Clinical Isolates Of Enterococcus Faecalis Research Articles

Characterization and antimicrobial activity of fungal endophytes from Crocus caspius (Iridaceae)

Plant endophytes are known as one of the newest natural sources for producing specialized metabolites and novel antibiotics. In the present study, endophytic fungi isolated from the corms of Crocus caspius Fisch. & C.A.Mey. ex Hohen. (Iridaceae) were characterized for the first time. Six fungal endophytes were isolated and their antibacterial activity was studied against human pathogens including Staphylococcus aureus ATCC 25923, Escherichia coli ATCC 25922, Enterococcus faecium (Clinical isolate), and Candida albicans ATCC 10231 by determination of the minimum inhibitory-minimum bactericidal concentration (MIC-MBC) assays. Thin-layer chromatography (TLC)-bioautography technique was applied to investigate the antifungal activity of the methanolic extracts of fungal endophytes against ochratoxin A-producing fungus, Aspergillus ochraceus Strain 168. Based on morphological and ITS1-5.8S-ITS2 sequencing analysis, isolated endophytes from the plant corms were identified as Xenoacremonium falcatus (SBU-1), Fusarium oxysporum (SBU-2), Cadophora luteo-olivacea (SBU-3), Phialocephala sp. (SBU-4) and Talaromyces purpureogenus (SBU-5 and SBU-6). The extracts of T. purpureogenus SBU-6 (MIC = 1 mg/ml) and Phialocephala sp. SBU-4 (MIC = 0.25 mg/ml) showed a considerable antibacterial activity against E. coli and S. aureus, respectively. The obtained extract from SBU-5 showed the highest antifungal activity (MIC = 4 mg/ml) against C. albicans. Furthermore, both SBU-2 and SBU-5 at a concentration of 16 mg/ml showed bactericidal and fungicidal effects against E. coli and C. albicans. The extracts of SBU-2, SBU-4, and SBU-5 showed significant antifungal activity against A. ochraceous. These findings could be interesting for further exploitation of these endophytes to produce antibiotics and biological fungicides.

Comparison of antibacterial activities and resistance mechanisms of omadacycline and tigecycline against Enterococcus faecium.

This study aims to compare the antimicrobial activity of omadacycline with tigecycline against clinical isolates of Enterococcus faecium and investigate their resistance mechanisms. Non-duplicate clinical E. faecium isolates (n = 224) were collected and the minimal inhibitory concentrations (MICs) of omadacycline and tigecycline were determined by broth microdilution method. The tet genes and the genetic mutations in 16 S rRNA genes and 30 S ribosomal protein S10 were determined by PCR and sequence alignment. The global protein abundances of the omadacycline-induced and parent isolates were determined by a Q Exactive plus mass spectrometer. The MIC50/MIC90 of omadacycline and tigecycline against the 224 E. faecium isolates were 0.25/0.5 mg l-1 and 0.125/0.25 mg l-1, respectively. Among these E. faecium isolates, the frequency of the isolates with omadacycline MICs ≥ 0.25 mg l-1 were significantly higher than that with tigecycline MICs ≥ 0.25 mg l-1. Moreover, the T1473C and/or G1468A mutations in the 16 S rRNA and Lys98Glu mutation in the 30 S ribosomal protein S10 were identified in the 3 series of tigecycline or omadacycline- nonsusceptible isolates selected in vitro. The abundances of 32 proteins changed in the omadacycline-induced isolate, of which 10 increased and 22 decreased. The abundance of tet(M) increased significantly in the omadacycline-induced isolate, and the abundance of proteins included in cellular process and metabolic process decreased. In conclusion, Omadacycline and tigecycline exhibits excellent activities against clinical isolates of E. faecium and exposure to omadacycline and tigecycline can result in significant cross-resistance to both antibiotics. The high-level expression of tet(M) in E. faecium may confer resistance to omadacycline.

