Clinical Isolates Of Enterococci Research Articles

Analysis of molecular epidemiological characteristics and antimicrobial susceptibility of vancomycin-resistant and linezolid-resistant Enterococcus in China

BackgroundThis study investigates the distribution and characteristics of linezolid and vancomycin susceptibilities among Enterococcus faecalis (E. faecalis) and Enterococcus faecium (E. faecium) and explores the underlying resistance mechanisms.MethodsA total of 2842 Enterococcus clinical isolates from patients were retrospectively collected, and their clinical data were further analyzed. The minimum inhibitory concentrations (MICs) of vancomycin and linezolid were validated by broth dilution method. The resistance genes optrA, cfr, vanA, vanB and vanM were investigated using polymerase chain reaction (PCR). Housekeeping genes and resistance genes were obtianed through whole-genome sequencing (WGS).ResultsOf the 2842 Enterococcus isolates, 88.5% (2516) originated from urine, with E. faecium accounted for 60.1% of these. The vanA gene was identified in 27/28 vancomycin resistant Enterococcus (VRE) isolates, 4 of which carried both vanA and vanM genes. The remaining strain was vanM positive. The optrA gene was identified in all E. faecalis isolates among linezolid resistant Enterococcus (LRE). E. faecium showed a higher multiple antibiotic resistance index (MAR index) compared to E. faecalis. The multi-locus sequence typing (MLST) showed the sequence type of E. faecium mainly belongs to clonal complex (CC) 17, nearly E. faecalis isolates analyzed were differentiated into 7 characteristics of sequence types (STs), among which ST16 of CC16 were the major lineage.ConclusionUrine was the primary source of VRE and LRE isolates in this study. E. faecium showed higher levels of resistance compared to E. faecalis. OptrA gene was detected in 91.6% of LRE, which could explain linezolid resistance, and van genes were detected in all vancomycin resistant Enterococcus strains, while vanA was a key resistance mechanism in VRE identified in this study.

A novel molecular assay conducted on the BD Max system to facilitate reflex testing for vanA and vanB in clinical isolates of enterococci

Infections caused by vancomycin-resistant enterococci (VRE) are common. Real-time PCR assays targeting vanA and vanB facilitate screening of patients in healthcare settings to limit the risk of dissemination, especially amongst those at high-risk of infection or with limited treatment options. Such assays are commonly performed as reflex testing procedures where they augment phenotypic techniques and shorten turnaround time to benefit timely clinical management. 'Random access' and 'sample-to-result' real-time PCR platforms are suited for this application as they are of low complexity and less technically demanding. Modelled on these attributes, we configured a real-time PCR assay (VRE BD) for detection of vanA/B in clinical isolates of enterococci, adapted for the BD Max System (Becton Dickinson). We applied an unconventional approach by testing suspensions of microorganisms in water to circumvent the traditional pre-analytical genomic extraction process. Our objective of this study was to assess the performance of this assay for detection of VRE in cultures by validating against a traditional real-time PCR assay based on the LightCycler 2.0 platform (Roche, VRE RO). A high level of analytical sensitivity and specificity (≥99.0%) for both genes was obtained when testing suspensions derived from blood agar. Results for suspensions obtained from chromID VRE (Edwards Group) showed a similar level of performance for vanA detection (100%), but not for the vanB target (≥90.9%) where a lesser number of isolates were available for testing. However, our results for VRE detection in isolates from these media were repeatable and reproducible, and equated to positive and negative predictive values of ≥95.2% and ≥97.8%, respectively. Furthermore, the VRE BD assay was also able to accurately detect VRE in clinical and spiked BacT/ALERT (bioMérieux) blood cultures. Thus, the technical simplicity, short turnaround time and robustness of this high performing assay for VRE is suitable for reflex testing. In addition, the format developed for the BD Max platform has potential application for reflex testing other molecular targets of clinical importance.

