Rapid Direct Identification of Microbial Pathogens and Antimicrobial Resistance Genes in Positive Blood Cultures Using a Fully Automated Multiplex PCR Assay.

This study assessed the performance of the BioFire Blood Culture Identification 2 (BCID2) panel in identifying microorganisms and antimicrobial resistance (AMR) profiles in positive blood cultures (BCs) and its influence on turnaround time (TAT) compared with conventional culture methods. We obtained 117 positive BCs, of these, 102 (87.2%) were correctly identified using BCID2. The discordance was due to off-panel pathogens detected by culture (n = 13), and additional pathogens identified by BCID2 (n = 2). On-panel pathogen concordance between the conventional culture and BCID2 methods was 98.1% (102/104). The conventional method detected 19 carbapenemase-producing organisms, 14 extended-spectrum beta-lactamase-producing Enterobacterales, 18 methicillin-resistant Staphylococcus spp., and four vancomycin-resistant Enterococcus faecium. BCID2 correctly predicted 53 (96.4%) of 55 phenotypic resistance patterns by detecting AMR genes. The TAT for BCID2 was significantly lower than that for the conventional method. BCID2 rapidly identifies pathogens and AMR genes in positive BCs.

Identification of an Enterococcus faecium strain isolated from raw bovine milk co-harbouring the oxazolidinone resistance genes optrA and poxtA in China.

Oxazolidinones are potent antimicrobial agents used to treat human infections caused by multidrug-resistant Gram-positive bacteria. The growing resistance to oxazolidinones poses a significant threat to public health. In August 2021, a linezolid-resistant Enterococcus faecium BN83 was isolated from a raw milk sample of cow in Inner Mongolia, China. This isolate exhibited a multidrug resistance phenotype and was resistant to most of drugs tested including linezolid and tedizolid. PCR detection showed that two mobile oxazolidinones resistance genes, optrA and poxtA, were present in this isolate. Whole genome sequencing analysis revealed that the genes optrA and poxtA were located on two different plasmids, designated as pBN83-1 and pBN83-2, belonging to RepA_N and Inc18 families respectively. Genetic context analysis suggested that optrA gene on plasmid pBN83-1 was located in transposon Tn6261 initially found in E. faecalis. Comprehensive analysis revealed that Tn6261 act as an important horizontal transmission vector for the spread of optrA in E. faecium. Additionally, poxtA-bearing pBN83-2 displayed high similarity to numerous plasmids from Enterococcus of different origin and pBN83-2-like plasmid represented a key mobile genetic element involved in movement of poxtA in enterococcal species. The presence of optrA- and poxtA-carrying E. faecium in raw bovine milk represents a public health concern and active surveillance is urgently warranted to investigate the prevalence of oxazolidinone resistance genes in animal-derived food products.

Genomic analysis of inter-hospital transmission of vancomycin-resistant Enterococcus faecium sequence type 80 isolated during an outbreak in Hiroshima, Japan.

Outbreaks caused by vancomycin-resistant enterococci that transcend jurisdictional boundaries are occurring worldwide. This study focused on a vancomycin-resistant enterococcus outbreak that occurred between 2018 and 2021 across two cities in Hiroshima, Japan. The study involved genetic and phylogenetic analyses using whole-genome sequencing of 103 isolates of vancomycin-resistant enterococci to identify the source and transmission routes of the outbreak. Phylogenetic analysis was performed using core genome multilocus sequence typing and core single-nucleotide polymorphisms; infection routes between hospitals were inferred using BadTrIP. The outbreak was caused by Enterococcus faecium sequence type (ST) 80 carrying the vanA plasmid, which was derived from strain A10290 isolated in India. Of the 103 isolates, 93 were E. faecium ST80 transmitted across hospitals. The circular vanA plasmid of the Hiroshima isolates was similar to the vanA plasmid of strain A10290 and transferred from E. faecium ST80 to other STs of E. faecium and other Enterococcus species by conjugation. The inferred transmission routes across hospitals suggest the existence of a central hospital serving as a hub, propagating vancomycin-resistant enterococci to multiple hospitals. Our study highlights the importance of early intervention at the key central hospital to prevent the spread of the infection to small medical facilities, such as nursing homes, with limited medical resources and a high number of vulnerable individuals.

