Genomics investigation of the potentially invasive firefly Photinus signaticollis Blanchard 1845: Complete mitochondrial genome, multigene phylogenies and obtention of the luciferase and luciferin-regenerating genes.

A genomic investigation of the potentially invasive firefly Photinus signaticollis Blanchard1845 has been performed and led to the obtention of its complete 16,411 bp long mitochondrial genome. The mitogenome encodes 13 protein-coding genes, 22 tRNA genes and 2 rRNA genes. With other species of the Photinus complex it shares several premature terminations of some protein-coding genes and also an overlap between cox1 and tRNA-Tyr. By data-mining, the complete luciferase and luciferin-regenerating genes were also identified from the contigs file and compared with existing data, in addition to WG and CAD, two genes used in pioneering phylogenetic studies on fireflies. Three maximum likelihood phylogenies were derived from all these data. The multigene phylogeny based on all mitochondrial protein-coding genes strongly associates P. signaticollis with Photinus pyralis Linnaeus, 1758 and the lantern-less daily "winter firefly", Photinus corruscus Linnaeus, 1767. A second phylogeny based on concatenated sequences of the cox1, WG and CAD genes positions P. signaticollis as a sister clade to a large cluster of species containing the 7 sub-groups previously evidenced among the North American species of the Photinus complex. A third phylogeny based on the amino-acid sequence of the luciferase protein associates P. signaticollis to Photinus scintillans. The analysis presented here will most certainly help to come to a better understanding of the very complex inter-relationships in the very large Photinus genus.

Surveillance of vancomycin-resistant enterococci reveals shift in dominating clusters from vanA to vanB Enterococcus faecium clusters, Denmark, 2015 to 2022.

BackgroundVancomycin-resistant enterococci (VRE) are increasing in Denmark and Europe. Linezolid and vancomycin-resistant enterococci (LVRE) are of concern, as treatment options are limited. Vancomycin-variable enterococci (VVE) harbour the vanA gene complex but are phenotypically vancomycin-susceptible.AimThe aim was to describe clonal shifts for VRE and VVE in Denmark between 2015 and 2022 and to investigate genotypic linezolid resistance among the VRE and VVE.MethodsFrom 2015 to 2022, 4,090 Danish clinical VRE and VVE isolates were whole genome sequenced. We extracted vancomycin resistance genes and sequence types (STs) from the sequencing data and performed core genome multilocus sequence typing (cgMLST) analysis for Enterococcus faecium. All isolates were tested for the presence of mutations or genes encoding linezolid resistance.ResultsIn total 99% of the VRE and VVE isolates were E. faecium. From 2015 through 2019, 91.1% of the VRE and VVE were vanA E. faecium. During 2020, to the number of vanB E. faecium increased to 254 of 509 VRE and VVE isolates. Between 2015 and 2022, seven E. faecium clusters dominated: ST80-CT14 vanA, ST117-CT24 vanA, ST203-CT859 vanA, ST1421-CT1134 vanA (VVE cluster), ST80-CT1064 vanA/vanB, ST117-CT36 vanB and ST80-CT2406 vanB. We detected 35 linezolid vancomycin-resistant E. faecium and eight linezolid-resistant VVEfm.ConclusionFrom 2015 to 2022, the numbers of VRE and VVE increased. The spread of the VVE cluster ST1421-CT1134 vanA E. faecium in Denmark is a concern, especially since VVE diagnostics are challenging. The finding of LVRE, although in small numbers, ia also a concern, as treatment options are limited.

Evaluation of Antimicrobial Resistancein Clinical Isolates of Enterococcus spp. Obtained from Hospital Patients in Latvia.

