Clinical Enterococcus Faecium Research Articles

Complete genome sequence of the linezolid-resistant clinical Enterococcus faecalis N23-3408 linked to a livestock lineage in Switzerland.

Here, we report the complete genome of human clinical linezolid-resistant Enterococcus faecalis N23-3408. N23-3408 harbored a 59.5 kb plasmid with antimicrobial resistance genes cat, erm(B), fexA, optrA, tet(L), and tet(M). Closely related E. faecalis harboring this plasmid was previously obtained from livestock animals and pet food in Switzerland.

Vancomycin heteroresistance caused by unstable tandem amplifications of the vanM gene cluster on linear conjugative plasmids in a clinical Enterococcus faecium.

Vancomycin heteroresistance is prone to missed detection and poses a risk of clinical treatment failure. We encountered one clinical Enterococcus faecium strain, SRR12, that carried a complete vanM gene cluster but was determined as susceptible to vancomycin using the broth microdilution method. However, distinct subcolonies appeared within the clear zone of inhibition in the E-test assay, one of which, named SRR12-v1, showed high-level resistance to vancomycin. SRR12 was confirmed as heteroresistant to vancomycin using population analysis profiling and displayed "revive" growth curves with a lengthy lag phase of over 13 hours when exposed to 2-32 mg/L vancomycin. The resistant subcolony SRR12-v1 was found to carry an identical vanM gene cluster to that of SRR12 but a significantly increased vanM copy number in the genome. Long-read whole genome sequencing revealed that a one-copy vanM gene cluster was located on a pELF1-like linear plasmid in SRR12. In comparison, tandem amplification of the vanM gene cluster jointed with IS1216E was seated on a linear plasmid in the genome of SRR12-v1. These amplifications of the vanM gene cluster were demonstrated as unstable and would decrease accompanied by fitness reversion after serial passaging for 50 generations under increasing vancomycin pressure or without antibiotic pressure but were relatively stable under constant vancomycin pressure. Further, vanM resistance in resistant variants was verified to be carried by conjugative plasmids with variable sizes using conjugation assays and S1-pulsed field gel electrophoresis blotting, suggesting the instability/flexibility of vanM cluster amplification in the genome and an increased risk of vanM resistance dissemination.

Comparative genomic analysis of clinical Enterococcus faecalis distinguishes strains isolated from the bladder

BackgroundEnterococcus faecalis is the most commonly isolated enterococcal species in clinical infection. This bacterium is notorious for its ability to share genetic content within and outside of its species. With this increased proficiency for horizontal gene transfer, tremendous genomic diversity within this species has been identified. Many researchers have hypothesized E. faecalis exhibits niche adaptation to establish infections or colonize various parts of the human body. Here, we hypothesize that E. faecalis strains isolated from the human bladder will carry unique genomic content compared to clinical strains isolated from other sources.ResultsThis analysis includes comparison of 111 E. faecalis genomes isolated from bladder, urogenital, blood, and fecal samples. Phylogenomic comparison shows no association between isolation source and lineage; however, accessory genome comparison differentiates blood and bladder genomes. Further gene enrichment analysis identifies gene functions, virulence factors, antibiotic resistance genes, and plasmid-associated genes that are enriched or rare in bladder genomes compared to urogenital, blood, and fecal genomes. Using these findings as training data and 682 publicly available genomes as test data, machine learning classifiers successfully distinguished between bladder and non-bladder strains with high accuracy. Genes identified as important for this differentiation were often related to transposable elements and phage, including 3 prophage species found almost exclusively in bladder and urogenital genomes.ConclusionsE. faecalis strains isolated from the bladder contain unique genomic content when compared to strains isolated from other body sites. This genomic diversity is most likely due to horizontal gene transfer, as evidenced by lack of phylogenomic clustering and enrichment of transposable elements and prophages. Investigation into how these enriched genes influence host-microbe interactions may elucidate gene functions required for successful bladder colonization and disease establishment.

Characterization of a non-functional erm(T) gene in a clinical Enterococcus faecium strain.