Dramatic increase in antimicrobial resistance in ESKAPE clinical isolates over the 2010-2020 decade in India.

RATIONALE AND OBJECTIVES: ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) constitute a threat to humans worldwide. India is now the most populous country. The goal was to investigate the evolution of the rates of antimicrobial resistance in ESKAPE pathogens across India over the 2010-20 decade. METHODS: The data (89 studies) were retrieved from the Medline PubMed repository using specific keywords. RESULTS: The study of 20 177 ESKAPE isolates showed that A. baumannii isolates were the most represented (35.9%, n = 7238), followed by P. aeruginosa (25.3%, n = 5113), K. pneumoniae (19.5%, n = 3934), S. aureus (16.3%, n = 3286), E. faecium (2.6%, n = 517) and Enterobacter spp. (0.4%, n = 89). A notable increase in the resistance rates to antimicrobial agents occurred over the 2010-20 decade. The most important levels of resistance were observed in 2016-20 for A. baumannii (90% of resistance to the amoxicillin-clavulanate combination) and K. pneumoniae (81.6% of resistance to gentamycin). The rise in ß-lactamase activities was correlated with an increase in the positivity of Gram-negative isolates for ß-lactamase genes. CONCLUSIONS: This review highlighted that, in contrast to developed countries that kept resistance levels under control, a considerable increase in resistance to various classes of antibiotics occurred in ESKAPE pathogens in India over the 2010-2020 decade.

Distinction between Enterococcus faecium and Enterococcus lactis by a gluP PCR-Based Assay for Accurate Identification and Diagnostics.

It was recently proposed that Enterococcus faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis. Our goals were to develop a PCR assay to rapidly differentiate these species and to discuss the main phenotypic and genotypic differences from a clinical perspective. The pan-genome of 512 genomes of E. faecium and E. lactis strains was analyzed to assess diversity in genes between the two species. Sequences were aligned to find the best candidate gene for designing species-specific primers, and their accuracy was tested with a collection of 382 enterococci. E. lactis isolates from clinical origins were further characterized by whole-genome sequencing (Illumina). Pan-genome analysis resulted in 12 gene variants, with gene gluP (rhomboid protease) being selected as the candidate for species differentiation. The nucleotide sequence of gluP diverged by 90 to 92% between sets, which allowed species identification through PCR with 100% specificity and no cross-reactivity. E. lactis strains were greatly pan-susceptible and not host specific. Hospital E. lactis isolates were susceptible to clinically relevant antibiotics, lacked infection-associated virulence markers, and were associated with patients presenting risk factors for enhanced bacterial translocation. Here, we propose a PCR-based assay using gluP for easy routine differentiation between E. faecium and E. lactis that could be implemented in different public health contexts. We further suggest that E. lactis, a dominant human gut species, can cross the gut barrier in severely ill, immunodeficient, and surgical patients. Knowing that bacterial translocation may be a sepsis promoter, the relevance of infections caused by E. lactis strains, even if they are pan-susceptible, should be explored. IMPORTANCE Enterococcus faecium is a WHO priority pathogen that causes severe and hard-to-treat human infections. It was recently proposed that E. faecium colonizing the human gut (previous clade B) actually corresponds to Enterococcus lactis; therefore, some of the human infections occurring globally are being misidentified. In this work, we developed a PCR-based rapid identification method for the differentiation of E. faecium and E. lactis and discussed the main phenotypic and genotypic differences of these species from a clinical perspective. We identified the gluP gene as the best candidate, based on the phylogenomic analysis of 512 published pan-genomes, and validated the PCR assay with a comprehensive collection of 382 enterococci obtained from different sources. Further detailed analysis of clinical E. lactis strains showed that they are highly susceptible to antibiotics and lack the typical virulence markers of E. faecium but are able to cause severe human infections in immunosuppressed patients, possibly in part due to gut barrier translocation.

Rare transmission of commensal and pathogenic bacteria in the gut microbiome of hospitalized adults.