Background and Objective: Enterococci are typically found in a healthy human gastrointestinal tract but can cause severe infections in immunocompromised patients. Such infections are treated with antibiotics. This study addresses the rising concern of antimicrobial resistance (AMR) in Enterococci, focusing on the prevalence of vancomycin-resistant enterococcus (VRE) strains. Materials and Methods: The pilot study involved 140 Enterococci isolates collected between 2021 and 2022 from two multidisciplinary hospitals (with and without local therapeutic drug monitoring protocol of vancomycin) in Latvia. Microbiological assays and whole genome sequencing were used. AMR gene prevalence with resistance profiles were determined and the genetic relationship and outbreak evaluation were made by applying core genome multi-locus sequence typing (cgMLST). Results: The acquired genes and mutations were responsible for resistance against 10 antimicrobial classes, including 25.0% of isolates expressing resistance to vancomycin, predominantly of the vanB type. Genetic diversity among E. faecalis and E. faecium isolates was observed and seven potential outbreak clusters were identified, three of them containing sequence types ST6, ST78 and ST80. The prevalence of vancomycin resistance was highest in the hospital without a therapeutic drug-monitoring protocol and in E. faecium. Notably, a case of linezolid resistance due to a mutation was documented. Conclusions: The study illustrates the concerning prevalence of multidrug-resistant Enterococci in Latvian hospitals, showcasing the rather widespread occurrence of vancomycin-resistant strains. This highlights the urgency of implementing efficient infection control mechanisms and the need for continuous VRE surveillance in Latvia to define the scope and pattern of the problem, influencing clinical decision making and planning further preventative measures.

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

OBJECTIVES: Linezolid-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. METHODS: Linezolid susceptibility testing was performed upon request on 2458 submitted enterococci strains. Whole-genome sequencing was performed on all LRE strains. RESULTS: Seventy-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. CONCLUSIONS: E. 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.

Characterization of a Tigecycline-, Linezolid- and Vancomycin-Resistant Clinical Enteroccoccus faecium Isolate, Carrying vanA and vanB Genes.

INTRODUCTION: Increasing incidence of Enterococcus faecium resistant to key antimicrobials used in therapy of hospitalized patients is a worrisome phenomenon observed worldwide. Our aim was to characterize a tigecycline-, linezolid- and vancomycin-resistant E. faecium isolate with the vanA and vanB genes, originating from a hematoma of a patient hospitalized in an intensive care unit in Poland. METHODS: Antimicrobial susceptibility (a broad panel) was tested using gradient tests with predefined antibiotic concentrations. The complete genome sequence was obtained from a mixed assembly of Illumina MiSeq and Oxford Nanopore's MinION reads. The genome was analyzed with appropriate tools available at the Center for Genomic Epidemiology, PubMLST and GenBank. Transferability of oxazolidinone, tigecycline and vancomycin resistance genes was investigated by conjugation, followed by PCR screen of transconjugants for antimicrobial resistance genes and plasmid rep genes characteristic for the donor and genomic sequencing of selected transconjugants. RESULTS: The isolate was resistant to most antimicrobials tested; susceptibility to daptomycin, erythromycin and chloramphenicol was significantly reduced, and only oritavancin retained the full activity. The isolate represented sequence type 18 (ST18) and carried vanA, vanB, poxtA, fexB, tet(L), tet(M), aac(6')-aph(2''), ant(6)-Ia and ant(6')-Ii. The vanA, poxtA and tet(M) genes located on ~ 40-kb plasmids were transferable by conjugation yielding transconjugants resistant to vancomycin, linezolid and tigecycline. The substitutions in LiaS, putative histidine kinase, SulP, putative sulfate transporter, RpoB and RpoC were potential determinants of an elevated daptomycin MIC. Comparative analyses of the studied isolate with E. faecium isolates from other countries revealed its similarity to ST18 isolates from Ireland and Uganda from human infections. CONCLUSIONS: We provide the detailed characteristics of the genomic determinants of antimicrobial resistance of a clinical E. faecium demonstrating the concomitant presence of both vanA and vanB and resistance to vancomycin, linezolid, tigecycline and several other compounds and decreased daptomycin susceptibility. This isolate is a striking example of an accumulation of resistance determinants involving various mechanisms by a single hospital strain.