Characterization of a non-functional erm(T) gene in a clinical Enterococcus faecium strain.

Genetic characterization of MDR genomic elements carrying two aac(6')-aph(2″) genes in feline-derived clinical Enterococcus faecalis isolate.

Multidrug-resistant Enterococcus faecalis (E. faecalis) often cause intestinal infections in cats. The aim of this study was to investigate a multidrug-resistant E. faecalis isolate for plasmidic and chromosomal antimicrobial resistance and their genetic environment. E. faecalis strain ESC1 was obtained from the feces of a cat. Antimicrobial susceptibility testing was carried out using the broth microdilution method. Conjugation experiments were performed using Escherichia coli and Staphylococcus aureus as receptors. Complete sequences of chromosomal DNA and plasmids were generated by whole genome sequencing (WGS) and bioinformatics analysis for the presence of drug resistance genes and mobile elements. Multidrug-resistant E. faecalis ESC1 contained a chromosome and three plasmids. The amino acid at position 80 of the parC gene on the chromosome was mutated from serine to isoleucine, and hence the amino acid mutation at this site led to the resistance of ESC1 strain to fluoroquinolones. Eleven antibiotic resistance genes were located on two plasmids. We identified a novel composite transposon carrying two aminoglycoside resistance genes aac(6')-aph(2″). This study reported the coexistence of a novel 5.4 kb composite transposon and a resistance plasmid with multiple homologous recombination in an isolate of E. faecalis ESC1. This data provides a basis for understanding the genomic signature and antimicrobial resistance mechanisms of this pathogen.

Occult Vancomycin-Resistant Enterococcus faecium ST117 Displaying a Highly Mutated vanB2 Operon

Rare information is available on clinical Enterococcus faecium encountered in Sardinia, Italy. This study investigated the antimicrobial susceptibility profiles and genotypic characteristics of E. faecium isolated at the University Hospital of Sassari, Italy, using the Vitek2 system and PCR, MLST, or WGS. Vitek2 revealed two VanB-type vancomycin-resistant Enterococcus faecium (VREfm) isolates (MICs mg/L = 8 and ≥32) but failed to detect vancomycin resistance in one isolate (MIC mg/L ≤ 1) despite positive genotypic confirmation of vanB gene, which proved to be vancomycin resistant by additional phenotypic methods (MICs mg/L = 8). This vanB isolate was able to increase its vancomycin MIC after exposure to vancomycin, unlike the "classic" occult vanB-carrying E. faecium, becoming detectable by Vitek 2 (MICs mg/L ≥ 32). All three E. faecium had highly mutated vanB2 operons, as part of a chromosomally integrated Tn1549 transposon, with common missense mutations in VanH and VanB2 resistance proteins and specific missense mutations in the VanW accessory protein. There were additional missense mutations in VanS, VanH, and VanB proteins in the vanB2-carrying VREfm isolates compared to Vitek2. The molecular typing revealed a polyclonal hospital-associated E. faecium population from Clade A1, and that vanB2-VREfm, and nearly half of vancomycin-susceptible E. faecium (VSEfm) analyzed, belonged to ST117. Based on core genome-MLST, ST117 strains had different clonal types (CT), excluding nosocomial transmission of specific CT. Detecting vanB2-carrying VREfm isolates by Vitek2 may be problematic, and alternative methods are needed to prevent therapeutic failure and spread.

Determination of Enterococcus faecalis and Enterococcus faecium Antimicrobial Resistance and Virulence Factors and Their Association with Clinical and Demographic Factors in Kenya.