A pharmacokinetic-pharmacodynamic (PKPD) model-based analysis of tedizolid against enterococci using the hollow-fibre infection model

Tedizolid is a novel oxazolidinone antibiotic. Considering the higher antibacterial effect in immunocompetent compared with immunosuppressed animals, it is not recommended in immunocompromised patients. In this study, we assessed the 'pure' pharmacokinetic-pharmacodynamic (PKPD) relationship for tedizolid against Enterococcus in the hollow-fibre infection model (HFIM). Unbound plasma concentration time profiles (200-5000 mg/day IV) were simulated in the HFIM over 120 h against an Enterococcus faecalis strain and two clinical isolates of Enterococcus faecium (VRE-vanB and VRE-vanA). Next, a PKPD model describing tedizolid efficacy against bacterial isolates was developed. A population PK model was linked to the developed PKPD model and utilized to predict the bacterial kinetics in plasma and in target tissues [adipose, muscle, epithelial lining fluid (ELF) and sputum] over 120 h of therapy. The PKPD model adequately described the bacterial kill kinetics for all bacterial populations. At the human recommended dose of 200 mg/day, bacterial growth was predicted in plasma and all tissues, except for ELF. Bacteriostasis was observed only at a higher dose of 1200 mg/day over 120 h. An fAUC/MIC of 80 related to stasis over 120 h. Subpopulations resistant to 3 × MIC were amplified in plasma and target tissues, except for ELF, at doses of 200-800 mg/day. The human dose of 200 mg/day was insufficient to suppress bacterial growth in the HFIM, indicating that further components contribute to the clinical effect of tedizolid. This study supports the warning/precaution for tedizolid to limit its use in immunocompromised patients.

In vitro activity of tigecycline and proteomic analysis of tigecycline adaptation strategies in clinical Enterococcus faecalis isolates from China

This study aimed to investigate the in vitro activities of tigecycline (TGC) and the underlying molecular mechanisms of TGC stress response and resistance in clinical Enterococcus faecalis isolates from China. Antimicrobial susceptibility and antibiofilm activities of TGC in 399 E. faecalis isolates were evaluated. Heteroresistance was evaluated by population analysis profiling. Resistance and heteroresistance mechanisms were investigated by identifying genetic mutations in tetracycline (tet) target sites and through analysis of efflux protein inhibitors (EPIs). Furthermore, quantitative proteomics was used to investigate the global proteomic response of E. faecalis to TGC stress, as well as the resistance mechanisms of TGC within in vitro induced resistant isolate. TGC minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates were ≤0.5 mg/L. TGC displayed remarkable inhibitory activity against biofilm formation. The occurrence rate of TGC heteroresistance was 1.75% (7/399), and the increased TGC MIC values of heteroresistance-derived clones could be reversed by EPI. TGC resistance was associated with mutations in the 16S rRNA site or 30S ribosomal protein S10. A total of 105 and 356 differentially expressed proteins was identified after being exposed to 1/2× MIC concentrations of TGC, while 356 differentially expressed proteins was identified in TGC-resistant isolate. The differentially expressed proteins were enriched in the translation and DNA replication process. In addition, multiple adenosine triphosphate (ATP)-binding cassette (ABC) transporters were upregulated. TGC exhibited excellent activity against a substantial proportion of clinical isolates from China. However, E. faecalis exhibited a strong adaptation mechanism during TGC exposure: mutation of TGC target sites and elevated expression of efflux pumps under TGC selection, resulting in TGC resistance.

Human Plasma Significantly Reduces Bacteriophage Infectivity Against Staphylococcus aureus Clinical Isolates.