Genomic insights into linezolid-resistant Enterococci revealed its evolutionary diversity and poxtA copy number heterogeneity.

OBJECTIVES: This study aimed to determine the molecular mechanisms of linezolid-resistant enterococci (LRE) in swine slaughterhouses in China and apply the "One Health" perspective to analyse the evolutionary dynamics of poxtA-positive E. faecium in clinical and non-clinical settings worldwide. METHODS: The phenotypic and genomic characteristics of multiple LRE isolates were systematically investigated using antimicrobial susceptibility testing, transfer assays, evolutionary experiments, quantitative RT-PCR assays, whole-genome sequencing, and bioinformatics analyses. RESULTS: Swine faeces served as a significant reservoir for LRE isolates, and optrA and poxtA were the primary contributors to linezolid resistance. Co-occurrence network analysis revealed a significant interconnection between optrA and several other ARGs. The poxtA copy number heterogeneity and polymorphism were initially observed in E. faecium parental and evolved isolates. The poxtA-carrying tandem repeat region exhibits high mobility and has undergone extensive duplication owing to linezolid pressure. The poxtA copy number varies from four copies on the plasmid of E. faecium IC25 to 11 copies on the plasmid and six copies on the chromosome in the evolved isolate IC25-50_poxtA. Furthermore, phylogenetic analysis of 185 poxtA-positive E. faecium strains worldwide found that one isolate from a French patient in 2018 shared only two SNPs with CC17 E. faecium isolates IC25 and IC7-2 from this study, highlighting the potential global transmission of CC17 poxtA-positive E. faecium between humans and animals. CONCLUSION: This study identified amplification of poxtA as a response of E. faecium to linezolid pressure. Phylogenetic analysis shed light on the potential global transmission of hospital-associated CC17 poxtA-positive E. faecium in clinical and non-clinical settings.

Vancomycin and Linezolid-Resistant Enterococcus Isolates from a Tertiary Care Center in India.

INTRODUCTION: There is increasing development of antibiotic resistance among the Enterococcus species. OBJECTIVES: This study was performed to determine prevalence and characterize the vancomycin-resistant and linezolid-resistant enterococcus isolates from a tertiary care center. Moreover, the antimicrobial susceptibility pattern of these isolates was also determined. MATERIALS AND METHODS: A prospective study was performed in Medical College, Kolkata, India, over a period of two years (from January 2018 to December 2019). After obtaining clearance from the Institutional Ethics Committee, Enterococcus isolates from various samples were included in the present investigation. In addition to the various conventional biochemical tests, the VITEK 2 Compact system was used to identify the Enterococcus species. The isolates were tested for antimicrobial susceptibility to different antibiotics using the Kirby-Bauer disk diffusion method and VITEK 2 Compact to determine the minimum inhibitory concentration (MIC). The Clinical and Laboratory Standards Institute (CLSI) 2017 guidelines were used to interpret susceptibility. Multiplex PCR was performed for genetic characterization of the vancomycin-resistant Enterococcus isolates and sequencing was performed for characterization of the linezolid-resistant Enterococcus isolates. RESULTS: During the period of two years, 371 isolates of Enterococcus spp. were obtained from 4934 clinical isolates showing a prevalence of 7.52%. Among these isolates, 239 (64.42%) were Enterococcus faecalis, 114 (30.72%) Enterococcus faecium, and others were Enterococcus durans, Enterococcus casseliflavus, Enterococcus gallinarum, and Enterococcus avium. Among these, 24 (6.47%) were VRE (Vancomycin-Resistant Enterococcus) of which 18 isolates were Van A type and six isolates of Enterococcus casseliflavus and Enterococcus gallinarum were resistant VanC type. There were two linezolid-resistant Enterococcus, and they were found to have the G2576T mutation. Among the 371 isolates, 252 (67.92%) were multi-drug resistant. CONCLUSION: This study found an increasing prevalence of vancomycin-resistant Enterococcus isolates. There is also an alarming prevalence of multidrug resistance among these isolates.

