetic analysis using the neighbor-joining method. The high SA concentrations of 50.2 and 66.9 g/L were produced by B. velezensis and E. gallinarum with an SA yield of 0.836 and 1.12 g/g glucose, respectively. The high SA concentration from these newly isolated strains was achieved with a low formation of unwanted acids compared with those from Actinobacillus succinogenes ATCC 55618. Moreover, E. gallinarum was cultured in palm oil mill wastewater (POMW) and molasses, which were cheap substrates. The high SA production of 73.9 g/L with low other acids (the ratio of SA to total acids = 0.917) was achieved using POMW and molasses (80:20) as substrates.

Enterococcus gallinarum group meningitis after transanal migration of the ventriculoperitoneal shunt: a pediatric case report.

In the literature, only 11 Enterococcus gallinarum group meningitis has been reported so far. The Enterococcus gallinarum group was shown for the first time in a pediatric patient presenting with meningitis after bowel perforation, a complication of a ventriculoperitoneal shunt. A 30-month-old male patient presented with vomiting and fever, with the ventriculoperitoneal shunt distal catheter protruding from the anal orifice. The patient was diagnosed with intestinal perforation and meningitis. Enterococcus gallinarum group bacterial yield in cerebrospinal fluid culture. A total of 6 weeks of intravenous antibiotic treatment was given in the hospital. After the treatment, the patient was re-ventriculoperitoneal shunt and was discharged. Among the shunt complications, meningitis with intestinal perforation is rare. It should be kept in mind that meningitis in such patients may be caused by very rare microbiological factors such as Enterococcus gallinarum group. Antibiotherapy should be given according to the culture result. Then planning should be made for a permanent shunt.

Isolation and identification of Enterococcus gallinarum P581a, a strain of intestinal bacteria deglycosylating flavone C-glycosides.

Flavone C-glycosides are not easily degraded because of their strong C-C bond between sugar moieties and aglycones. However, some bacteria such as intestinal species can produce specific enzymes to degrade them. In this study, a bacterial strain P581a, which is capable of deglycosylating flavone C-glycosides, was isolated from human intestinal bacteria and was identified as Enterococcus gallinarum by morphological examination, physiological and biochemical analysis and 16S rRNA gene sequencing. This strain may produce a specific flavonoside glycosidase. The activity of the enzyme in the culture medium containing different quantity of carbon sources was also studied, and it was found that the content of carbon sources is negatively correlated with the deglycosylation efficiency of this strain.

Enterococcus gallinarum causing cervical vertebral osteomyelitis: Imagery detecting the process of rapid progression of degeneration-like change in 3 months.

We present a series of images of X-rays and MRI of vertebral osteomyelitis caused by Enterococcus gallinarum in a 65-year-old patient with persistent neck pain and fever accompanied by preceding transient biliary enzymes elevation. Images detected progression of degeneration-like changes of C5-7 in three months, which is too rapid for true degeneration and relatively slow for vertebral osteomyelitis of common pathogens. Though initial imagery evaluation detected merely degenerative change, the patient was followed up monthly because of persistent fever. Three months later, the images detected the typical imagery of vertebral osteomyelitis i.e., the destruction of vertebral bone: narrowing of intervertebral spaces with focal osteosclerosis and osteolysis on C5-7 became prominent. At this point, consultation to general internal medicine was made. With grade 3 regurgitation murmur, transthoracic echocardiography was performed and revealed 14-mm-in-diameter vegetation on aortic valve. Blood cultures detected Enterococcus gallinarum of which suspected entry was biliary tract. No previous case reports of Enterococcus gallinarum referred to vertebral osteomyelitis. While this case showed a typical clinical course of infective endocarditis, the course of progression of vertebral osteomyelitis and perhaps endocarditis was much slower comparing to common pathogens. This might reflect the relatively non-life-threatening features of this organism. Enterococcus gallinarum shows mild resistance to vancomycin and cephalosporins, initial therapy sometimes fails. Enterococcus gallinarum should be newly added to pathogenic candidates of vertebral osteomyelitis especially when feverish patients shows back or neck pain with preceding biliary tract problems.