Bacteriophage therapy has been regaining interest as a potential therapeutic in treating a wide range of infections. However, there is a paucity of knowledge regarding numerous aspects of bacteriophage therapy, thereby hindering the development of proper treatment protocols and effective clinical trials. In this report, the activities of three bacteriophages are evaluated against clinical bacterial isolates in the presence and absence of human plasma (HP). The bacteriophages used in this experiment were residual therapeutic doses from the United States Food and Drug Administration (FDA) approved compassionate use cases to treat recalcitrant prosthetic joint infections (PJIs). Herein we demonstrate that in the presence of HP, the infectivity of these Staphylococcal bacteriophages was significantly reduced compared to the infectivity in the absence of HP. Inhibition of infectivity ranged from 48% to 81% for two methicillin-resistant Staphylococcus aureus (MRSA) clinical isolates independently infected with the same bacteriophage and 98% for a third MRSA clinical isolate infected with a different bacteriophage. In contrast, bacteriophage infectivity of an Enterococcus faecalis clinical isolate was not affected by the presence of HP. We hypothesize that the inhibition is correlated with plasma proteins binding to Staphylococcal surface proteins masking the receptors associated with bacteriophage attachment, thereby reducing infectivity. This has clinical ramifications for bacteriophage therapy use in treating Staphylococcal bacteremia and periprosthetic joint infections.

Impact of PBP4 Alterations on β-Lactam Resistance and Ceftobiprole Non-Susceptibility Among Enterococcus faecalis Clinical Isolates.

Penicillin-resistance among Enterococcus faecalis clinical isolates has been recently associated with overexpression or aminoacidic substitutions in low-affinity PBP4. Ceftobiprole (BPR), a new-generation cephalosporin, is a therapeutic option against E. faecalis. Here, we present evidence that pbp4 gene sequence alterations may influence the expression level of the gene and ceftobiprole binding to PBP4 in E. faecalis clinical isolates showing remarkable MDR-phenotypes, and how this could interfere with BPR in vitro antibacterial and bactericidal activity. Seven E. faecalis strains from bloodstream infections were analyzed for their antibiotic and β-lactam resistance. BPR bactericidal activity was assessed by time-kill analysis; pbp4 genes were sequenced and pbp4 relative expression levels of transcription were performed by RT-qPCR. Five penicillin-resistant ampicillin-susceptible (PRAS) isolates were detected, 4 of which were also BPR non-susceptible (BPR-NS). In the time-kill experiments, BPR exposure resulted in a potent bactericidal activity (3-5 log10 reduction) at the different concentrations tested. pbp4 gene sequence analysis revealed some mutations that may account for the changes in PBP4 affinity and MIC increase in the 4 BPR-NS strains (MICs 4-16 mg/L): the deletion of an adenine (delA) in the promoter region in all PRAS/BPR-NS strains; 12 different amino acid substitutions, 7 of which were next to the PBP catalytic-sites. The most significant were: T418A, located 6 amino acids (aa) upstream of the catalytic-serine included in the 424STFK427 motif I; L475Q, 7 aa upstream of the 482SDN484 motif II; V606A and the novel Y605H, 13/14 aa upstream of the 619KTGT622 motif III. Taken together, our data showed that elevated BPR MICs were attributable to increased transcription of pbp4 - associated with a single upstream adenine deletion and PBP4 alterations in the catalytic-site motifs – which might interfere with the formation of the BPR/PBP4 complex. pbp4 molecular alterations may account for the changes in PBP4 affinity and MIC increase, without affecting BPR cidal activity. Indeed, our in vitro dynamic analysis by time-kill assays showed that BPR exerted a bactericidal activity against E. faecalis clinical isolates, despite their MDR phenotypes.

Antimicrobial and Antibiofilm Activity of Biosynthesized Silver Nanoparticles Against Beta-lactamase-Resistant Enterococcus faecalis.