Epidemiology and genetic diversity of linezolid-resistant Enterococcus clinical isolates in Belgium from 2013 to 2021

ObjectivesLinezolid-resistant opportunistic human pathogens Enterococcus faecalis and Enterococcus faecium are emerging health threats as limited therapeutic options remain. The aim of this study was to investigate the epidemiology, resistance mechanisms, and genetic diversity of linezolid-resistant enterococci (LRE) isolated between 2013 and 2021 and received at the Belgian National Reference Centre (NRC) for Enterococci. MethodsLinezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. ResultsSeventy-eight LRE human isolates, of which 63 (81%) E. faecalis and 15 (19%) E. faecium strains, were submitted to the Belgian NRC for Enterococci. Of the linezolid-resistant E. faecalis strains, 97% harboured the optrA gene (56% wild-type pE349) and 3% the poxtA gene. Of the linezolid-resistant E. faecium strains, 54% harboured the G2576T point mutation in the V domain of the 23S rRNA genes, 23% the poxtA, and 23% the optrA gene. Furthermore, two E. faecium strains were identified with a combination of two resistance mechanisms ([i] optrA and poxtA, and [ii] cfr(B) and G2576T point mutation, respectively). Vancomycin resistance was observed in 15% (n = 12) of the LRE. ST480 (n = 42/63 typed strains, 67%) was the most frequently detected sequence type (ST) in linezolid-resistant E. faecalis strains, while ST203 (n = 5/15 typed strains, 33%) was the most frequently detected ST in linezolid-resistant E. faecium strains. ConclusionsE. faecalis isolates harbouring optrA were the predominant LRE in Belgium, with ST480 as the most prominent multilocus sequence typing. Linezolid resistance in E. faecium could be attributed to either chromosomal mutations or transferable resistance determinants.

Repurposing fusidic acid as an antimicrobial against enterococci with a low probability of resistance development.

Drug repurposing constitutes a strategy to combat antimicrobial resistance, by using agents with known safety, pharmacokinetics, and pharmacodynamics. Previous studies have implemented new fusidic acid (FA) front-loading-dose regimens, allowing higher serum levels than those achievable with ordinary doses. As susceptibility breakpoints are affected by serum level, we evaluated the repurposing of FA as an antimicrobial product against enterococci. FA minimum inhibitory concentrations (MICs) against standard enterococci strains; Enterococcus faecalis ATCC 29212 and Enterococcus faecium ATCC 27270 were 2 and 4µg/mL, respectively. The MIC against 98 enterococcal clinical isolates was ≤ 8µg/mL; all would be susceptible if categorized according to recalculated breakpoints (≥ 16µg/mL), based on the serum level achieved using the front-loading regimen. FA administration in vivo, using the BALB/c mouse infection model, significantly reduced bacterial burden by two to three log10 units in the liver and spleen of mice infected with vancomycin-susceptible and -resistant strains. Exposure of the standard enterococcal strains to increasing, but not fixed, FA concentrations resulted in resistant strains (MIC = 128µg/mL), with thicker cell walls and slower growth rates. Only one mutation (M651I) was detected in the fusA gene of the resistant strain derived from serial passage of E. faecium ATCC 27270, which was retained in the revertant strain after passage in the FA-free medium. In conclusion, FA can be repurposed as an antimicrobial drug against enterococci with a low probability of mutational resistance development, and can be employed for treatment of infections attributable to vancomycin-resistant enterococci.

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.

State-of-the-Art Review: Persistent Enterococcal Bacteremia.

Persistent enterococcal bacteremia is a commonly encountered and morbid syndrome without a strong evidence base for optimal management practices. Here we highlight reports on the epidemiology of enterococcal bacteremia to better describe and define persistent enterococcal bacteremia, discuss factors specific to Enterococcus species that may contribute to persistent infections, and describe a measured approach to diagnostic and therapeutic strategies for patients with these frequently complicated infections. The diagnosis of persistent enterococcal bacteremia is typically clinically evident in the setting of repeatedly positive blood culture results; instead, the challenge is to determine in an accurate, cost-effective, and minimally invasive manner whether any underlying nidus of infection (eg, endocarditis or undrained abscess) is present and contributing to the persistent bacteremia. Clinical outcomes for patients with persistent enterococcal bacteremia remain suboptimal. Beyond addressing host immune status if relevant and pursuing source control for all patients, management decisions primarily involve the selection of the proper antimicrobial agent(s). Options for antimicrobial therapy are often limited in the setting of intrinsic and acquired antimicrobial resistance among enterococcal clinical isolates. The synergistic benefit of combination antimicrobial therapy has been demonstrated for enterococcal endocarditis, but it is not clear at present whether a similar approach will provide any clinical benefit to some or all patients with persistent enterococcal bacteremia.