Milk oligosaccharide-mediated cross-feeding between Enterococcus gallinarum and lactobacilli in the gut microbiota of infant rats.

We investigated bacteria that have a nutritional symbiotic relationship with respect to milk oligosaccharides in gut microbiota of suckling rats, with specific reference to sialyllactose (SL) degrading Enterococcus gallinarum. Our next generation sequencing analysis of the colonic contents of 12-day-old suckling rats revealed that almost half of the bacteria in the microbiota belonged to the Lactobacillaceae family. Major Lactobacillus species in the contents were identified as L. johnsonii, L. murinus, and L. reuteri. We then monitored changes in numbers of the above Lactobacillus species, E. gallinarum, and the bacteria belonging to the family Enterobacteriaceae (i.e., enterobacteria) in the colonic contents of infant rats at 7, 12, 21, 28, and 35 days of age by using real-time PCR assays targeting these bacterial groups. The 7-day-old infant rats had a gut microbiota in which enterobacteria were predominant. Such dominance was replaced by L. johnsonii and the concomitant E. gallinarum markedly increased in those of 12 and 21 days of ages. During this period, the number of enterobacteria declined dramatically, but that of L. reuteri surged dramatically. Our separate in vitro experiment showed that supplementation of culture media with SL promoted the growth of L. johnsonii and E. gallinarum, with marked production of lactic acid. These findings revealed possible milk oligosaccharide-mediated cross-feeding between E. gallinarum and L. johnsonii, with the former degrading SL to release lactose to be utilized by the latter.

Non-Alcoholic Pearl Millet Beverage Innovation with Own Bioburden: Leuconostoc mesenteroides, Pediococcus pentosaceus and Enterococcus gallinarum.

The appropriate solution to the problem of quality variability and microbial stability of traditional non-alcoholic pearl millet fermented beverages (NAPMFB) is the use of starter cultures. However, potential starter cultures need to be tested in the production process. We aimed to identify and purify bioburden lactic acid bacteria from naturally fermented pearl millet slurry (PMS) and assess their effectiveness as cultures for the production of NAPMFB. Following the traditional Kunun-zaki process, the PMS was naturally fermented at 37 °C for 36 h. The pH, total titratable acidity (TTA), lactic acid bacteria (LAB), total viable count (TVC) and the soluble sugar were determined at 3 h interval. The presumptive LAB bacteria were characterized using a scanning electron microscope, biochemical tests and identified using the VITEK 2 Advanced Expert System for microbial identification. The changes in pH and TTA followed a non-linear exponential model with the rate of significant pH decrease of 0.071 h-1, and TTA was inversely proportional to the pH at the rate of 0.042 h-1. The Gompertz model with the mean relative deviation modulus, 0.7% for LAB and 2.01% for TVC explained the variability in microbial growth during fermentation. The LAB increased significantly from 6.97 to 7.68 log cfu/mL being dominated by Leuconostoc, Pediococcus, Streptococcus and Enterococcus with an optimum fermentation time of 18 h at 37 °C and 4.06 pH. L. mesenteroides and P. pentosaceus created an acidic environment while E. gallinarum increased the pH of the pearl millet extract (PME). Innovative NAPMFB was produced through assessment of LAB from PMS to PME fermented with L. mesentoroides (0.05%) and P. pentosaceus (0.025%) for 18 h, thereby reducing the production time from the traditional 24 h.

Isolation and identification of milk oligosaccharide-degrading bacteria from the intestinal contents of suckling rats.

