Elemental Analysis and In Vitro Evaluation of Antibacterial and Antifungal Activities of Usnea barbata (L.) Weber ex F.H. Wigg from Călimani Mountains, Romania

This study aims to complete our research on Usnea barbata (L.) Weber ex F.H. Wigg (U. barbata) from the Călimani Mountains, Romania, with an elemental analysis and to explore its antibacterial and antifungal potential. Thus, we analyzed twenty-three metals (Ca, Fe, Mg, Mn, Zn, Al, Ag, Ba, Co, Cr, Cu, Li, Ni, Tl, V, Mo, Pd, Pt, Sb, As, Pb, Cd, and Hg) in dried U. barbata lichen (dUB) by inductively coupled plasma mass spectrometry (ICP-MS). For the second study, we performed dried lichen extraction with five different solvents (ethyl acetate, acetone, ethanol, methanol, and water), obtaining five U. barbata dry extracts (UBDE). Then, using an adapted disc diffusion method (DDM), we examined their antimicrobial activity against seven bacterial species—four Gram-positive (Staphylococcus aureus, Enterococcus casseliflavus, Streptococcus pyogenes, and Streptococcus pneumoniae) and three Gram-negative (Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa)—and two fungi species (Candida albicans and Candida parapsilosis). Usnic acid (UA) was used as a positive control. The ICP-MS data showed a considerable Ca content (979.766 µg/g), followed by, in decreasing order, Mg, Mn, Al, Fe, and Zn. Other elements had low levels: Ba, Cu, Pb, and Cr (3.782–1.002 µg/g); insignificant amounts (<1 µg/g) of Hg and V were also found in dUB. The trace elements Ag, As, Cd, Co, Li, Tl, Mo, Pd, Pt, and Sb were below detection limits (<0.1 µg/g). The DDM results—expressed as the size (mm) of the inhibition zone diameter (IZs)—proved that the water extract did not have any inhibitory activity on any pathogens (IZs = 0 mm). Gram-positive bacteria displayed the most significant susceptibility to all other UBDE, with Enterococcus casseliflavus showing the highest level (IZs = 20–22 mm). The most susceptible Gram-negative bacterium was Pseudomonas aeruginosa (IZs = 16–20 mm); the others were insensitive to all U. barbata dry extracts (IZs = 0 mm). The inhibitory activity of UBDE and UA on Candida albicans was slightly higher than on Candida parapsilosis.

Novel genomic islands and a new vanD-subtype in the first sporadic VanD-type vancomycin resistant enterococci in Norway

Background Vancomycin-resistant enterococci (VRE) represent several types of transferable vancomycin resistance gene clusters. The vanD type, associated with moderate to high level vancomycin resistance, has only sporadically been described in clinical isolates. The aim of this study was to perform a genetic characterization of the first VanD-type VRE strains detected in Norway. Methods The VanD-type VRE-strains (n = 6) from two patient cases were examined by antimicrobial susceptibility testing and whole genome sequencing (WGS) to uncover Van-phenotype, strain phylogeny, the vanD gene clusters, and their genetic surroundings. The putative transferability of vanD was examined by circularization PCR and filter mating. Results The VanD-type Enterococcus faecium (n = 4) and Enterococcus casseliflavus (n = 2) strains recovered from two cases (A and B), expressed moderate to high level vancomycin resistance (MIC 64—>256 mg/L) and various levels of teicoplanin susceptibility (MIC 2—>256 mg/L). WGS analyses revealed phylogenetically different E. faecium strains (A1, A2, and A3 of case A and B1 from case B) as well as vanD gene clusters located on different novel genomic islands (GIs). The E. casseliflavus strains (B2 and B3 of case B) were not clonally related, but harbored nearly identical novel GIs. The vanD cluster of case B strains represents a novel vanD-subtype. All the vanD-GIs were integrated at the same chromosomal site and contained genes consistent with a Clostridiales origin. Circular forms of the vanD-GIs were detected in all strains except B1. Transfer of vanD to an E. faecium recipient was unsuccessful. Conclusions We describe the first VanD-type E. casseliflavus strains, a novel vanD-subtype, and three novel vanD-GIs with a genetic content consistent with a Clostridiales order origin. Despite temporal occurrence, case A and B E. faecium strains were phylogenetically diverse and harbored different vanD subtypes and vanD-GIs.

MILKING ANTISEPTICS AGAINST BACTERIA MULTIDRUG-RESISTANT BOVINE MASTITIS

This work aims to verify the in vitro action of antiseptics used in the pre- and post-dipping against multidrug-resistant bacteria from bovine mastitis. Antiseptic solutions in the concentration of use of iodine, chlorhexidine and sodium hypochlorite were tested against Staphylococcus aureus (n=12), Staphylococcus sciuri (n=1), Staphylococcus lentus (n=1), Streptococcus sp. (n=1), Enterococcus faecalis (n=1), Enterococcus casseliflavus (n=1), Kocuria kristinae (n=2), Kocuria varians (n=1). Was verified the Minimal Inhibitory and Bactericidal Concentration. The cytotoxicity test complemented the work. It was found that the Minimal Inhibitory Concentration and Minimal Bactericidal Concentration of the iodine, sodium hypochlorite and chlorhexidine was effective in the values recommended for use, however the presence of organic material reduced the activity of the tested products. Chlorhexidine showed higher efficiency of the other disinfectants.

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.

Monocular loss of vision following an open tibial fracture: a case of Enterococcus casseliflavus endogenous endophthalmitis

Rare intraocular complication in a healthy immunocompetent patient with an atypical organism following presumed haematogenous spread after a highly contaminated open tibial fracture.

Genomic Based Characterization of Enterococcus Spp-An Emerging Pathogen Isolated from Human Gut

Background Enterococci are ubiquitous microorganisms having diverse ecological niches but mostly prominently in gastrointestinal tract of humans and animals. Production of enterocins make them used as probiotics, but in last few years their role as probiotic become ambiguous. This ambiguity in their probiotic role is related to presence of virulence factors and antibiotic resistance genes. Moreover, these virulence traits are also known to be transfer genetically which make them opportunistic pathogens in gastrointestinal track. These reports suggest serious concerns related to enterococcus before using them as probiotics. In present study Whole-genome sequencing (WGS) of Enterococcus spp was done for checking presence of resistance and virulence genes, isolated from human gut.Methods and resultsFour human origin Enterococcus spp including Enterococcus faecalis, Enterococcus casseliflavus, and two Enterococcus gallinarum were isolated from human fecal samples, further cultured on blood and MacConkey agar. Sanger sequencing was done using Applied Biosystems 3730xl DNA Analyzer. These strains were further subjected to WGS using oxford nano pore technology MinION. Raw data was analyzed using free online tool epi2me. The Comprehensive Antibiotic Resistance Database (CARD) and RAST software’s were used to look for presence of antibiotic resistance genes in these strains. Resistance determinants for clinically important antibiotics (vancomycin) and functional virulence factor genes were detected. G-view server was used for comparative genomics of all strains.Conclusion:The draft genomic sequencing of enterococcus suggested that Enterococcus faecalis, Enterococcus casseliflavus and Enterococcus gallinarum strains are opportunistic pathogens, having antibiotic resistance genes. All isolates have vancomycin resistance genes which they also expressed phenotypically. Some genes related to bacteriocin resistance were also present in E. casseliflavus and E. gallinarum.

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.