734. Trend Analysis of Oritavancin and Comparator Agents Activity against Enterococcus Causing Infections in US Medical Centers between 2017–2019 and 2022

Abstract Background A new formulation of oritavancin (ORI) to be infused over 1 hour for the treatment of skin and skin structure infections was approved in 2021 by the US FDA. The activity of ORI and its comparators against Enterococcus (ENT) and resistant (R) subsets from US medical centers was evaluated comparatively between 2022 and 2017–2019. Methods 4,462 ENT, including 246/2,556 E. faecalis (EF) from 2022 and 2017–2019, respectively, 148/1,348 E. faecium (EFM), and 14/150 other ENT were collected (1/patient) from 34 US medical centers. Isolates were identified by MALDI-TOF MS and standard microbiology tests and susceptibility (S) tested by CLSI broth microdilution. CLSI clinical breakpoints (BPs) and VanA/VanB phenotypes were used. ORI BPs against vancomycin (VAN)-S EF were applied to all ENT. Results ORI activity against ENT from 2022 (MIC50/90, 0.015/0.03 mg/L) was similar to 2017–2019 (MIC50/90, 0.015/0.06 mg/L; Table). ORI inhibited 97.5%/98.9% of ENT from 2022/2017–2019 at ≤ 0.12 mg/L. VAN and linezolid (LZD) inhibited 97.2%/98.4% and 99.6%/99.6% of ENT from 2022/2017–2019, at the respective BPs. ORI, VAN, LZD and daptomycin (DAP) showed stable S rates ( > 96%) against EF. ORI remained active against 57.1/53.1% of VAN-R EF. VanA rates in VAN-R EF from 2022 and 2017–2019 were 85.7%/93.8%. ORI inhibited the 3 VanB EF isolates at ≤ 0.03 mg/L. LZD and DAP remained active against VAN-R EF (100%). Similar activity was noted for ORI against EFM from 2022 (MIC50/90, 0.008/0.06 mg/L) and 2017–2019 (MIC50/90, 0.008/0.03 mg/L). The EFM S rate to VAN was 35.1% in 2017–2019 and 39.2% in 2022. The S rates to ORI (VAN-S EF BPs) and LZD remained stable ( > 98%) as well as rates of DAP susceptible dose-dependent (SDD; 96.6% in 2022 and 99.5% in 2017–2019). The VanB phenotype increased from 7.8% in 2017–2019 to 20.0% in 2022. ORI inhibited all VanB and 98.5% of VanA EFM at ≤ 0.12 mg/L. LZD inhibited 98.6%/99.4% of VanA EFM isolates in 2022/2017–2022. The DAP SDD rate slightly decreased against VanA EFM (99.7% to 93.1%). ORI and comparators were active against other ENT. Conclusion ORI exhibited potent and stable activity against ENT clinical isolates, including VAN-R EFM in US. An increase in VanB EFM phenotype and a slight decrease in the DAP SDD rates in VAN-R EFM subsets were noted over time. Disclosures Cecilia G. Carvalhaes, MD, PhD, AbbVie: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Melinta: Grant/Research Support|Pfizer: Grant/Research Support Rodrigo E. Mendes, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|Cipla: Grant/Research Support|Entasis: Grant/Research Support|GSK: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support Dee Shortridge, PhD, Melinta: Grant/Research Support|Shionogi: Grant/Research Support Mariana Castanheira, PhD, AbbVie: Grant/Research Support|Basilea: Grant/Research Support|bioMerieux: Grant/Research Support|Cipla: Grant/Research Support|CorMedix: Grant/Research Support|Entasis: Grant/Research Support|Melinta: Grant/Research Support|Paratek: Grant/Research Support|Pfizer: Grant/Research Support|Shionogi: Grant/Research Support