Enterococci are clinically significant because of their increasing antibiotic resistance and their ability to cause severe infections due to an arsenal of virulence genes. Few studies in the developing world have examined virulence factors that may significantly impact patient outcomes. This study describes the antimicrobial resistance profiles and prevalence of five key Enterococcal virulence genes gelE, asa, cylA, esp, and hyl in forty-four clinical Enterococcus faecalis and E. faecium isolates in Kenya and their association with patients' demographic and clinical characteristics. All E. faecium isolates were obtained from hospital-acquired skin and soft tissue infections. While E. faecalis was associated with community-acquired urinary tract infections. All isolates were resistant to erythromycin, whereas 11/44 (27.5%), 25/44 (56.8%), 28/44 (63.6%), 37/44 (84.1%), 40/44 (90.0%), and 43/44 (97.5%) were susceptible to tetracycline, levofloxacin, gentamicin, ampicillin, nitrofurantoin, and teicoplanin, respectively. All isolates were susceptible to tigecycline, vancomycin, and linezolid. There was little difference in the antibiotic resistance profiles between E. faecalis and E. faecium. The prevalence of the virulence genes among the 44 isolates were 27 (61.4%) for gelE, 26 (59.1%) for asa1, 16 (36.3%) for esp, 11 (25.0%) for cylA, and 1 (2.3%) for hyl. 72.9% of E. faecalis isolates had multiple virulence genes compared to 57% of E. faecium isolates with no virulence genes. The hyl gene was only detected in E. faecium, while cylA and asa1 were only detected in E. faecalis. A significant correlation was observed between the presence of asa1 and esp virulence genes and tetracycline resistance (P=0.0305 and 0.0363, respectively). A significant correlation was also observed between the presence of virulence genes gelE and asa1 and nitrofurantoin resistance (P=0.0175 and 0.0225, respectively) and ampicillin resistance (P=0.0005 and 0.0008, respectively). The study highlights the high levels of erythromycin resistance in E. faecalis and E. faecium, the demographic factors influencing the species distribution among patients, and the accumulation of multiple virulence genes in E. faecalis. The significant association of gelE, asa1, and esp virulence genes with drug resistance could explain the pathogenic success of E. faecalis and provides a guide for future studies.

In vitro activity and adaptation strategies of eravacycline in clinical Enterococcus faecium isolates from China.

Eravacycline (Erava) is a synthetic fluorocycline with potent antimicrobial activity against a wide range of Gram-positive bacteria. This study aimed to investigate the in vitro antimicrobial activity and resistance mechanism of Erava in clinical E. faecium isolates from China. Erava minimum inhibitory concentrations (MICs) against clinical E. faecium isolates-including those resistant to linezolid (LZD) or harboring the tetracycline (Tet) resistance genes was ≤0.25 mg l-1. Moreover, our data indicated that clinical isolates of E. faecium with Erava MIC 0.25 mg l-1 were predominantly shown to belong to Sequence-type 78 (ST78) and ST80. The prevalence of Erava heteroresistance in clinical E. faecium strain was 2.46% (3/122). The increased Erava MIC values of heteroresistance-derived E. faecium clones could be significantly reduced by efflux pump inhibitors (EPIs). Furthermore, comparative proteomics results showed that efflux pumps lmrA, mdlA, and mdlB contributed significantly to the acquisition of Erava resistance in E. faecium. In addition, a genetic mutation in 16 S rRNA (G190A) were detected in resistant E. faecium isolates induced by Erava. In summary, Erava exhibits potent in vitro antimicrobial activity against E. faecium, but mutation of Tet target sites and elevated expression of efflux pumps under Erava selection results in Erava resistance.

New Mutations in cls Lead to Daptomycin Resistance in a Clinical Vancomycin- and Daptomycin-Resistant Enterococcus faecium Strain