Due to the presence of antibiotic-resistant genes, treatment options of clinical isolates are exceedingly limited. This study was aimed to fabricate, optimize, characterize, and evaluate the action of silver nanoparticles (AgNPs) against a clinical isolate of Enterococcus faecalis. A combination of cell-free supernatant (C-FS) of the filamentous fungus Fusarium solani and Gram-negative Comamonas aquatica for AgNP formation was proposed; the antigrowth and antibiofilm of AgNPs against E. faecalis harboring blaTEM and blaCTX-M genes were assessed. The ratio of 1:2 v/v (C-FS:AgNO3) at pH 9.0 for 72h in 1mM AgNO3 were the optimal conditions for AgNP formation. UV-vis absorption peak appeared at 425nm and the crystalline nature of synthesized particles was verified by X-ray diffraction (XRD). Fourier transform infrared spectroscopy (FTIR) analysis confirmed the interaction of protein molecules with the AgNPs. Transmission electron microscopy (TEM) analysis demonstrated that fabricated AgNPs were relatively monodispersed, approximately spherical, and of size 2-7.5nm. blaTEM and blaCTX-M were detected in E. faecalis; the growth and biofilm of E. faecalis were significantly decreased by the action of 12.5μg/mL AgNPs. This is the first study proposing alternative sources to form AgNPs via synergistic metabolites of F. solani and C. aquatica. The results here offer a foundation for developing an effective therapy using AgNPs against clinical pathogens.

Ciprofloxacin-Tethered 1,2,3-Triazole Conjugates: New Quinolone Family Compounds to Upgrade Our Antiquated Approach against Bacterial Infections.

A newer ciprofloxacin series containing 1,2,3-triazole conjugates of ciprofloxacin was designed, synthesized, and well characterized using modern analytical techniques by reacting diversified anilines with ciprofloxacin obtained from ciprofloxacin hydrochloride. The newer conjugates were evaluated for their antimicrobial activity against various strains, viz. Staphylococcus aureus (ATCC25923), Enterococcus faecalis (clinical isolate), Staphylococcus epidermidis (ATCC3594), Escherichia coli (ATCC25922), Pseudomonas aeruginosa (ATCC27853), Salmonella typhi (clinical isolate), Salmonella typhimurium (clinical isolate), Acinetobacter baumannii (ATCC19606), Aeromonas hydrophila (ATCC7966), Plesiomonas shigelloides (ATCC14029), and Sphingo biumpaucimobilis (MTCC6362) in vitro. Interestingly, some of the conjugates showed superior antimicrobial activity as compared to the control drug ciprofloxacin. The three compounds 4i, 4j, and 4n showed strong activity with minimum inhibitory concentration (MIC) 0.78 μM, while the compound 4g showed MIC 1.56 μM against S. typhi (clinical). The compound 4a showed good efficacy against S. aureus (ATCC25923) and S. typhi (clinical) with MIC 3.12 μM, while the compound 4b exhibited efficacy with MIC 3.12 μM against S. aureus (ATCC25923) and the control drug ciprofloxacin showed MIC 6.25 μM. Among all of the synthesized compounds, 4e, 4f, 4g, 4h, 4p, 4q, 4t, and 4u displayed less than 20% hemolysis, while the rest of the compounds showed hemolysis in the range of 21–48%. Moreover, the structure of compound 4b was also established by single-crystal X-ray diffraction studies.

Detection of vancomycin variable enterococci (VVE) among clinical isolates of Enterococcus faecium collected across India-first report from the subcontinent

PurposeEmergence of vancomycin variable enterococci (VVE) poses a challenge to empiric vancomycin therapy. Vancomycin-variable enterococci (VVE) are vanA-positive, yet phenotypically vancomycin-susceptible enterococci that can switch to a vancomycin-resistant phenotype when exposed to vancomycin. The aim of the present study was to determine the prevalence of VVE in India. MethodsIsolates of phenotypically vancomycin susceptible Enterococcus faecium from 20 tertiary care hospitals across India were collected and tested for the presence of vanA, vanR, vanS, vanB and vanC genes by conventional PCR using previously published primers. Isolates positive for vanA gene were considered as VVE. ResultsThe prevalence of VVE was 1.5% (5/340). Only one VVE isolate was positive for vanR and vanS, and all the isolates were negative for vanB and vanC. ConclusionsAlthough the prevalence is low, our finding emphasizes the importance of routinely screening for van genes in enterococci that are phenotypically susceptible. Silenced vanA able to escape detection and revert to resistance during vancomycin therapy represents a new challenge in clinical settings.

Inhibitory Effects of Carvacrol on Biofilm Formation and Expression of Biofilm Related Genes in Clinical Isolates of Enterococcus faecalis.