Virulence Factors of Clinical and Fecal Isolates of Enterococci Species

Enterococci species are known commensals of the gastrointestinal flora; however, in recent years, they have emerged as important nosocomial pathogens that possess many virulence factors that are attributed to the pathogenesis of diseases caused by them. The study evaluated and compared the virulence factors of Enterococci isolated from fecal and clinical samples. From the obtained isolates, the clinical enterococcal isolates produced 35%, 20%, and 50%, and fecal isolates produced 23%, 13%, and 13% gelatinase, hemolysin, and biofilm, respectively. Biofilm production determined by the Congo Red agar, tube, and microtiter plate methods was 23%, 39%, and 49%, respectively. The sensitivity of the Congo Red agar and tube method compared to the microtiter plate method was 27% and 46%, respectively, whereas the specificity of both tests was 79%. This study showed that biofilm production plays a significant role in the pathogenesis of diseases caused by Enterococci. Detection of biofilm production using the microtiter plate method is more sensitive and specific than the Congo Red agar and tube method.

Clinical Resistant Strains of Enterococci and Their Correlation to Reduced Susceptibility to Biocides: Phenotypic and Genotypic Analysis of Macrolides, Lincosamides, and Streptogramins.

Enterococci are troublesome nosocomial, opportunistic Gram-positive cocci bacteria showing enhanced resistance to many commonly used antibiotics. This study aims to investigate the prevalence and genetic basis of antibiotic resistance to macrolides, lincosamides, and streptogramins (MLS) in Enterococci, as well as the correlation between MLS resistance and biocide resistance. From 913 clinical isolates collected from King Khalid Hospital, Hail, Saudi Arabia, 131 isolates were identified as Enterococci spp. The susceptibility of the clinical enterococcal isolates to several MLS antibiotics was determined, and the resistance phenotype was detected by the triple disk method. The MLS-involved resistance genes were screened in the resistant isolates. The current results showed high resistance rates to MLS antibiotics, and the constitutive resistance to all MLS (cMLS) was the most prevalent phenotype, observed in 76.8% of resistant isolates. By screening the MLS resistance-encoding genes in the resistant isolates, the erythromycin ribosome methylase (erm) genes that are responsible for methylation of bacterial 23S rRNA were the most detected genes, in particular, ermB. The ereA esterase-encoding gene was the most detected MLS modifying-encoding genes, more than lnuA (adenylation) and mphC (phosphorylation). The minimum inhibitory concentrations (MICs) of commonly used biocides were detected in resistant isolates and correlated with the MICs of MLS antibiotics. The present findings showed a significant correlation between MLS resistance and reduced susceptibility to biocides. In compliance with the high incidence of the efflux-encoding genes, especially mefA and mefE genes in the tolerant isolates with higher MICs to both MLS antibiotics and biocides, the efflux of resistant isolates was quantified, and there was a significant increase in the efflux of resistant isolates with higher MICs as compared to those with lower MICs. This could explain the crucial role of efflux in developing cross-resistance to both MLS antibiotics and biocides.

A Study on Glycopeptide-Resistant Genotypes among Clinical Isolates of Enterococcus with Van B Phenotype.

Enterococci, although remarked as harmless commensals of the intestinal tract of humans and animals, have become a significant source of infection in hospitalized patients worldwide.The present study aimed to isolate and identify enterococci from clinical samples and to determine the genotypic characteristics of vancomycin-resistant enterococcus isolates. A total of 774 isolates of enterococci from clinical samples were identified to the species level, and their anti-microbial susceptibility pattern was determined by Kirby Bauer Disk Diffusion method and Vitek 2 automated system. Screening for vancomycin resistance was performed by using brain heart infusion agar containing 6 micrograms/ml of vancomycin. The minimum inhibitory concentration (MIC) of vancomycin was determined by the agar dilution method and Epsilometer test (E test). Genotyping was carried out for resistant isolates by multiplex polymerase chain reaction. Thirty (3.88%) isolates were resistant to vancomycin by agar screen method. Five isolates showed intermediate resistance with anMIC of 8-16 micrograms/ml for vancomycin by agar dilution. By the E test, two isolates displayed an MIC of ≥32 micrograms/ml for vancomycin and one isolate showed an MICof ≥32 micrograms/ml for teicoplanin.Van A was the common genotype isolated. The present study reports isolates of Enterococcus faecalis with a susceptible MIC for glycopeptide and the presence of the Van A gene. Heterogeneous resistance among clinical isolates of Enterococcus faecalis was observed in our study. The predominant phenotype and genotype detected among clinical isolates were Van A.

