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.

VanA-Enterococcus faecalis in Poland: hospital population clonal structure and vanA mobilome.

The aim of our study was to characterize the epidemiological situation concerning nosocomial vancomycin-resistant Enterococcus faecalis of VanA-phenotype (VREfs-VanA) in Poland by investigating their clonal relationships and the vanA-associated mobilome. One-hundred twenty-five clinical isolates of VREfs-VanA collected between 2004 and 2016 were studied by phenotypic assays, multilocus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), PCR detection of plasmid-specific genes, and Tn1546 structure and localization mapping. Selected isolates were subjected to PFGE-S1, Southern hybridization, genomic sequencing and conjugation experiments. The majority of isolates (97.6%) belonged to clonal complexes CC2 and CC87 of E. faecalis. All isolates were resistant to vancomycin and teicoplanin, and resistance to ciprofloxacin and aminoglycosides (high level) was very prevalent in this group. VanA phenotype was associated with 16 types of Tn1546, carrying insertion sequences IS1216, ISEfa4, IS1251 and IS1542, located on repUS1pVEF1, rep1pIP501, rep2pRE25, rep9pAD1/pTEF2/pCF10 and rep6pS86 replicons. The most common Tn1546 B- and BB-type transposons, harbouring one or two copies of IS1216, were inserted between rep18ap200B and repUS1pVEF1 genes and located on ~ 20 kb and 150-200 kb plasmids. VREfs-VanA in Poland represent a polyclonal group, indicating a number of acquisitions of the vanA determinant. The repUS1pVEF1-vanA plasmids, unique for Poland, were the main factor beyond the acquisition of vancomycin resistance by E. faecalis, circulating in Polish hospitals.

Complete Genome Sequence of a Polish Enterococcus faecalis vanA-Positive Hospital Isolate.

Enterococcus faecalis is an important human pathogen involved in health care-associated infections, and its increasing resistance to vancomycin is worrisome. Here, we report the complete genome sequence of a Polish hospital vanA-positive isolate of E. faecalis, consisting of a 3,264,821-bp chromosome and six plasmids.

Molecular analysis of vanA outbreak of Enterococcus faecium in two Warsaw hospitals: the importance of mobile genetic elements.

Vancomycin-resistant Enterococcus faecium represents a growing threat in hospital-acquired infections. Two outbreaks of this pathogen from neighboring Warsaw hospitals have been analyzed in this study. Pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA, multilocus VNTR analysis (MLVA), and multilocus sequence typing (MLST) revealed a clonal variability of isolates which belonged to three main lineages (17, 18, and 78) of nosocomial E. faecium. All isolates were multidrug resistant and carried several resistance, virulence, and plasmid-specific genes. Almost all isolates shared the same variant of Tn1546 transposon, characterized by the presence of insertion sequence ISEf1 and a point mutation in the vanA gene. In the majority of cases, this transposon was located on 50 kb or 100 kb pRUM-related plasmids, which lacked, however, the axe-txe toxin-antitoxin genes. 100 kb plasmid was easily transferred by conjugation and was found in various clonal backgrounds in both institutions, while 50 kb plasmid was not transferable and occurred solely in MT159/ST78 strains that disseminated clonally in one institution. Although molecular data indicated the spread of VRE between two institutions or a potential common source of this alert pathogen, epidemiological investigations did not reveal the possible route by which outbreak strains disseminated.

Abundance and diversity of plasmid-associated genes among clinical isolates of Enterococcus faecalis.