Nowadays, novel antimicrobial strategies are being developed which focus on debilitating, rather than killing the microorganisms. In this regard, anti-biofilm therapy is one of the important ways to combat bacterial infections. Therefore, the aim of the current study was to evaluate the anti-biofilm activity of Carvacrol against E. faecalis by means of its effects on biofilm formation as well as on the gene expression levels of the two biofilm related genes, Epa and Esp. A total of 40 clinical strains of E. faecalis were collected from three hospitals in Tehran, Iran during 2020. These isolates were confirmed by biochemical and genotypic methods. Antibacterial and anti-biofilm activity of Carvacrol essence were determined according the standard protocol. Finally, expression level of the biofilm related genes (Epa and Esp) were evaluated before and after the treatment with Carvacrol. A total of 14 isolates were considered as strong biofilm producers and were used for analysis. Carvacrol essence showed the best antibacterial activity at 2,500 μg/mL concentration against all the isolates, the biofilm formation capacity was decreased by Carvacrol essence, and it was statistically significant (p < 0.05). Expression levels of the Esp gene were decreased in 5 isolates while increased in 3 isolates following the Carvacrol treatment. Ex-pression levels of the EpaI gene was significantly decreased (p < 0.05) in 4 isolates following the Carvacrol treatment. In conclusion, the results presented in this study suggest that carvacrol extract exhibits significant antimicrobial and anti-biofilm properties against E. faecalis, even against vancomycin resistant isolates.

Sesquiterpene Lactones from Cotula cinerea with Antibiotic Activity against Clinical Isolates of Enterococcus faecalis.

Cotula cinerea, belonging to the tribe Anthemideae, is a plant widespread in the Southern hemisphere. It is frequently used in folk medicine in North African countries for several of its medical properties, shown by its extracts and essential oils. The dichloromethane extract obtained from its aerial parts demonstrated antibiotic activity against Enterococcus faecalis and was fractionated by bioguided purification procedures affording five main sesquiterpene lactones. They were identified by spectroscopic methods (NMR and ESIMS data) as guaiantrienolides, i.e., 6-acetoxy-1β-,6-acetoxy-1α-, and 6-acetoxy-10-β-hydroxyguaiantrienolide (1–3), and germacrenolides, i.e., haagenolide and 1,10-epoxyhaagenolide (4 and 5). The absolute configuration was assigned by applying the advanced Mosher’s method to haagenolide and by X-ray diffraction analysis to 1,10-epoxyhaagenolide. The specific antibiotic and antibiofilm activities were tested toward the clinical isolates of Enterococcus faecalis. The results showed that compounds 3–5 have antibacterial activity against all the strains of E. faecalis, while compound 2 exhibited activity only toward some strains. Compound 1 did not show this activity but had only antibiofilm properties. Thus, these metabolites have potential as new antibiotics and antibiofilm against drug resistant opportunistic pathogens.

Whole-genome sequencing analysis reveals the spread of a vanB-carrying transposon among different vancomycin-resistant Enterococcus faecium clinical isolates in a non-endemic setting

Vancomycin-resistant enterococci (VRE), in particular Enterococcus faecium (VREfm), can cause serious nosocomial infections, and have been responsible for healthcare-associated outbreaks. Spreading of VREfm can occur both clonally and by the dissemination of mobile genetic elements. In this study, we report on prospective analysis of WGS data, including both cgMLST and transposon analysis during a vanB VREfm outbreak. Screening for vanB positive VREfm isolates was performed by real-time PCR on an overnight enriched broth and, if positive, subculture was performed. vanB positive VREfm isolates were whole-genome sequenced. Generated data were used for molecular typing that was performed by cgMLST using SeqSphere. For transposon characterization, sequence data were mapped against the reference sequence of transposon Tn1549 using CLC Genomics workbench or de novo assemblies were used for BLASTN comparisons. A total of 1358 real-time PCRs were performed. Two hundred fifty-one specimens from 207 patients tested PCR positive for vanB, of which 13 specimens obtained from 6 different patients were identified as vanB VREfm positive by culture. These 6 patients harbored 7 unique isolates, belonging to 4 different cluster types: CT118 (n=2), CT2483 (n=3), CT2500 and CT2501. Transposon analysis revealed the presence of an identical vanB-carrying transposon in the isolates cultured from all 6 patients, that could be linked based on epidemiological data. A vanB VREfm outbreak in our hospital occurred, including 6 patients with isolates belonging to 4 different cluster types. In-depth transposon analysis revealed dissemination of the transposon Tn1549 rather than clonal spread being the cause of the outbreak.