Incidence and Predictors of Liver-Related Events in Patients With Nonalcoholic Fatty Liver Disease.

Background: Nonalcoholic fatty liver disease (NAFLD) is the commonest type of liver disease worldwide. We aimed to assess the incidence and predictors of liver-related events (LREs) and mortality in NAFLD patients. Methods: NAFLD patients (n = 957) evaluated between January 2000 and November 2021 were included. Patients were categorised as noncirrhosis (NC), compensated cirrhosis (CC) and decompensated cirrhosis (DC), and the incidence of LRE and mortality were estimated and compared. Results: The proportions of NC, CC and DC were 87.8% (n = 840), 8.8% (n = 84) and 3.4% (n = 33), respectively. The median follow-up duration was 3.9 (3.0-5.7) years, and the total cumulative duration was 4633 person-years. The incidence of LRE per 100 person-years was 0.14, 2.72 and 10.24 in patients with NC, CC and DC, respectively. The incidence of mortality was 0.12, 1.05 and 4.24 per 100 person-years, respectively, in the 3 groups. The causes of mortality in the 3 groups were liver related in 1/5 (20%), 3/4 (75%) and 6/9 (66.7%), respectively. Overall, the mortality rate was higher in those with diabetes than those without diabetes (log-rank P value = 0.005). On further analysis, diabetes was associated with poor outcomes only in NC group (log-rank P value = 0.036), and not in CC (log-rank P value = 0.353) or DC groups (log-rank P value = 0.771). On multivariate Cox proportional hazard analysis, age (hazard ratio [HR] 1.070), hypertension (HR 4.361) and DC (HR 15.036) were independent predictors of poor outcomes. Liver stiffness measurement, bilirubin, CC and DC were independent predictors of LRE. Conclusion: In our study of NAFLD from India, the incidence of LRE was found to be similar to that seen in Western studies. In NC NAFLD, diabetes was associated with poor outcomes.

Evaluation of the anticarcinogenic potential of the endophyte, Streptomyces sp. LRE541 isolated from Lilium davidii var. unicolor (Hoog) Cotton.

BACKGROUND: Endophytic actinomycetes, as emerging sources of bioactive metabolites, have been paid great attention over the years. Recent reports demonstrated that endophytic streptomycetes could yield compounds with potent anticancer properties that may be developed as chemotherapeutic drugs. RESULTS: Here, a total of 15 actinomycete-like isolates were obtained from the root tissues of Lilium davidii var. unicolor (Hoog) Cotton based on their morphological appearance, mycelia coloration and diffusible pigments. The preliminary screening of antagonistic capabilities of the 15 isolates showed that isolate LRE541 displayed antimicrobial activities against all of the seven tested pathogenic microorganisms. Further in vitro cytotoxicity test of the LRE541 extract revealed that this isolate possesses potent anticancer activities with IC50 values of 0.021, 0.2904, 1.484, 4.861, 6.986, 8.106, 10.87, 12.98, and 16.94 µg/mL against cancer cell lines RKO, 7901, HepG2, CAL-27, MCF-7, K562, Hela, SW1990, and A549, respectively. LRE541 was characterized and identified as belonging to the genus Streptomyces based on the 16S rRNA gene sequence analysis. It produced extensively branched red substrate and vivid pink aerial hyphae that changed into amaranth, with elliptic spores sessile to the aerial mycelia. To further explore the mechanism underlying the decrease of cancer cell viability following the LRE541 extract treatment, cell apoptosis and cell cycle arrest assays were conducted in two cancer cell lines, RKO and 7901. The result demonstrated that LRE541 extract inhibited cell proliferation of RKO and 7901 by causing cell cycle arrest both at the S phase and inducing apoptosis in a dose-dependent manner. The chemical profile of LRE541 extract performed by the UHPLC-MS/MS analysis revealed the presence of thirty-nine antitumor compounds in the extract. Further chemical investigation of the LRE541 extract led to the discovery of one prenylated indole diketopiperazine (DKP) alkaloid, elucidated as neoechinulin A, a known antitumor agent firstly detected in Streptomyces; two anthraquinones 4-deoxy-ε-pyrromycinone (1) and epsilon-pyrromycinone (2) both displaying anticancer activities against RKO, SW1990, A549, and HepG2 with IC50 values of 14.96 ± 2.6 - 20.42 ± 4.24 µg/mL for (1); 12.9 ± 2.13, 19.3 ± 4.32, 16.8 ± 0.75, and 18.6 ± 3.03 µg/mL for (2), respectively. CONCLUSION: Our work evaluated the anticarcinogenic potential of the endophyte, Streptomyces sp. LRE541 and obtained one prenylated indole diketopiperazine alkaloid and two anthraquinones. Neoechinulin A, as a known antitumor agent, was identified for the first time in Streptomyces. Though previously found in Streptomyces, epsilon-pyrromycinone and 4-deoxy-ε-pyrromycinone were firstly shown to possess anticancer activities.