Detection of Genes Related to Linezolid Resistance (poxtA, cfr, and optrA) in Clinical Isolates of Enterococcus spp. from Humans: A First Report from Iran.

Enterococci may develop resistance to linezolid through chromosomal mutations that involve specific linezolid resistance genes, such as cfr, optrA, and poxtA. The objective of this study was to evaluate the antibiotic susceptibility of enterococcal isolates and identify cfr, optrA, and poxtA genes in MDR isolates. Enterococcal isolates were collected from various clinical specimens at Al-Zahra, Amin, and Khorshid Hospitals in Isfahan. The Enterococcus isolates were identified as belonging to the E. faecalis and E. faecium species by using specific gene (D alanine D alanine ligase ddl) sets in PCR. To detect cfr, optrA, and poxtA genes among the species, a multiplex-PCR assay was performed. Out of 175 isolates, E. faecalis predominated 129/175 (73.7%). Furthermore, the prevalence of vancomycin-resistant Enterococci (VRE) and linezolid-resistant Enterococci (LRE) was 29.7% and 4%, respectively. The overall prevalence of MDR was 91.1%, 68.9%, and 66.6% of E. faecium, E. faecalis, and other Enterococcus spp., respectively. Interestingly, the frequency of optrA (71.4%) in E. faecium and poxtA and crf (42.8%) in E. faecalis were detected among LRE species. A statistically significant relationship (P < 0.05) was found between the presence of the three genes and the occurrence of LRE. This is the first study to report the detection of linezolid resistance genes (cfr, optrA, and poxtA) in clinical Enterococcus spp. isolates from Iran, conducted at Isfahan University of Medical Sciences hospitals. The emergence of enterococcal strains that resist linezolid is concerning as it can lead to the spread of resistant strains among patients, resulting in treatment failure.

Comparative metabolite profiling of four polyphenol rich Morus leaves extracts in relation to their antibiofilm activity against Enterococcus faecalis

Enterococci are a common cause of urinary tract infections. The severity of enterococcal infections is associated with their ability to form biofilms. Morus leaves are known as a natural antibacterial, however, their antibiofilm activity against Enterococcus remains unveiled. This study aimed to evaluate the ability of four polyphenol-rich Morus leaves extracts (Morus nigra, M. rubra, M. macroura, and M. alba) to inhibit biofilm formed by enterococcal clinical isolates in relation to their metabolic profiling. Results revealed that 48% of the isolates formed strong biofilm, 28% formed moderate biofilm, 20% formed weak biofilm, and only 4% did not form a biofilm. The strong biofilm-forming isolates were E. faecalis, and hence were chosen for this study. The antibiofilm activity of the four polyphenol-rich Morus leaves extracts revealed that the M. nigra extract exhibited the highest percentage of biofilm inhibition followed by M. rubra then M. macroura and the least inhibition was detected in M. alba, and these results were in accordance with the phenolic and flavonoid contents of each extract. UPLC-ESI-MS/MS identified 61 polyphenolic compounds in the four extracts. Further, multivariate analysis confirmed clear segregation of M. nigra from the other species suggesting disparity in its metabolome, with accumulation of flavonoids, anthocyanidins, phenolic acids and coumarin derivatives. Quercetin and kaempferol glycosides were found to be positively and significantly correlated to the antibiofilm activity. In conclusion, M. nigra ethanolic extracts showed the highest phenolic content and antibiofilm activity and they could be developed as a complementary treatment for the development of antimicrobial agents.

Web-based prediction of antimicrobial resistance in enterococcal clinical isolates by whole-genome sequencing.