Enterococcus phage Nonaheksakonda infecting clinical isolates of Enterococcus faecalis represents a new lineage in the family Siphoviridae.

Enterococcus phage Nonaheksakonda was isolated from wastewater, using a vancomycin-resistant strain of the opportunistic pathogen Enterococcus faecalis (VRE) as a host. Nonaheksakonda is a lytic phage infecting E. faecalis V583 and clinical isolates with at least four different multi-locus sequence types (MLSTs). The genome is a 41.9-kb double-stranded DNA molecule (34.6% GC) with 74 coding sequences. Comparative analysis revealed only one close relative, Enterococcus phage heks. All other phages had low protein similarity and shared less than 54% nucleotide sequence identity with phage Nonaheksakonda. The most similar phages were all classified and unclassified efquatroviruses. We propose that the phages Nonaheksakonda and heks represent a novel genus within the family Siphoviridae, order Caudovirales, for which we propose the name "Nonaheksakondavirus".

Design, synthesis, and antibacterial activity of N-(trifluoromethyl)phenyl substituted pyrazole derivatives.

Design and synthesis of N-(trifluoromethyl)phenyl substituted pyrazole derivatives and their potency as antimicrobial agents are described. Several of these novel compounds are effective growth inhibitors of antibiotic-resistant Gram-positive bacteria and prevent the development of biofilms by methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus faecalis. These compounds eradicated the preformed biofilms effectively and were found to be more effective than the control antibiotic vancomycin. Potent compounds showed low toxicity to cultured human embryonic kidney cells with a selectivity factor of >20. The most promising compound is very potent against meropenem, oxacillin, and vancomycin-resistant clinical isolates of Enterococcus faecium. Investigations into the mode of action by performing macromolecular synthesis inhibition studies showed a broad range of inhibitory effects, suggesting targets that have a global effect on bacterial cell function.

Nosiheptide analogues as potential antibacterial agents via dehydroalanine region modifications: Semi-synthesis, antimicrobial activity and molecular docking study

The frequent and inappropriate use of antibiotics aggravate the variation and evolution of multidrug-resistant bacteria, posing a serious threat to public health. Nosiheptide (NOS) has excellent lethality against a variety of Gram-positive bacteria, however the physical and chemical drawbacks hamper its routine application in clinical practice. In this study, by using NOS as the starting material, a total of 15 NOS analogues (2a-4e) were semi-synthesized via its dehydroalanine residue reacting with monosubstituted anilines. In vitro antimicrobial susceptibilities of NOS and its analogues against two methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecium (VRE) clinical isolates were determined by broth microdilution assay to determine the minimum inhibitory concentration (MIC). Antimicrobial susceptibility testing data shown that most of the NOS analogues had a better antibacterial effect than the parent compound, with compound 3c exhibiting the highest antibacterial activity against VRE (MIC = 0.0078 mg/L) and MRSA (MIC < 0.0039 mg/L). Molecular docking of synthetic compounds was also performed to verify the binding interactions of NOS analogues with the target. Our data indicated that compound 3c possesses stronger and more complex intermolecular force than other analogues, which is consistent with the results of the biological activity evaluation. Overall, this study identified a number of potential antibacterial NOS analogues that could act as potent therapeutic agents for multidrug-resistant bacterial infections.

Biofilm Formation on Breast Implant Surfaces by Major Gram-Positive Bacterial Pathogens.