The Soluble Adenylyl Cyclase Inhibitor LRE1 Prevents Hepatic Ischemia/Reperfusion Damage Through Improvement of Mitochondrial Function.

Hepatic ischemia/reperfusion (I/R) injury is a leading cause of organ dysfunction and failure in numerous pathological and surgical settings. At the core of this issue lies mitochondrial dysfunction. Hence, strategies that prime mitochondria towards damage resilience might prove applicable in a clinical setting. A promising approach has been to induce a mitohormetic response, removing less capable organelles, and replacing them with more competent ones, in preparation for an insult. Recently, a soluble form of adenylyl cyclase (sAC) has been shown to exist within mitochondria, the activation of which improved mitochondrial function. Here, we sought to understand if inhibiting mitochondrial sAC would elicit mitohormesis and protect the liver from I/R injury. Wistar male rats were pretreated with LRE1, a specific sAC inhibitor, prior to the induction of hepatic I/R injury, after which mitochondria were collected and their metabolic function was assessed. We find LRE1 to be an effective inducer of a mitohormetic response based on all parameters tested, a phenomenon that appears to require the activity of the NAD+-dependent sirtuin deacylase (SirT3) and the subsequent deacetylation of mitochondrial proteins. We conclude that LRE1 pretreatment leads to a mitohormetic response that protects mitochondrial function during I/R injury.

Low Radiation Environment Switches the Overgrowth-Induced Cell Apoptosis Toward Autophagy.

Low radiation doses can affect and modulate cell responses to various stress stimuli, resulting in perturbations leading to resistance or sensitivity to damage. To explore possible mechanisms taking place at an environmental radiation exposure, we set-up twin biological models, one growing in a low radiation environment (LRE) laboratory at the Gran Sasso National Laboratory, and one growing in a reference radiation environment (RRE) laboratory at the Italian National Health Institute (Istituto Superiore di Sanità, ISS). Studies were performed on pKZ1 A11 mouse hybridoma cells, which are derived from the pKZ1 transgenic mouse model used to study the effects of low dose radiation, and focused on the analysis of cellular/molecular end-points, such as proliferation and expression of key proteins involved in stress response, apoptosis, and autophagy. Cells cultured up to 4 weeks in LRE showed no significant differences in proliferation rate compared to cells cultured in RRE. However, caspase-3 activation and PARP1 cleavage were observed in cells entering to an overgrowth state in RRE, indicating a triggering of apoptosis due to growth-stress conditions. Notably, in LRE conditions, cells responded to growth stress by switching toward autophagy. Interestingly, autophagic signaling induced by overgrowth in LRE correlated with activation of p53. Finally, the gamma component of environmental radiation did not significantly influence these biological responses since cells grown in LRE either in incubators with or without an iron shield did not modify their responses. Overall, in vitro data presented here suggest the hypothesis that environmental radiation contributes to the development and maintenance of balance and defense response in organisms.