Daptomycin (DAP), a last-resort antibiotic for treating Gram-positive bacterial infection, has been widely used in the treatment of vancomycin-resistant enterococci (VRE). Resistance to both daptomycin and vancomycin leads to difficulties in controlling infections of enterococci. A clinical multidrug-resistant Enterococcus faecium EF332 strain that shows resistance to both daptomycin and vancomycin was identified, for which resistance mechanisms were investigated in this work. Whole-genome sequencing and comparative genomic analysis were performed by third-generation PacBio sequencing, showing that E. faecium EF332 contains four plasmids, including a new multidrug-resistant pEF332-2 plasmid. Two vancomycin resistance-conferring gene clusters vanA and vanM were found on this plasmid, making it the second reported vancomycin-resistant plasmid containing both clusters. New mutations in chromosomal genes cls and gdpD that, respectively, encode cardiolipin synthase and glycerophosphoryl diester phosphodiesterase were identified. Their potential roles in leading to daptomycin resistance were further investigated. Through molecular cloning and phenotypic screening, two-dimensional thin-layer chromatography, fluorescence surface charge test, and analysis of cardiolipin distribution patterns, we found that mutations in cls decrease surface negative charges of the cell membrane (CM) and led to redistribution of lipids of CM. Both events contribute to the DAP resistance of E. faecium EF332. Mutation in gdpD leads to changes in CM phospholipid compositions, but cannot confer DAP resistance. Neither mutation could result in changes in cellular septa. Therefore, we conclude that the daptomycin resistance of E. faecium EF332 is conferred by new cls mutations. This work reports the genetic basis for vancomycin and daptomycin resistance of a multidrug-resistant E. faecium strain, with the finding of new mutations of cls that leads to daptomycin resistance.

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.

Metabolomics and Genomics Approach for the Discovery of Serrawettin W2 Lipopeptides from Serratia marcescens NP2.

A metabolomics/peptidomics and genomics approach, using UPLC-MSE, molecular networking, and genome mining, was used to describe the serrawettin W2 lipopeptide family produced by Serratia marcescens NP2. Seven known serrawettin W2 analogues were structurally elucidated along with 17 new analogues, which varied based on the first (fatty acyl length of C8, C10, C12, or C12:1), fifth (Phe, Tyr, Trp, or Leu/Ile), and sixth (Leu, Ile, or Val) residues. Tandem MS results suggested that the previously classified serrawettin W3 may be an analogue of serrawettin W2, with a putative structure of cyclo(C10H18O2-Leu-Ser-Thr-Leu/Ile-Val). Chiral phase amino acid analysis enabled the distinction between l/d-Leu and l-Ile residues within nine purified compounds. 1H and 13C NMR analyses confirmed the structures of four purified new analogues. Additionally, genome mining was conducted using Serratia genome sequences available on the NCBI database to identify the swrA gene using the antiSMASH software. NRPSpredictor2 predicted the specificity score of the adenylation-domain within swrA with 100% for the first, second, and third modules (Leu-Ser-Thr), 60-70% for the fourth module (Phe/Trp/Tyr/Val), and 70% for the fifth module (Val/Leu/Ile), confirming MSE data. Finally, antibacterial activity was observed for compounds 6 and 11 against a clinical Enterococcus faecium strain.

Distinguishing Clinical Enterococcus faecium Strains and Resistance to Vancomycin Using a Simple In-House Screening Test.

Vancomycin-resistant enterococci (VRE) are a major concern as microorganisms with antimicrobial resistance and as a public health threat contributing significantly to morbidity, mortality, and socio-economic costs. Among VREs, vancomycin-resistant Enterococcus faecium (VREfm) is frequently isolated and is resistant to many antibiotics used to treat patients with hospital-acquired infection. Accurate and rapid detection of VREfm results in effective antimicrobial therapy, immediate patient isolation, dissemination control, and appropriate disinfection measures. An in-house VREfm screening broth was developed and compared to the broth microdilution method and multiplex polymerase chain reaction for the detection of 105 enterococci, including 81 VRE isolates (61 E. faecium, 5 E. faecalis, 10 E. gallinarum, and 5 E. casseliflavus). Verification of this screening broth on 61 VREfm, 20 other VRE, and 24 non-VRE revealed greater validity for VREfm detection. The accuracy of this broth was 100% in distinguishing E. faecium from other enterococcal species. Our test revealed 93.3% accuracy, 97.5% sensitivity, and 79.2% specificity compared with broth microdilution and PCR detecting van genes. The kappa statistic to test interrater reliability was 0.8, revealing substantial agreement for this screening test to the broth microdilution method. In addition, the in-house VREfm screening broth produced rapid positivity after at least 8 h of incubation. Application of this assay to screen VREfm should be useful in clinical laboratories and hospital infection control units.