Besides phenotypic antimicrobial susceptibility testing (AST), whole genome sequencing (WGS) is a promising alternative approach for detection of resistance phenotypes. The aim of this study was to investigate the concordance between WGS-based resistance prediction and phenotypic AST results for enterococcal clinical isolates using a user-friendly online tools and databases. A total of 172 clinical isolates (34 E. faecalis, 138 E. faecium) received at the French National Reference Center for enterococci from 2017 to 2020 were included. AST was performed by disc diffusion or MIC determination for 14 antibiotics according to CA-SFM/EUCAST guidelines. The genome of all strains was sequenced using the Illumina technology (MiSeq) with bioinformatic analysis from raw reads using online tools ResFinder 4.1 and LRE-finder 1.0. For both E. faecalis and E. faecium, performances of WGS-based genotype to predict resistant phenotypes were excellent (concordance > 90%), particularly for antibiotics commonly used for treatment of enterococcal infections such as ampicillin, gentamicin, vancomycin, teicoplanin, and linezolid. Note that 100% very major errors were found for quinupristin-dalfopristin, tigecycline, and rifampicin for which resistance mutations are not included in databases. Also, it was not possible to predict phenotype from genotype for daptomycin for the same reason. WGS combined with online tools could be easily used by non-expert clinical microbiologists as a rapid and reliable tool for prediction of phenotypic resistance to first-line antibiotics among enterococci. Nonetheless, some improvements should be made such as the implementation of resistance mutations in the database for some antibiotics.

Prevalence and Genetic Analysis of Resistance Mechanisms of Linezolid-Nonsusceptible Enterococci in a Tertiary Care Hospital Examined via Whole-Genome Sequencing.

(1) Background: Linezolid plays an important role in the treatment of invasive infections caused by vancomycin-resistant enterococci after its introduction to clinical practice. However, a detailed examination of linezolid-nonsusceptible enterococci (LNSE) is required. In this study, we attempted to analyze the mechanisms of LNSE strains isolated from a tertiary care hospital. (2) Methods: From 2019 to 2020, 18 Enterococcus faecalis, 14 E. faecium, and 2 E. gallinarum clinical isolates were collected at Severance Hospital. Agar dilution was performed to evaluate precise linezolid minimum inhibitory concentrations (MICs). Short-read whole-genome sequencing (WGS) was used to analyze resistance determinants. (3) Results: The presence of the optrA gene was likely the primary resistance mechanism in these three species, typically demonstrating a MIC value of 8 μg/mL. The co-existence of the cfr(D) and poxtA2 gene was the second major mechanism, primarily predicting a phenotype showing intermediate susceptibility to linezolid. G2576U mutation on 23S rRNA was only found in E. faecium; it mediated the most significant increase in linezolid MIC. (4) Conclusion: This is the first report examining poxtA2-cfr(D) co-harboring clinical enterococcal isolates in Korea and demonstrating the poxtA EF9F6-harboring clinical E. gallinarum strain worldwide. The comparison with resistance-gene-containing fragments in the isolates obtained from different countries and different sources demonstrated the spread of linezolid-resistance genes and suggested the possibility of foodborne transmission.

Influence of ZP2 peptide, a synthetic analogue of the GM-GSF active center on the anticytokine activity of bacteria from &lt;i&gt;Enterococcus&lt;/i&gt; genus and their ability to produce cytokine-like substances

Peptide ZP2, a synthetic analogue of the active center of granulocyte-macrophage colony stimulating factor (GM-CSF), exhibits a wide range of immunobiological effects, including antibacterial activity. At the same time, its effect (in sub-inhibitory concentrations) on the biological properties of Enterococcus spp., the causative agents of many infectious and inflammatory diseases remains poorly understood. The aim of our study was to analyze the nature of the effect of synthetic peptide ZP2 on anticytokine activity (ACA) and its ability to produce cytokine-like substances (CLS) in Enterococcus spp.&#x0D; 18 clinical isolates of Enterococcus spp. were used. Over the experiments, the bacterial strains were cultured in Schaedlers broth with ZP2 peptide at 37 C for 24 hours. No specific peptide was added in the control. ACA for IL-8, TNF and IL-17A and the production of the corresponding CLS were determined by the ELISA method. To assess ACA, the proportion of cytokine inactivation in the experiment relative to the control was calculated and expressed in pg/ml; CLS production was evaluated by the level of cytokines in the experiment and control, expressed as pg/ml. The data were subjected to statistical processing.&#x0D; It was revealed that Enterococcus spp. strains are capable of secreting compounds that inactivate cytokines IL-8, IL-17A and TNF, and produce CLS in the culture medium. Intragenital and intraspecific variability was noted in the presence and frequency of occurrence and in the severity of these properties. It was found that the ZP2 peptide in E. faecium increases ACA with respect to all studied cytokines. When tested with E. faecalis, it either did not affect their ACA against TNF and IL-8, or completely inhibited ACA for IL-17A. At the same time, ZP2 blocked the production of CLS, e.g., IL-17A and IL-8 in E. faecium, but increased the production of CLS similar to TNF, and, with E. faecalis, it increased the number of IL-17A-producing isolates twofold, although the average level of production of these CLS was lower than in the control.&#x0D; Enterococcus spp strains are capable of secreting compounds that inactivate cytokines IL-8, TNF and IL- 17A, and may produce substances similar to these cytokines. The synthetic peptide ZP2 has a modifying effect on the manifestation of these properties by Enterococci. Further studies of biological and pathogenetic features of Enterococci and other bacterial species, as well as modifying effects of the ZP2 peptide are required.