Bacterial bloodstream infections are a major cause of morbidity and mortality among patients undergoing hematopoietic cell transplantation (HCT). Although previous research has demonstrated that pathogens may translocate from the gut microbiome into the bloodstream to cause infections, the mechanisms by which HCT patients acquire pathogens in their microbiome have not yet been described. Here, we use linked-read and short-read metagenomic sequencing to analyze 401 stool samples collected from 149 adults undergoing HCT and hospitalized in the same unit over three years, many of whom were roommates. We use metagenomic assembly and strain-specific comparison methods to search for high-identity bacterial strains, which may indicate transmission between the gut microbiomes of patients. Overall, the microbiomes of patients who share time and space in the hospital do not converge in taxonomic composition. However, we do observe six pairs of patients who harbor identical or nearly identical strains of the pathogen Enterococcus faecium, or the gut commensals Akkermansia muciniphila and Hungatella hathewayi. These shared strains may result from direct transmission between patients who shared a room and bathroom, acquisition from a common hospital source, or transmission from an unsampled intermediate. We also identify multiple patients with identical strains of species commonly found in commercial probiotics, including Lactobacillus rhamnosus and Streptococcus thermophilus. In summary, our findings indicate that sharing of identical pathogens between the gut microbiomes of multiple patients is a rare phenomenon. Furthermore, the observed potential transmission of commensal, immunomodulatory microbes suggests that exposure to other humans may contribute to microbiome reassembly post-HCT.

Optimising genomic approaches for identifying vancomycin-resistant Enterococcus faecium transmission in healthcare settings.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major nosocomial pathogen. Identifying VREfm transmission dynamics permits targeted interventions, and while genomics is increasingly being utilised, methods are not yet standardised or optimised for accuracy. We aimed to develop a standardized genomic method for identifying putative VREfm transmission links. Using comprehensive genomic and epidemiological data from a cohort of 308 VREfm infection or colonization cases, we compared multiple approaches for quantifying genetic relatedness. We showed that clustering by core genome multilocus sequence type (cgMLST) was more informative of population structure than traditional MLST. Pairwise genome comparisons using split k-mer analysis (SKA) provided the high-level resolution needed to infer patient-to-patient transmission. The more common mapping to a reference genome was not sufficiently discriminatory, defining more than three times more genomic transmission events than SKA (3729 compared to 1079 events). Here, we show a standardized genomic framework for inferring VREfm transmission that can be the basis for global deployment of VREfm genomics into routine outbreak detection and investigation.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Recovery of probiotic bacteria from the intestinal tract of broilers after noninvasive pre-hatch application.

This paper reports the success of intestinal colonisation of chickens and foetuses by probiotics after different methods of pre-hatch application. Hatcheries not using in ovo injection of probiotics or wish to avoid the reduced hatchability associated with in ovo injections prefer using alternatives to in ovo technologies. Therefore, we used noninvasive pre-hatch application methods. This included the vertical transmission of probiotics from the mother hen to offspring, application of probiotic late in incubation and transmission of probiotics during hatch. Enterococcus faecium (NCIMB11181) and Lactobacillus animalis (DSM33570) were used as probiotics. Probiotics were applied either through drinking water for the mother hens, by dipping the eggs in a probiotic solution on days 16-18 of incubation or through drops/spray on the eggshell of the fertilised eggs. Similarly, intestinal colonisation of the probiotic in chickens was investigated either before hatch (pre-hatch) or immediately after hatch (post-hatch). Based on the performed experiments, it is concluded that E. faecium was vertically transmitted from the mother hen to the offspring, as E. faecium was recovered in 20 and 33% of the offspring pre- and post-hatch, respectively. When applied on the eggshell, the recovery of E. faecium before hatch depended on the application method and ranged from 0 to 9%. In contrast, L. animalis was not recovered before hatch. Moreover, when sampling post-hatch 100% of the chickens were colonised when E. faecium was used and 54% were colonised when L. animalis was used. Furthermore, spray application with E. faecium was the most successful application method as 9% of the foetuses were colonised pre-hatch and 100% became colonised post-hatch. Therefore, pre-hatch application by, for example, spray of probiotics on the eggshell can be used as an easy-to-use, noninvasive method for early life colonisation of chicken gut.

Direct detection of ESKAPEc pathogens from whole blood using the T2Bacteria Panel allows early antimicrobial stewardship intervention in patients with sepsis.