Safety and efficacy of stereoelectroencephalography in pediatric focal epilepsy: a single-center experience.

OBJECTIVE: Patients with medically refractory localization-related epilepsy (LRE) may be candidates for surgical intervention if the seizure onset zone (SOZ) can be well localized. Stereoelectroencephalography (SEEG) offers an attractive alternative to subdural grid and strip electrode implantation for seizure lateralization and localization; yet there are few series reporting the safety and efficacy of SEEG in pediatric patients. METHODS: The authors review their initial 3-year consecutive experience with SEEG in pediatric patients with LRE. SEEG coverage, SOZ localization, complications, and preliminary seizure outcomes following subsequent surgical treatments are assessed. RESULTS: Twenty-five pediatric patients underwent 30 SEEG implantations, with a total of 342 electrodes placed. Ten had prior resections or ablations. Seven had no MRI abnormalities, and 8 had multiple lesions on MRI. Based on preimplantation hypotheses, 7 investigations were extratemporal (ET), 1 was only temporal-limbic (TL), and 22 were combined ET/TL investigations. Fourteen patients underwent bilateral investigations. On average, patients were monitored for 8 days postimplant (range 3-19 days). Nearly all patients were discharged home on the day following electrode explantation. There were no major complications. Minor complications included 1 electrode deflection into the subdural space, resulting in a minor asymptomatic extraaxial hemorrhage; and 1 in-house and 1 delayed electrode superficial scalp infection, both treated with local wound care and oral antibiotics. SEEG localized the hypothetical SOZ in 23 of 25 patients (92%). To date, 18 patients have undergone definitive surgical intervention. In 2 patients, SEEG localized the SOZ near eloquent cortex and subdural grids were used to further delineate the seizure focus relative to mapped motor function just prior to resection. At last follow-up (average 21 months), 8 of 15 patients with at least 6 months of follow-up (53%) were Engel class I, and an additional 6 patients (40%) were Engel class II or III. Only 1 patient was Engel class IV. CONCLUSIONS: SEEG is a safe and effective technique for invasive SOZ localization in medically refractory LRE in the pediatric population. SEEG permits bilateral and multilobar investigations while avoiding large craniotomies. It is conducive to deep, 3D, and perilesional investigations, particularly in cases of prior resections. Patients who are not found to have focally localizable seizures are spared craniotomies.

Measuring E. coli and bacteriophage DNA in cell sonicates to evaluate the CAL1 reaction as a synthetic biology standard for qPCR.

We measured the impact of the presence of total Escherichia coli (E. coli) cellular material on the performance of the Linear Regression of Efficiency (LRE) method of absolute quantitative PCR (LRE qPCR), which features the putatively universal CAL1 calibration reaction, which we propose as a synthetic biology standard. We firstly used a qPCR reaction in which a sequence present in the lone genomic BirA locus is amplified. Amplification efficiency for this reaction, a key metric for many quantitative qPCR methods, was inhibited by cellular material from bioreactor cultivation to a greater extent than material from shake flask cultivation. We then compared LRE qPCR to the Standard Curve method of absolute qPCR (SC qPCR). LRE qPCR method matched the performance of the SC qPCR when used to measure 417-4.17 × 107 copies of the BirA target sequence present in a shake flask-derived cell sonicates sample, and for 97-9.7 × 105 copies in the equivalent bioreactor-derived sample. A plasmid-encoded T7 bacteriophage sequence was next used to compare the methods. In the presence of cell sonicates from samples of up to OD600 = 160, LRE qPCR outperformed SC qPCR in the range of 1.54 × 108-1.54 × 1010 copies of the T7 target sequence and matched SC qPCR over 1.54 × 104-1.54 × 107 copies. These data suggest the CAL1 standard, combined with the LRE qPCR method, represents an attractive choice as a synthetic biology qPCR standard that performs well even when unpurified industrial samples are used as the source of template material.