We report the isolation of bacteria capable of degrading milk oligosaccharides from suckling infant rats. The bacteria were successfully isolated via a selective enrichment method, in which the serially diluted intestinal contents of infant rats were individually incubated in an enrichment medium containing 3'-sialyllactose (3'-SL), followed by the isolation of candidate strains from streaked agar plates and selection of 3'-SL-degrading strains using thin-layer chromatography. Subsequent genomic and phenotypic analyses identified all strains as Enterococcus gallinarum. The strains were capable of degrading both 3'-SL and 6'-SL, which was not observed with the type strain of E. gallinarum used as a reference. Furthermore, a time-course study combining high-performance anion-exchange chromatography with pulsed amperometric detection revealed that the representative strain AH4 degraded 3'-SL completely to yield an equimolar amount of lactose and an approximately one-fourth equimolar amount of sialic acid after 24 hr of anaerobic incubation. These findings point to a possibility that the enterococci degrade rat milk oligosaccharides to "cross-feed" their degradants to other members of concomitant bacteria in the gut of the infant rat.

Enterococcus gallinarum endophthalmitis and meningitis in an allogeneic hematopoietic stem cell transplant patient: A case report and literature review.

Endogenous endophthalmitis caused by Enterococcus gallinarum, an organism with intrinsic resistance to vancomycin, has rarely been reported. We present a case of persistent E. gallinarum bacteremia in a female recipient of hematopoietic stem cell transplant (HSCT) complicated by endophthalmitis and meningoventriculitis, resulting in a fatal outcome despite treatment with intravenous ampicillin and daptomycin. Treatment of endophthalmitis often presents a challenge due to the lack of options for antimicrobials with reliable ocular penetration. Therapeutic decisions can become particularly complex with the involvement of drug-resistant pathogens and host characteristics that limit the choice of antimicrobials due to drug toxicity. This case illustrates a rare manifestation of an opportunistic pathogen.

L'endophtalmite endogène causée par l'Enterococcus gallinarum, un organisme ayant une résistance intrinsèque à la vancomycine, est rarement déclarée. Les auteurs présentent un cas de bactériémie à E. gallinarum persistante chez une femme après une greffe de cellules souches hématopoïétiques compliquée par une endophtalmite et une méningoventriculite dont l'issue a été fatale malgré un traitement à l'ampicilline et à la daptomycine par voie intraveineuse. Il est souvent difficile de traiter l'endophtalmite, à cause du manque d'antimicrobiens ayant une pénétration oculaire fiable. Les décisions thérapeutiques peuvent devenir particulièrement complexes en raison des agents pathogènes pharmacorésistants et des caractéristiques de l'hôte qui limitent le choix d'antimicrobiens, dont la toxicité est élevée. Ce cas illustre une manifestation rare d'agent pathogène opportuniste.

Whole genome sequencing data of a clinical Enterococcus gallinarum strain EGR748 from Sabah, Malaysia.

Enterococcus gallinarum is a gram positive facultatively anaerobic bacteria that is typically found in mammalian intestinal tracts. It is generally not considered pathogenic to humans and is rarely reported. Here, we present the draft genome sequence data of Enterococcus gallinarum strain EGR748 isolated from a human clinical sample, and sequenced using the Illumina HiSeq 4000 system. The estimated whole genome size of the strain was 3,730,000 bp with a G + C content of 40.43%. The de novo assembly of the genome generated 55 contigs with an N50 of 208,509 bp. In addition, the Maximum Likelihood phylogenetic analysis based on the 16S rRNA sequence data accurately clustered EGR748 with other E. gallinarum strains. The data may be useful to demonstrate the capacity of this enterococcal species becoming the causal agents of nosocomial blood-stream infections. The genome dataset has been deposited at DDBJ/ENA/GenBank under the accession number JAABOR000000000.

Whole genome sequence of Enterococcus gallinarum EG81, a porcine strain harbouring the oxazolidinone-phenicol resistance gene optrA with chromosomal and plasmid location.