In the microbiological diagnosis of bloodstream infections (BSI), blood culture (BC) is considered the gold standard test despite its limitations such as low sensitivity and slow turnaround time. A new FDA-cleared and CE-marked platform utilizing magnetic resonance to detect amplified DNA of the six most common and/or problematic BSI pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli; referred to as ESKAPEc) is available and may shorten the time to diagnosis and potentially improve antimicrobial utilization. Whole blood samples from hospitalized patients with clinical signs of sepsis were analyzed using the T2Bacteria Panel (T2Biosystems) and compared to simultaneously collected BC. Discrepant results were evaluated based on clinical infection criteria, combining supporting culture results and the opinion of treating physicians. A total of 55 samples from 53 patients were evaluated. The sensitivity and specificity of the T2Bacteria panel was 94% (16 out of 17 detections of T2Bacteria-targeted organisms) and 100%, respectively, with 36.4% (8 of 22) causes of BSI detected only by this method. The T2Bacteria Panel detected pathogens on average 55 hours faster than standard BC. In our study, 9 of 15 patients with positive T2Bacteria Panel results received early-targeted antibiotic therapy and/or modification of antimicrobial treatment based on T2Bacteria Panel findings. Given the high reliability, faster time to detection, and easy workflow, the technique qualifies as a point-of-care testing approach.

Antimicrobial susceptibility of bacteremic vancomycin-resistant Enterococcus faecium to eravacycline, omadacycline, lipoglycopeptides, and other comparator antibiotics: Results from the 2019-2020 Nationwide Surveillance of Multicenter Antimicrobial Resistance in Taiwan (SMART).

Multicenter surveillance of antimicrobial susceptibility was performed for 235 vancomycin-resistant Enterococcus faecium (VREfm) isolates from 18 Taiwanese hospitals. The minimum inhibitory concentrations (MICs) of eravacycline, omadacycline, lipoglycopeptides, and other comparator antibiotics were determined using the broth microdilution method. Nearly all isolates of VREfm were not susceptible to teicoplanin, dalbavancin, and telavancin, with susceptibility rates of 0.5%, 1.7% and 0.5%, respectively. Tigecycline and eravacycline were active against 93.2% and 89.7% of the VREfm isolates, respectively. Moreover, the susceptibility rates of quinupristin/dalfopristin, tedizolid, and linezolid were 59.1%, 84.2%, and 77.4%, respectively. Additionally, 94% of the VREfm isolates were classified as susceptible to daptomycin, and the MICs of omadacycline required to inhibit VREfm growth by 50% and 90% were 0.12 and 0.5 mg/L, respectively. Susceptibility rates of VREfm isolates to synthetic tetracyclines and daptomycin were slightly lower and to oxazolidinone-class antibiotics were much lower in Taiwan than those in other parts of the world. Continuous monitoring of VREfm resistance to novel antibiotics, including synthetic tetracyclines, oxazolidinone-class antibiotics, and daptomycin, is needed in Taiwan.

Occurrence of multidrug-resistant Enterococcus faecium isolated from environmental samples.

Enterococcus species are present in the microbiota of humans and animals and have also been described in the environment. Among the species, Enterococcus faecium is one of the main pathogens associated with nosocomial infections worldwide. Enterococcus faecium isolates resistant to different classes of antimicrobials have been increasingly reported, including multidrug-resistant (MDR) isolates in environmental sources, which is worrying. Therefore, this study aimed to characterize E. faecium isolates obtained from soil and water samples regarding antimicrobial resistance and virulence determinants. A total 40 E. faecium isolates were recovered from 171 environmental samples. All isolates were classified as MDR, highlighting the resistance to the fluoroquinolones class, linezolid and vancomycin. Furthermore, high-level aminoglycoside resistance and high-level ciprofloxacin resistance were detected in some isolates. Several clinically relevant antimicrobial resistance genes were found, including vanC1, ermB, ermC, mefAE, tetM, tetL, ant(6')-Ia, ant(4')-Ia, aph(3')-IIIa and aac(6')-Ie-aph(2″)-Ia. Three virulence genes were detected among the MDR E. faecium isolates, such as esp, gelE and ace. The results of this study contribute to a better understanding of MDR E. faecium isolates carrying antimicrobial resistance and virulence genes in environmental sources and report for the first time in the world the presence of vanC1-producing E. faecium isolated from soil.