In vitro activities of thiazolidione derivatives combined with daptomycin against clinical Enterococcus faecium strains

BackgroundPrevious reports have demonstrated two thiazolidione derivatives (H2-60 and H2-81) can robustly inhibit the planktonic growth and biofilm formation of S. epidermidis and S. aureus by targeting the histidine kinase YycG. Whereas the antibacterial and anti-biofilm activity of these two thiazolidione derivatives (H2-60 and H2-81) against Enterococcus faecium remains elusive. Here, the pET28a-YycG recombinant plasmid were in vitro expressed in E. coli competent cell BL21 (DE3) and induced to express YycG’ protein (conding HisKA and HATPase_c domain) by 0.5 mM IPTG and was purified by Ni – NTA agarose and then for the autophosphorylation test. Antimicrobial testing and time-killing assay were also be determined. Anti-biofilm activity of two derivatives with sub-MIC concentration towards positive biofilm producers of clinical E. faecium were detected using polystyrene microtiter plate and CLSM.ResultsThe MICs of H2-60 and H2-81 in the clinical isolates of E. faecium were in the range from 3.125 mg/L to 25 mg/L. Moreover, either H2-60 or H2-81 showed the excellent bactericidal activity against E. faecium with monotherapy or its combination with daptomycin by time-killing assay. E. faecium planktonic cells can be decreased by H2-60 or H2-81 for more than 3 × log10 CFU/mL after 24 h treatment when combined with daptomycin. Furthermore, over 90% of E. faecium biofilm formation could markedly be inhibited by H2-60 and H2-81 at 1/4 × MIC value. In addition, the frequency of the eradicated viable cells embedded in mature biofilm were evaluated by the confocal laser microscopy, suggesting that of H2-60 combined with ampicillin or daptomycin was significantly high when compared with single treatment (78.17 and 74.48% vs. 41.59%, respectively, P < 0.01).ConclusionThese two thiazolidione derivatives (H2-60 and H2-81) could directly impact the kinase phosphoration activity of YycG of E. faecium. H2-60 combined with daptomycin exhibit the excellent antibacterial and anti-biofilm activity against E. faecium by targeting YycG.

Prediction of Antimicrobial Resistance in Clinical Enterococcus faecium Isolates Using a Rules-Based Analysis of Whole-Genome Sequences.

Enterococcus faecium is a major cause of clinical infections, often due to multidrug-resistant (MDR) strains. Whole-genome sequencing (WGS) is a powerful tool to study MDR bacteria and their antimicrobial resistance (AMR) mechanisms. In this study, we used WGS to characterize E. faecium clinical isolates and test the feasibility of rules-based genotypic prediction of AMR. Clinical isolates were divided into derivation and validation sets. Phenotypic susceptibility testing for ampicillin, vancomycin, high-level gentamicin, ciprofloxacin, levofloxacin, doxycycline, tetracycline, and linezolid was performed using the Vitek 2 automated system, with confirmation and discrepancy resolution by broth microdilution, disk diffusion, or gradient diffusion when needed. WGS was performed to identify isolate lineage and AMR genotype. AMR prediction rules were derived by analyzing the genotypic-phenotypic relationship in the derivation set. Phylogenetic analysis demonstrated that 88% of isolates in the collection belonged to hospital-associated clonal complex 17. Additionally, 12% of isolates had novel sequence types. When applied to the validation set, the derived prediction rules demonstrated an overall positive predictive value of 98% and negative predictive value of 99% compared to standard phenotypic methods. Most errors were falsely resistant predictions for tetracycline and doxycycline. Further analysis of genotypic-phenotypic discrepancies revealed potentially novel pbp5 and tet(M) alleles that provide insight into ampicillin and tetracycline class resistance mechanisms. The prediction rules demonstrated generalizability when tested on an external data set. In conclusion, known AMR genes and mutations can predict E. faecium phenotypic susceptibility with high accuracy for most routinely tested antibiotics, providing opportunities for advancing molecular diagnostics.