Comprehensive insight into anti-staphylococcal and anti-enterococcal action of brominated and chlorinated pyrazine-based chalcones.

The greatest threat and medicinal impact within gram-positive pathogens are posed by two bacterial genera, Staphylococcus and Enterococcus. Chalcones have a wide range of biological activities and are recognized as effective templates in medicinal chemistry. This study provides comprehensive insight into the anti-staphylococcal and anti-enterococcal activities of two recently published brominated and chlorinated pyrazine-based chalcones, CH-0y and CH-0w. Their effects against 4 reference and 12 staphylococcal and enterococcal clinical isolates were evaluated. Bactericidal action, the activity in combination with selected conventional antibiotics, the study of post-antimicrobial effect (PAE, PAE/SME), and in vitro and in vivo toxicity, were included. In CH-0y, anti-staphylococcal activity ranging from MIC = 15.625 to 62.5 μM, and activity against E. faecium from 31.25 to 62.5 μM was determined. In CH-0w, anti-staphylococcal activity ranging from 31.25 to 125 μM, and activity against E. faecium and E. faecalis (62.5 μM) was revealed. Both CH-0y and CH-0w showed bactericidal action, beneficial impact on bacterial growth delay within PAE and PAE/SME studies, and non/low toxicity in vivo. Compared to CH-0w, CH-0y seems to have higher anti-staphylococcal and less toxic potential. In conclusion, chalcones CH-0y and CH-0w could be considered as structural pattern for future adjuvants to selected antibiotic drugs.

Species prevalence, antimicrobial susceptibility and detection of virulence factors of enterococci isolated from tertiary care hospital

Introduction: Emergence of multidrug resistant nosocomial enterococcus strains emphasizes the need for further investigating enterococci. Objectives: To characterize enterococci from various clinical specimens, to determine the antimicrobial susceptibility pattern and to explore the association between virulence factors and antimicrobial resistance. Material and Methods: Two hundred and eighty three clinical isolates of enterococcus were speciated and subjected to antimicrobial susceptibility testing using Kirby Bauer disc diffusion method. They were screened for vancomycin resistance by vancomycin screening agar method as recommended by Clinical Laboratory Standards Institute 2014, and confirmed by determination of minimum inhibitory concentration using agar dilution and E test. Genotypic confirmation was done by polymerase chain reaction. Virulence factors (haemolysin, gelatinase and biofilm production) were detected phenotypically. Results: Of the 283 enterococci isolated, 12 species were identified; predominant species was Enterococcus faecalis (82.33%). High level gentamicin resistance (HLGR) and vancomycin resistance were observed among 55.57% and 6.01% of Enterococcus isolates respectively. All vancomycin resistant enterococci (VRE) were Enterococcus faecalis and had vanA phenotype and genotype. Sensitivity to linezolid was 100 per cent among enterococci. Hemolysin, gelatinase and biofilm production were seen in 15.90%, 12.36% and 13.43% of enterococcal isolates respectively.