OBJECTIVE: The aim of this study was to characterise the whole genome sequence of linezolid-intermediate Enterococcus gallinarum strain EG81 of swine origin in China. METHODS: Whole genome of EG81 was sequenced using Illumina MiSeq platform combined with the Nanopore PromethION platform, and assembled de novo using Canu v1.5. NCBI Prokaryotic Genome Annotation Pipeline (PGAP) was used to annotate the genome of EG81. Antimicrobial resistance genes were identified using CGE ResFinder 3.2. RESULTS: The genome of EG81 consists of one 3,433,237-bp chromosome and two plasmids, pEG81-1 (51,632 bp) and pEG81-2 (3425 bp). A total of 3285 coding sequences and 80 RNA genes were predicted by PGAP. The oxazolidinone-phenicol resistance gene optrA is located on both the chromosome and plasmid pEG81-1 associated with Tn554 and Tn558, respectively. In addition, EG81 harbours vanC1XY (vancomycin resistance), fexA (phenicol), dfrG (trimethoprim), aadD, ant(6)-Ia and ant(9)-Ia (aminoglycoside), erm(A) and erm(B) (macrolide), and tet(L) and tet(M) (tetracycline). CONCLUSION: Here, we first report the oxazolidinone-phenicol gene optrA in E. gallinarum that is intrinsically resistant to vancomycin, which poses a great threat to public health. The genome sequence of E. gallinarum EG81 provides valuable information for the dissemination of optrA among vancomycin-resistant enterococci.

Enterococcus gallinarum Causing Native Valve Endocarditis.

Endocarditis due to Enterococcus gallinarum is a rare condition, usually affecting older patients. The most frequent source of infection is the gastrointestinal or genitourinary tracts; it frequently involves the aortic valve and tends to produce heart failure. We present a case of Enterococcus gallinarum endocarditis developing on a normal native heart valve. Enterococcus gallinarum is intrinsically resistant to vancomycin. Antibiotic susceptibility patterns indicate that most isolates are penicillin and ampicillin-susceptible. LEARNING POINTS: Endocarditis due to Enterococcus gallinarum is rare.Enterococcus gallinarum is intrinsically resistant to vancomycin.Most isolates are penicillin and ampicillin-susceptible.

Enterococcus gallinarum meningitis: a case report and literature review.

BACKGROUND: As an opportunistic pathogen, E. gallinarum mainly leads to nosocomial infections, and it's multi-drug resistance has gained more and more attention. Central nervous system infections caused by E. gallinarum are rare, but have been reported more often in recent years. The previous cases were generally secondary to neurosurgery, especially ventriculoperitoneal shunts. In recent years, the cases largely occurred in patients with impaired immune function. The patient in our report may have had dual risk factors (immune impairment and an invasive surgical procedure). CASE PRESENTATION: The patient, a 35-year-old female, was admitted to our hospital for headaches of 3 days duration accompanied by nausea and vomiting for 2 days. The patient had fevers and chills for 3 days before admission; the peak body temperature was 38.5 °C. The patient had a splenectomy in our hospital 2 years earlier for thrombocytopenia and was thought to be immunocompromised. The abnormal findings on physical examination and laboratory testing were as follows: neck stiffness, present; lumbar puncture: pressure, 300 mmH2O; Pandy's test, positive; white blood cell (WBC) count, 1536 × 106/L; monocyte count, 602 × 106/L; monocyte percentage, 39.2%; multinucleate cell count, 934 × 106/L; multinucleate cell percentage, 60.8%; protein, 1.08 g/L; WBC count, 21.1 × 109/ L; neutrophil percentage, 85.3%; neutrophil count, 20.55 × 109/L; C reactive protein (CRP): 136.4 mg/L; procalcitonin, 6.70 ng/mL. The patient was given meropenem (2.0 g, intravenous infusion, every 8 h) for anti-infection supplemented with other symptomatic support treatments. The patient's fever and headache had no significant relief. CONCLUSIONS: Central nervous system infections caused by E. gallinarum are rare, but should be suspected, particularly inpatients with impaired immune function or ineffective treatment. Avoiding long-term invasive treatment and improving immunity are helpful to reduce the occurrence of E. gallinarum infections. Early detection and diagnosis, as well as rational antibiotic use, are the keys to achieve satisfactory efficacy.