Novel vancomycin resistance gene cluster in Enterococcus faecium ST1486, Belgium, June 2021.

We identified a novel van gene cluster in a clinical Enterococcus faecium isolate with vancomycin minimum inhibitory concentration (MIC) of 4 µg/mL. The ligase gene, vanP, was part of a van operon cluster of 4,589 bp on a putative novel integrative conjugative element located in a ca 98 kb genomic region presumed to be acquired by horizontal gene transfer from Clostridium scidens and Roseburia sp. 499. Screening for van genes in E. faecium strains with borderline susceptibility to vancomycin is important.

Antibacterial activity of grapefruit peel extracts and green-synthesized silver nanoparticles.

Background and Aim:The gradual loss of efficacy of conventional antibiotics is a global issue. Plant material extracts and green-synthesized nanoparticles are among the most promising options to address this problem. Therefore, the aim of this study was to assess the antibacterial properties of aqueous and hydroalcoholic extracts of grapefruit peels as well as their inclusion in green-synthesized silver nanoparticles (AgNPs).Materials and Methods:Aqueous and hydroalcoholic extracts (80% v/v) were prepared, and the volume and mass yields were determined. The synthesis of AgNPs was done in an eco-friendly manner using AgNO3 as a precursor. The nanoparticles were characterized by ultraviolet–vis spectrometry and photon cross-correlation spectroscopy. The antibacterial activity of the extracts was tested on three Gram-positive bacteria (Staphylococcus aureus ATCC 6538, clinical Enterococcus faecalis, and S. aureus) and two Gram-negative bacteria (two clinical Escherichia coli) using various concentrations of extracts (100, 50, 25, 12, and 5 mg/mL and 5% dimethyl sulfoxide as negative control). Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined using the microdilution method. Modulation of cefazoline and ampicillin on resistant E. coli and S. aureus strains was added to the mixture design response surface methodology with extreme vertices design, with the diameters of inhibition and the fractional inhibitory concentration index as responses and factors, respectively. The antibiotic, the ethanolic extract, and water varied from 0.1 MIC to 0.9 MIC for the first two and from 0 to 0.8 in proportion for the third. Validating the models was done by calculating the absolute average deviation, bias factor, and accuracy factor.Results:The volume yield of the EE and aqueous extract (AE) was 96.2% and 93.8% (v/v), respectively, whereas their mass yields were 7.84% and 9.41% (m/m), respectively. The synthesized AgNPs were very uniform and homogeneous, and their size was dependent on the concentration of AgNO3. The antibacterial activity of the two extracts was dose-dependent, and the largest inhibition diameter was observed for the Gram-positive bacteria (S. aureus ATCC 6538; AE, 12; EE, 16), whereas AgNPs had a greater effect on Gram-negative bacteria. The MICs (mg/mL) of the AEs varied from 3.125 (S. aureus ATCC 6538) to 12.5 (E. coli 1 and E. coli 2), whereas the MICs of the EEs varied from 1.5625 (S. aureus 1, S. aureus ATCC 6538, and E. faecalis) to 6.25 (E. coli 1). There was a significant difference between the MICs of AEs and EEs (p=0.014). The MBCs (mg/mL) of the AEs varied from 12.5 (S. aureus ATCC 6538) to 50 (S. aureus 1), whereas those of the EEs varied from 6.25 (S. aureus 1) to 25 (E. coli 1 and E. faecalis). Ethanolic grapefruit extracts demonstrated an ability to modulate cefazolin on E. coli and S. aureus but were completely indifferent to ampicillin on E. coli.Conclusion:Grapefruit peel extracts and their AgNPs exhibit antibacterial properties that can be exploited for the synthesis of new antimicrobials and their EEs may be efficiently used synergistically with other antibiotics against bacteria with intermediate susceptibility.

A Metabolomics and Molecular Networking Approach to Elucidate the Structures of Secondary Metabolites Produced by Serratia marcescens Strains.