Bacterial biofilm on surfaces of mammary implants is a predisposing factor for several outcomes. Because Gram-positive bacteria are potential agents of biomaterial-associated infections (BAIs), their abilities to form biofilm on breast implants should be elucidated. The aim of this study was to evaluate biofilm formation on different mammary prosthesis surfaces by major Gram-positive bacterial pathogens involved in BAIs. We initially evaluated biofilm formation on polystyrene plates with and without fibrinogen or collagen for 1 reference strain and 1 clinical isolate of Enterococcus faecalis, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. We also tested the ability of clinical isolates to form biofilm on 4 different implant surfaces: polyurethane foam and smooth, microtextured, and standard textured silicone. Biofilm structure and cell viability were observed by scanning electron microscopy and confocal laser scanning microscopy. All strains showed strong biofilm formation on polystyrene. After fibrinogen or collagen treatment, biofilm formation varied. With fibrinogen, reference strains of S. aureus and S. pyogenes increased biofilm formation (P < 0.05). Reference strains of all species and the clinical isolate of S. pyogenes increased biofilm formation after collagen treatment (P < 0.05). In general, S. aureus showed higher capacity to produce biofilm. Scanning electron microscopy showed that biofilm attached to all surfaces tested, with the presence of extracellular polymeric substances and voids. Viable cells were more frequent for E. faecalis and S. pyogenes. All species produced biofilm on all prosthesis surfaces and under different conditions. Micrographies indicated thicker bacterial biofilm formation on microtextured and/or standard textured silicone by all species, except E. faecalis.

Comparative In Vitro Activities of Ceftaroline and Tedizolid against Clinical Strains of Staphylococcus aureus and Enterococcus: Results from the China Antimicrobial Surveillance Network (CHINET) in 2018.

The in vitro activities of ceftaroline and tedizolid were compared against Staphylococcus aureus, Enterococcus faecalis, and Enterococcus faecium clinical isolates collected from the China Antimicrobial Surveillance Network. Ceftaroline demonstrated potent activity against S. aureus isolates (MIC50/90, ≤0.25/1 mg/liter). Tedizolid was also highly active against S. aureus (MIC50/90, 0.25/0.5 mg/liter) and Enterococcus (MIC50/90, 0.5/0.5 mg/liter) isolates. Our results support the clinical usefulness of ceftaroline and tedizolid in treating Gram-positive infections.

Attenuated Total Reflection Fourier Transform Infrared Spectroscopy combined with chemometric modelling for the classification of clinically relevant Enterococci.

Attenuated Total Reflection Fourier Transform Infrared (ATR-FT-IR) Spectroscopy and chemometric modelling, including soft independent modelling by class analogy (SIMCA), partial least squares discriminant analysis (PLS-DA) and support vector machine (SVM), were applied to attempt to discriminate 60 clinical isolates of Enterococcus faecium and Enterococcus faecalis and hence evaluate the performance of the spectroscopic approach in identifying enterococci infections. The bacterial samples were identified by polymerize chain reaction (PCR) amplification and their ATR-FT-IR spectra acquired. Spectra were processed to the second derivative using the Savitzky-Golay algorithm and normalized using extended multiplicative signal correction employing the UnscramblerX (CAMO, Norway) software package. Multivariate classification models and their performance were evaluated using Cohen's Kappa coefficient. Principal component analysis (PCA) score plots showed separate clusters of spectra related to membership to E. faecium and E. faecalis, with this explained by bands assigned to PO2 (1230cm-1 ), P-O-C (1114cm-1 ), monosubstituted alkene (997, 987cm-1 ) and C-O (1070, 1055, 1036cm-1 ) corresponding to teichoic acids, polysaccharides and peptidoglycan from the cell wall in PCA and PLS-DA loading plots. The best classification model for E. faecium and E. faecalis is SVM, indicating via highest Kappa score. The classification coefficient between SIMCA, PLS-DA, SVM and PCR as reference method were 0·59, 0·9 and 1, respectively, shown as the Kappa scores. The main spectral differences observed between the two clinically relevant enterococci species were associated with changes in the teichoic acid content of cell walls. With regard to the binary classification method, SVM was found to be the best performing classification model, providing the highest correlation with the PCR results. The study shows that ATR-FT-IR spectroscopy in combination with chemometric modelling can be applied for the phenotypic identification and discrimination of clinically relevant and similar enterococcal species.