Enterococcus faecalis, a normal compound of the human intestinal microbiome, plays an important role in hospital-acquired infections. Plasmids make a significant contribution to the acquisition of the novel traits such as antimicrobial resistance and virulence by this pathogen. The study investigated the plasmid content and the diversity of plasmid-associated genes in a group of 152 hospital isolates of E. faecalis. The majority of plasmids visualized by pulsed-field gel electrophoresis of S1 nuclease-digested DNA fell into the range of 50-100 kb. PCR-based screening allowed detection of genes of the rep1(pIP501), rep2(pRE25), rep4(pMBB1), rep6(pS86), rep7(pT181), rep8(pAM373), rep9(pAD1/pTEF2/pCF10), rep10(pIM13) and rep13(pC194) families in 29 different combinations. The par and ω-ε-ζ plasmid stabilization systems were ubiquitous (45 isolates, 29.6% and 88 isolates, 57.9%, respectively), while the axe-txe system was not found. The asa1 gene homologues encoding aggregation substance characteristic for the pAD1 and related group of pheromone-responsive plasmids were present in 106 isolates. A variety of sequence variants, including novel ones, of genes associated with pheromone-responsive plasmids, such as rep8(pAM373), rep9(pAD1/pTEF2/pCF10), par, and asa1 were observed. In conclusion, there is a big and only partially characterized pool of diverse plasmids in clinical E. faecalis.

Insights into the transposable mobilome of Paracoccus spp. (Alphaproteobacteria).

Several trap plasmids (enabling positive selection of transposition events) were used to identify a pool of functional transposable elements (TEs) residing in bacteria of the genus Paracoccus (Alphaproteobacteria). Complex analysis of 25 strains representing 20 species of this genus led to the capture and characterization of (i) 37 insertion sequences (ISs) representing 9 IS families (IS3, IS5, IS6, IS21, IS66, IS256, IS1182, IS1380 and IS1634), (ii) a composite transposon Tn6097 generated by two copies of the ISPfe2 (IS1634 family) containing two predicted genetic modules, involved in the arginine deiminase pathway and daunorubicin/doxorubicin resistance, (iii) 3 non-composite transposons of the Tn3 family, including Tn5393 carrying streptomycin resistance and (iv) a transposable genomic island TnPpa1 (45 kb). Some of the elements (e.g. Tn5393, Tn6097 and ISs of the IS903 group of the IS5 family) were shown to contain strong promoters able to drive transcription of genes placed downstream of the target site of transposition. Through the application of trap plasmid pCM132TC, containing a promoterless tetracycline resistance reporter gene, we identified five ways in which transposition can supply promoters to transcriptionally silent genes. Besides highlighting the diversity and specific features of several TEs, the analyses performed in this study have provided novel and interesting information on (i) the dynamics of the process of transposition (e.g. the unusually high frequency of transposition of TnPpa1) and (ii) structural changes in DNA mediated by transposition (e.g. the generation of large deletions in the recipient molecule upon transposition of ISPve1 of the IS21 family). We also demonstrated the great potential of TEs and transposition in the generation of diverse phenotypes as well as in the natural amplification and dissemination of genetic information (of adaptative value) by horizontal gene transfer, which is considered the driving force of bacterial evolution.

Complex nature of enterococcal pheromone-responsive plasmids.

Pheromone-responsive plasmids constitute a unique group of approximately 20 plasmids identified, as yet, only among enterococcal species. Several of their representatives, e.g. pAD1, pCF10, pPD1 and pAM373 have been extensively studied. These plasmids possess a sophisticated conjugation mechanism based on response to sex pheromones--small peptides produced by plasmid-free recipient cells. Detailed analysis of regulation and function of the pheromone response process revealed its great complexity and dual role--in plasmid conjugation and modulation of enterococcal virulence. Among other functional modules identified in pheromone plasmids, the stabilization/partition systems play a crucial role in stable maintenance of the plasmid molecule in host bacteria. Among them, the par locus of pAD1 is one of the exceptional RNA addiction systems. Pheromone-responsive plasmids contribute also to enterococcal phenotype being an important vehicle of antibiotic resistance in this genus. Both types of acquired vancomycin resistance determinants, vanA and vanB, as well many other resistant phenotypes, were found to be located on these plasmids. They also encode two basic agents of enterococcal virulence, i.e. aggregation substance (AS) and cytolysin. AS participates in mating-pair formation during conjugation but can also facilitate the adherence ofenterococci to human tissues during infection. The second protein, cytolysin, displays hemolytic activity and helps to invade eukaryotic cells. There are still many aspects of the nature of pheromone plasmids that remain unclear and more detailed studies are needed to understand their uniqueness and complexity.