An integrated approach that combines reverse-phase high-performance liquid chromatography (RP-HPLC), electrospray ionization mass spectrometry, untargeted ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MSE) and molecular networking (using the Global Natural Products Social molecular network platform) was used to elucidate the metabolic profiles and chemical structures of the secondary metabolites produced by pigmented (P1) and non-pigmented (NP1) Serratia marcescens (S. marcescens) strains. Tandem mass spectrometry-based molecular networking guided the structural elucidation of 18 compounds for the P1 strain (including 6 serratamolides, 10 glucosamine derivatives, prodigiosin and serratiochelin A) and 15 compounds for the NP1 strain (including 8 serratamolides, 6 glucosamine derivatives and serratiochelin A) using the MSE fragmentation profiles. The serratamolide homologues were comprised of a peptide moiety of two L-serine residues (cyclic or open-ring) linked to two fatty acid chains (lengths of C10, C12, or C12:1). Moreover, the putative structure of a novel open-ring serratamolide homologue was described. The glucosamine derivative homologues (i.e., N-butylglucosamine ester derivatives) consisted of four residues, including glucose/hexose, valine, a fatty acid chain (lengths of C13 – C17 and varying from saturated to unsaturated) and butyric acid. The putative structures of seven novel glucosamine derivative homologues and one glucosamine derivative congener (containing an oxo-hexanoic acid residue instead of a butyric acid residue) were described. Moreover, seven fractions collected during RP-HPLC, with major molecular ions corresponding to prodigiosin, serratamolides (A, B, and C), and glucosamine derivatives (A, C, and E), displayed antimicrobial activity against a clinical Enterococcus faecalis S1 strain using the disc diffusion assay. The minimum inhibitory and bactericidal concentration assays however, revealed that prodigiosin exhibited the greatest antimicrobial potency, followed by glucosamine derivative A and then the serratamolides (A, B, and C). These results provide crucial insight into the secondary metabolic profiles of pigmented and non-pigmented S. marcescens strains and confirms that S. marcescens strains are a promising natural source of novel antimicrobial metabolites.

Evaluation of the antibacterial and anticancer activities of marine Bacillus subtilis ESRAA3010 against different multidrug resistant Enterococci (MDRE) and cancer cell lines

Fifty nine isolates belonging to six species of Enterococci namely, Enterococcus faecalis, Enterococcus faecium, Enterococcus raffinosus, Enterococcus durans, Enterococcus mundtiiand Enterococcus avium (n = 35, 15, 4, 3, 1 and 1 isolates, respectively) were obtained from different clinical specimens including urine, pus, blood, wound, sputum and synovial fluid. The highest numbers of Enterococci were recorded from the pus (20 isolates, 33.90%) followed by urine (12 isolates, 20.34%) while the lowest frequency was observed with synovial fluid samples (2 isolates, 3.39%). These isolates showed different multidrug resistant patterns with the lowest resistant for linezolid (n = 5, 8.48%), followed by teicoplanin (n = 14, 23.73%) and vancomycin (n = 20, 33.90%) while they exhibited the highest resistant against penicillin (n = 53, 89.83%), oxacillin (n = 50, 84.75%), erythromycin (n = 49, 83.05%) and streptomycin (n = 47, 79.66 %). On the other hand, a free living marine bacterium under isolation code ESRAA3010 was isolated from seawater samples obtained from the fishing area Masturah, Red Sea, Jeddah, Saudi Arabia. The phenotypic, chemotaxonomic, 16S rRNA gene analyses and phylogenetic data proved that isolate ESRAA3010 is very close to Bacillus subtilis and then it was designated as Bacillus subtilis ESRAA3010. It gave the highest antagonistic activity against all clinical Enterococcus faecalis, Enterococcus faecium, Enterococcus raffinosus, Enterococcus durans, Enterococcus mundtiiand Enterococcus avium isolates under study with minimum inhibitory concentration (MIC) ranged from 4 to 56 µg/mL, 4 to 12 µg/mL, 4 to 8 µg/mL, 4 to 8 µg/mL, 8 µg/mL and 4 µg/mL, respectively as well as minimum bactericidal concentration (MBC) (8 to 64 µg/mL, 4 to 16 µg/mL, 4 to 12 µg/mL, 4 to 16 µg/mL, 12 µg/mL and 8 µg/mL, respectively). Moreover it showed anti-proliferative activity against colon (HCT-116), liver (HepG-2), breast (MCF-7) and lung (A-549) carcinomas with IC50 equal to 39, 50, 75 and 19 µg/mL, respectively which indicates its prospective usage in the upcoming decades.