Species prevalence, antimicrobial susceptibility of enterococci isolated from various clinical samples in tertiary care hospital

Introduction: Emergence of multidrug resistant nosocomial enterococcus strains emphasizes the need for further investigating enterococci. Objectives: To characterize enterococci from various clinical specimens, to determine the antimicrobial susceptibility pattern and to explore the association between virulence factors and antimicrobial resistance. Material and Methods: Two hundred and eighty three clinical isolates of enterococcus were speciated and subjected to antimicrobial susceptibility testing using Kirby Bauer disc diffusion method. They were screened for vancomycin resistance by vancomycin screening agar method as recommended by Clinical Laboratory Standards Institute 2020, and confirmed by determination of minimum inhibitory concentration using agar dilution and E test. Genotypic confirmation was done by polymerase chain reaction. Results: Of the 283 enterococci isolated, 12 species were identified; predominant species was Enterococcus faecalis (82.33%). High level gentamicin resistance (HLGR) and vancomycin resistance were observed among 55.57% and 6.01% of Enterococcus isolates respectively. All vancomycin resistant enterococci (VRE) were Enterococcus faecalis and had vanA phenotype and genotype. Sensitivity to linezolid was 100 per cent among enterococci. Conclusion: Our study reveals the occurrence of sizable number of HLGR isolates and emergence of VRE.

A virulence factor as a therapeutic: the probiotic Enterococcus faecium SF68 arginine deiminase inhibits innate immune signaling pathways

ABSTRACT The probiotic bacterial strain Enterococcus faecium SF68 has been shown to alleviate symptoms of intestinal inflammation in human clinical trials and animal feed supplementation studies. To identify factors involved in immunomodulatory effects on host cells, E. faecium SF68 and other commensal and clinical Enterococcus isolates were screened using intestinal epithelial cell lines harboring reporter fusions for NF-κB and JNK(AP-1) activation to determine the responses of host cell innate immune signaling pathways when challenged with bacterial protein and cell components. Cell-free, whole-cell lysates of E. faecium SF68 showed a reversible, inhibitory effect on both NF-κB and JNK(AP-1) signaling pathway activation in intestinal epithelial cells and abrogated the response to bacterial and other Toll-like receptor (TLR) ligands. The inhibitory effect was species-specific, and was not observed for E. avium, E. gallinarum, or E. casseliflavus. Screening of protein fractions of E. faecium SF68 lysates yielded an active fraction containing a prominent protein identified as arginine deiminase (ADI). The E. faecium SF68 arcA gene encoding arginine deiminase was cloned and introduced into E. avium where it conferred the same NF-κB inhibitory effects on intestinal epithelial cells as seen for E. faecium SF68. Our results indicate that the arginine deiminase of E. faecium SF68 is responsible for inhibition of host cell NF-κB and JNK(AP-1) pathway activation, and is likely to be responsible for the anti-inflammatory and immunomodulatory effects observed in prior clinical human and animal trials. The implications for the use of this probiotic strain for preventive and therapeutic purposes are discussed.

Assessing the Emergence of Resistance in vitro and Invivo: Linezolid Combined with Fosfomycin Against Fosfomycin-Sensitive and Resistant Enterococcus.

PurposeWe aimed to evaluate the synergistic effect of linezolid and fosfomycin on fosfomycin-sensitive and -resistant Enterococcus clinical isolates in vitro and in vivo and whether the emergence of fosfomycin resistance in Enterococcus is associated with changes in strain virulence, from the perspective of fitness cost.MethodsThe synergistic effect of linezolid and fosfomycin was studied via in vitro checkerboard and static time-kill assays, as well as based on the in vivo survival rate and hemolymph load of a Galleria mellonella infection model. Fosfomycin resistance was induced via a stepwise increase in concentration. Changes in the virulence of the strains after drug resistance were investigated using the G. mellonella infection model and reverse transcription quantitative polymerase chain reaction (RT-qPCR). In vitro and in vivo growth curves and competitive experiments were used to study the fitness cost of the strain. Finally, a static time-kill assay was performed to explore the effect of the combined medication.ResultsIn vitro and in vivo data showed that linezolid combined with fosfomycin had a good synergistic effect on Enterococcus treatment. The G. mellonella infection model and RT-qPCR data showed that the virulence of the resistant strains was weakened to varying degrees. A survival curve and competition experimental data showed that this was related to the fitness cost of strains while acquiring resistance and negatively impacted linezolid treatment; however, the combination still showed a good synergistic effect in drug-resistant strains.ConclusionLinezolid combined with fosfomycin had a synergistic effect on both fosfomycin-sensitive and -resistant Enterococcus strains. Strains incur fitness costs as they develop drug resistance, which leads to a decrease in virulence. There is an interaction between fitness cost, virulence, and drug resistance, which indirectly affects drug treatment.