Comparative study of multidrug-resistant Enterococcus faecium obtained from different hosts.

Introduction. The possible transfer of antimicrobial resistance genes between Enterococcus faecium isolates from humans and different animal species, including those not covered by monitoring programs (e.g. pet and wildlife), poses a serious threat to public health.Hypothesis/Gap Statement. Little is known about occurrence and mechanisms of phenomenon of multidrug resistance of E. faecium isolated from various host species in Poland.Aim. The aim of the study was to characterize multidrug-resistant E. faecium isolated from humans and animals (livestock, pets and wildlife) in terms of the occurrence of genetic markers determining resistance.Methodology. Bacterial isolates were tested for phenotypic resistance and the presence of genes encoding resistance to macrolides, tetracycline, aminoglycosides, aminocyclitols and phenicols as well as efflux pump (emeA), resolvase (tndX) and integrase (Int-Tn) genes. The quinolone resistance-determining regions of gyrA and parC were sequenced.Results. Human isolates of E. faecium were characterized by high-level resistance to: ciprofloxacin, enrofloxacin, erythromycin (100 %), as well, as aminoglycosides resistance (kanamycin - 100%, streptomycin - 78 %, gentamicin - 78%). Regardless of the animal species, high level of resistance of E. faecium to tetracycline (from 88-100 %), erythromycin (from 82-94 %) and kanamycin (from 36-100 %) was observed. All E. faecium isolates from wildlife were resistant to fluoroquinolones. However, full susceptibility to vancomycin was observed in all isolates tested. Phenotypic antimicrobial resistance of E. faecium was identified in the presence of the following resistance genes: erm(B) (70%), msr(A) (50 %), tet(L) (35 %), tet(K) (34 %), tet(M) (76 %), aac(6')-Ie-aph(2″)-Ia (25%), ant(6)-Ia (31%), aph(3)-IIIa (68 %), (tndX) (23 %), and integrase gene (Int-Tn) (34 %). A correlation between an amino acid substitution at positions 83 and 87 of gyrA and position 80 of parC and the high-level fluoroquinolone resistance in E. faecium has been observed as well.Conclusion. The level and range of antimicrobial resistance and the panel of resistance determinants is comparable between E. faecium isolates, despite host species.

Intestinal carriage of vancomycin-resistant Enterococcus spp. among high-risk patients in university hospitals in Serbia: first surveillance report.

BACKGROUND: The screening for intestinal carriage of vancomycin-resistant Enterococcus spp. (VRE) among high risk patients in the Balkan region and molecular epidemiology of VRE is insufficiently investigated, yet it could be of key importance in infection control. The aim of this study was to provide baseline data on VRE intestinal carriage among high-risk patients in Serbian university hospitals, to determine the phenotypic/genotypic profiles of the isolated VRE, to obtain knowledge of local resistance patterns and bridge the gaps in current VRE surveillance. METHODS: The VRE reservoir was investigated using stool samples from 268 inpatients. Characterization of isolated VRE stains consisted of BD Phoenix system, genotypic identification, glycopeptide and quinupristin-dalfopristin (Q-D) resistance probing, virulence gene (esp, hyl, efaA, asa1, gelE, cpd) detection and MLVA. Biofilm formation was evaluated by the microtiter plate method. RESULTS: VRE carriage prevalence among at-risk patients was 28.7%. All VRE strains were vanA positive multidrug-resistant Enterococcus faecium (VRfm), harboring ermB-1 (38.9%), esp (84%), efaA (71.2%), hyl (54.5%), asa1 (23.4%), gelE and cpd (11.6%) each. Ability of biofilm production was detected in 20.8%. Genetic relatedness of the isolates revealed 13 clusters, heterogeneous picture and 25 unique MTs profiles. CONCLUSION: The obtained prevalence of VRE intestinal carriage among high-risk inpatients in Serbia is higher than the European average, with high percentage of multidrug resistance. The emergence of resistance to Q-D is of particular concern. Close monitoring of pattern of resistance and strict adherence to specific guidelines are urgently needed in Serbia.

Liquid Extraction Surface Analysis Mass Spectrometry of ESKAPE Pathogens.