Mechanism of Eravacycline Resistance in Clinical Enterococcus faecalis Isolates From China.

Opportunistic infections caused by multidrug-resistant Enterococcus faecalis strains are a significant clinical challenge. Eravacycline (Erava) is a synthetic fluorocycline structurally similar to tigecycline (Tige) that exhibits robust antimicrobial activity against Gram-positive bacteria. This study investigated the in vitro antimicrobial activity and heteroresistance risk of Eravacycline (Erava) in clinical E. faecalis isolates from China along with the mechanism of Erava resistance. A total of 276 non-duplicate E. faecalis isolates were retrospectively collected from a tertiary care hospital in China. Heteroresistance to Erava and the influence of tetracycline (Tet) resistance genes on Erava susceptibility were examined. To clarify the molecular basis for Erava resistance, E. faecalis variants exhibiting Erava-induced resistance were selected under Erava pressure. The relative transcript levels of six candidate genes linked to Erava susceptibility were determined by quantitative reverse-transcription PCR, and their role in Erava resistance and heteroresistance was evaluated by in vitro overexpression experiments. We found that Erava minimum inhibitory concentrations (MICs) against clinical E. faecalis isolates ranged from ≤0.015 to 0.25 mg/l even in strains harboring Tet resistance genes. The detection frequency of Erava heteroresistance in isolates with MICs ≤ 0.06, 0.125, and 0.25 mg/l were 0.43% (1/231), 7.5% (3/40), and 0 (0/5), respectively. No mutations were detected in the 30S ribosomal subunit gene in Erava heteroresistance-derived clones, although mutations in this subunit conferred cross resistance to Tige in Erava-induced resistant E. faecalis. Overexpressing RS00630 (encoding a bone morphogenetic protein family ATP-binding cassette transporter substrate-binding protein) in E. faecalis increased the frequency of Erava and Tige heteroresistance, whereas RS12140, RS06145, and RS06880 overexpression conferred heteroresistance to Tige only. These results indicate that Erava has potent in vitro antimicrobial activity against clinical E. faecalis isolates from China and that Erava heteroresistance can be induced by RS00630 overexpression.

Evaluation of a Newly Developed Vacuum Dried Microtiter Plate for Rapid Biocide Susceptibility Testing of Clinical Enterococcus Faecium Isolates.

We investigated the suitability of a newly developed biocide susceptibility test system based on microtiter plates containing vacuum dried biocides as a fast and reliable screening method. The evaluated substances included the cationic biocides benzalkonium chloride (BAC), chlorhexidine dihydrochloride (CHX), cetylpyridinium chloride, didecyldimethylammonium chloride, and octenidine dihydrochloride. Testing a selection of Escherichia coli and enterococci, the biocide microtiter plates provided results comparable to those obtained from broth microdilution according to ISO 20776-1. Broad MIC ranges allowed for testing gram-positive and gram-negative species with the same plate design. In the second part of our study, we applied the established method to analyze the susceptibility of 90 clinical Enterococcus faecium isolates from a German university hospital, as previous studies have indicated a link between reduced susceptibility to substances such as CHX and BAC and vancomycin resistance. We therefore determined MIC and minimum bactericidal concentrations (MBC) for 48 non-clonal vancomycin susceptible and 42 non-clonal vancomycin resistant isolates, but MIC95 and MBC95 were quite similar in both groups. Our easy to handle and ready to use test system enables the routine surveillance of bacterial tolerance towards disinfectants in hospitals. As a result, hygiene measures can be adapted and nosocomial infections controlled despite increasing prevalence of antibiotic-resistant bacteria.