Drug Resistance Determinants in Clinical Isolates of Enterococcus faecalis in Bangladesh: Identification of Oxazolidinone Resistance Gene optrA in ST59 and ST902 Lineages.

Enterococcus faecalis is one of the major causes of urinary tract infection, showing acquired resistance to various classes of antimicrobials. The objective of this study was to determine the prevalence of drug resistance and its genetic determinants for E. faecalis clinical isolates in north-central Bangladesh. Among a total of 210 E. faecalis isolates, isolated from urine, the resistance rates to erythromycin, levofloxacin, and gentamicin (high level) were 85.2, 45.7, and 11.4%, respectively, while no isolates were resistant to ampicillin, vancomycin and teicoplanin. The most prevalent resistance gene was erm(B) (97%), and any of the four genes encoding aminoglycoside modifying enzyme (AME) were detected in 99 isolates (47%). The AME gene aac(6′)-Ie-aph(2”)-Ia was detected in 46 isolates (21.9%) and was diverse in terms of IS256-flanking patterns, which were associated with resistance level to gentamicin. Tetracycline resistance was ascribable to tet(M) (61%) and tet(L) (38%), and mutations in the quinolone resistance-determining region of both GyrA and ParC were identified in 44% of isolates. Five isolates (2.4%) exhibited non-susceptibility to linezolide (MIC, 4 μg/mL), and harbored the oxazolidinone resistance gene optrA, which was located in a novel genetic cluster containing the phenicol exporter gene fexA. The optrA-positive isolates belonged to ST59, ST902, and ST917 (CC59), while common lineages of other multiple drug-resistant isolates were ST6, ST28, CC16, and CC116. The present study first revealed the prevalence of drug resistance determinants of E. faecalis and their genetic profiles in Bangladesh.

Molecular and functional analysis of the novel cfr(D) linezolid resistance gene identified in Enterococcus faecium.

To characterize the novel cfr(D) gene identified in an Enterococcus faecium clinical isolate (15-307.1) collected from France. The genome of 15-307.1 was entirely sequenced using a hybrid approach combining short-read (MiSeq, Illumina) and long-read (GridION, Oxford Nanopore Technologies) technologies in order to analyse in detail the genetic support and environment of cfr(D). Transfer of linezolid resistance from 15-307.1 to E. faecium BM4107 was attempted by filter-mating experiments. The recombinant plasmid pAT29Ωcfr(D), containing cfr(D) and its own promoter, was transferred to E. faecium HM1070, Enterococcus faecalis JH2-2 and Escherichia coli AG100A. As previously reported, 15-307.1 belonged to ST17 and was phenotypically resistant to linezolid (MIC, 16 mg/L), vancomycin and teicoplanin. A hybrid sequencing approach confirmed the presence of several resistance genes including vanA, optrA and cfr(D). Located on a 103 kb plasmid, cfr(D) encoded a 357 amino acid protein, which shared 64%, 64%, 48% and 51% amino acid identity with Cfr, Cfr(B), Cfr(C) and Cfr(E), respectively. Both optrA and cfr(D) were successfully co-transferred to E. faecium BM4107. When expressed in E. faecium HM1070 and E. faecalis JH2-2, pAT29Ωcfr(D) did not confer any resistance, whereas it was responsible for an expected PhLOPSA resistance phenotype in E. coli AG100A. Analysis of the genetic environment of cfr(D) showed multiple IS1216 elements, putatively involved in its mobilization. Cfr(D) is a novel member of the family of 23S rRNA methyltransferases. While only conferring a PhLOPSA resistance phenotype when expressed in E. coli, enterococci could constitute an unknown reservoir of cfr(D).