The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae) represent clinically important bacterial species that are responsible for most hospital-acquired drug-resistant infections; hence, the need for rapid identification is of high importance. Previous work has demonstrated the suitability of liquid extraction surface analysis mass spectrometry (LESA MS) for the direct analysis of colonies of two of the ESKAPE pathogens (Staphylococcus aureus and Pseudomonas aeruginosa) growing on agar. Here, we apply LESA MS to the remaining four ESKAPE species (E. faecium E745, K. pneumoniae KP257, A. baumannii AYE, and E. cloacae S11) as well as E. faecalis V583 (a close relative of E. faecium) and a clinical isolate of A. baumannii AC02 using an optimized solvent sampling system. In each case, top-down LESA MS/MS was employed for protein identification. In total, 24 proteins were identified from 37 MS/MS spectra by searching against protein databases for the individual species. The MS/MS spectra for the identified proteins were subsequently searched against multiple databases from multiple species in an automated data analysis workflow with a view to determining the accuracy of identification of unknowns. Out of 24 proteins, 19 were correctly assigned at the protein and species level, corresponding to an identification success rate of 79%.

The global dissemination of hospital clones of Enterococcus faecium.

BACKGROUND: The hospital-adapted A1 group of Enterococcus faecium remains an organism of significant concern in the context of drug-resistant hospital-associated infections. How this pathogen evolves and disseminates remains poorly understood. METHODS: A large, globally representative collection of short-read genomic data from the hospital-associated A1 group of Enterococcus faecium was assembled (n = 973). We analysed, using a novel analysis approach, global diversity in terms of both the dynamics of the accessory genome and homologous recombination among conserved genes. RESULTS: Two main modes of genomic evolution continue to shape E. faecium: the acquisition and loss of genes, including antimicrobial resistance genes, through mobile genetic elements including plasmids, and homologous recombination of the core genome. These events lead to new clones emerging at the local level, followed by the erosion of signals of clonality through recombination, and in some identifiable cases producing new clonal clusters. These patterns lead to new, emerging lineages which are able to spread globally over relatively short timeframes. CONCLUSIONS: The ability of A1 E. faecium to continually present new combinations of genes for potential selection suggests that controlling this pathogen will remain challenging but establishing a framework for understanding genomic evolution is likely to aid in tracking the threats posed by newly emerging lineages.

High-Level Resistance to Erythromycin and Tetracycline and Dissemination of Resistance Determinants among Clinical Enterococci in Iran.

OBJECTIVES: The purpose of this study was to investigate the distribution pattern of genes responsible for erythromycin and tetracycline resistance and their association with resistance phenotypes in enterococcus isolates. MATERIALS AND METHODS: Eighty-six Enterococcus faecalis and 26 E. faecium isolates were collected from 2 hospitals in Kerman, Iran. Minimum inhibitory concentration of erythromycin and tetra-cycline was determined and then genes encoding resistance to erythromycin - erm (A-C), mef, and msr - and tetracycline - tet (M), tet (O), tet (S), tet (K), and tet (L) - were investigated. RESULTS: In all resistant isolates (n = 72, 64%), high-level resistance to both tested antibiotics was found. The most prevalent erm gene was erm (B) (77.7%), followed by erm (A) (15.2%) and erm (C) (8.3%). Genes mediating erythromycin efflux were detected in 70.8% (mef) and 9.7% (msr) of resistant isolates. Regarding tetracycline, tet (M) was detected at the highest rate (50%), followed by tet (O) (31%) and tet (S) (11%). Export of tetracycline was found in 31% (tet (K)) and 12% (tet (L)) of isolates. CONCLUSION: A high prevalence of high-level resistance to both erythromycin and tetracycline was documented. Alterations at the ribosomal level was more frequently detected in erythromycin and tetracycline resistance than efflux systems. Concurrent resistance mechanisms were more involved in resistance to erythromycin than tetracycline.

Ultraviolet-induced in situ gold nanoparticles for point-of-care testing of infectious diseases in loop-mediated isothermal amplification.

The present study investigated ultraviolet-induced in situ gold nanoparticles (AuNPs) coupled with loop-mediated isothermal amplification (LAMP) for the point-of-care testing (POCT) of two major infectious pathogens, namely, Coronavirus (COVID-19) and Enterococcus faecium (E. faecium spp.). In the process, gold ions in a gold chloride (HAuCl4) solution were reduced using trisodium citrate (Na3Ct), a reducing agent, and upon UV illumination, red-colored AuNPs were produced in the presence of LAMP amplicons. The nitrogenous bases of the target deoxyribonucleic acid (DNA) acted as a physical support for capturing gold ions dissolved in the sample. The high affinity of gold with the nitrogenous bases enabled facile detection within 10 min, and the detection limit of COVID-19 plasmid DNA was as low as 42 fg µL-1. To ensure POCT, we designed a portable device that contained arrays of reagent chambers and detection chambers. In the portable device, colorimetric reagents such as HAuCl4 and Na3Ct were contained in the reagent chambers; these reagents were subsequently transferred to the detection chambers where LAMP amplicons were present and thus allowed convenient sample delivery and multiplex detection. Owing to the high sensitivity of the in situ AuNPs, simplicity of portable device fabrication, and rapid colorimetric detection, we strongly believe that the fabricated portable device could serve as a kit for rapid POCT for instantaneous detection of infectious diseases, and could be readily usable at the bedside.

Vancomycin-resistant Enterococcus faecium in Algeria: phenotypic and genotypic characterization of clinical isolates.

INTRODUCTION: vancomycin-resistant Enterococcus faecium (VREfm) is a major public health problem worldwide. The aim of our study was to determine the microbiological, epidemiological and molecular characteristics of VREfm isolated in north-central, eastern and western Algeria. METHODOLOGY: a collection of 48 VREfm isolated from September 2010 to April 2017 in several Algerian hospitals were studied. Minimum inhibitory concentrations (MICs) were determined by E-test method according to CLSI guidelines. the detection of van genotype of all strains was performed by PCR. Clonal relationship of five VREfm targeted by region were characterized using multilocus sequence typing (MLST). RESULTS: All isolates have multidrug-resistance (MDR) and were resistant to at least five classes of antibiotics; however, all were susceptible to tigecycline and daptomycin with MIC50 at 0.094 µg/mL and 2 µg/mL respectively. All strains belonged to vanA genotype and have high level of resistance to vancomycin and teicoplanin. MLST revealed two sequence types (STs): ST80 (from the four regions of Algeria) and ST789, both belonging to the former hospital-adapted clonal complex CC17. CONCLUSIONS: the alarming dissemination of MDR E. faecium vanA and the ST80 in several regions of Algeria suggest a clonal spread of VREfm strains, which urgently require implementation of adequate infection control measures.

Characterization of a novel theta-type plasmid pSM409 of Enterococcus faecium RME isolated from raw milk.

Enterococcal plasmids have generated renewed interest for their indispensable role in pathogenesis and dissemination of multidrug-resistance. Recently, a novel plasmid pSM409 (4303-bp, GC% = 33.6%), devoid of antibiotic-resistance and virulence genes, has been identified in Enterococcus faecium RME, isolated from raw milk by us. pSM409 contains six open reading frames encoding a replication initiator protein (RepB) and five accessory proteins: antitoxin epsilon, bacteriocin immunity protein, HsdS, and two hypothetical proteins. Comparative sequence analysis of pSM409 reveals a mosaic pattern of similarity with different loci obtained from different theta plasmids, which dictates the plasmid to be heterogeneous or mosaic, possibly due to recombination. The pSM409 comprised of a typical theta-type origin of replication with four and a half direct repeats (iterons) of 22 nucleotides. The pSM409-RepB shared 76-82% homology with the RepB of reported theta plasmids from different genera, with dissimilarities mostly in its DNA-binding and C-terminal domain. The RepB sequence-based phylogenetic tree revealed its distinct position relative to the reported ones. The RepB grouped in the same clade has identical DNA-binding domains and their cognate iterons, possibly due to their sequence-specific interaction to initiate plasmid replication. Comparative analysis of the pSM409-iteron reveals that the repeats markedly differed from their closest homologues. This clade-specific relationship provides a new concept of classifying theta plasmids. The theta-type replicon identified in pSM409 has been found to be unique to E. faecium RME, prompting us to further investigate its utility as a vector for genetic manipulation of enterococci for health and industry.