Molecular Epidemiology of Underreported Emerging Zoonotic Pathogen Streptococcus suis in Europe.

Streptococcus suis, a zoonotic bacterial pathogen circulated through swine, can cause severe infections in humans. Because human S. suis infections are not notifiable in most countries, incidence is underestimated. We aimed to increase insight into the molecular epidemiology of human S. suis infections in Europe. To procure data, we surveyed 7 reference laboratories and performed a systematic review of the scientific literature. We identified 236 cases of human S. suis infection from those sources and an additional 87 by scanning gray literature. We performed whole-genome sequencing to type 46 zoonotic S. suis isolates and combined them with 28 publicly available genomes in a core-genome phylogeny. Clonal complex (CC) 1 isolates accounted for 87% of typed human infections; CC20, CC25, CC87, and CC94 also caused infections. Emergence of diverse zoonotic clades and notable severity of illness in humans support classifying S. suis infection as a notifiable condition.

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.

Mobile genetic elements beyond the VanB-resistance dissemination among hospital-associated enterococci and other Gram-positive bacteria.

An increasing resistance to vancomycin among clinically relevant enterococci, such as Enterococcus faecalis and Enterococcus faecium is a cause of a great concern, as it seriously limits treatment options. The vanB operon is one of most common determinants of this type of resistance. Genes constituting the operon are located in conjugative transposons, such as Tn1549-type transposons or, more rarely, in ICEEfaV583-type structures. Such elements show differences in structure and size, and reside in various sites of bacterial chromosome or, in the case of Tn1549-type transposons, are also occasionally associated with plasmids of divergent replicon types. While conjugative transposition contributes to the acquisition of Tn1549-type transposons from anaerobic gut commensals by enterococci, chromosomal recombination and conjugal transfer of plasmids appear to represent main mechanisms responsible for horizontal dissemination of vanB determinants among hospital E. faecalis and E. faecium. This review focuses on diversity of genetic elements harbouring vanB determinants in hospital-associated strains of E. faecium and E. faecalis, the mechanisms beyond vanB spread in populations of these bacteria, and provides an overview of the vanB-MGE distribution among other enterococci and Gram-positive bacteria as potential reservoirs of vanB genes.

Global emergence and population dynamics of divergent serotype 3 CC180 pneumococci.

Streptococcus pneumoniae serotype 3 remains a significant cause of morbidity and mortality worldwide, despite inclusion in the 13-valent pneumococcal conjugate vaccine (PCV13). Serotype 3 increased in carriage since the implementation of PCV13 in the USA, while invasive disease rates remain unchanged. We investigated the persistence of serotype 3 in carriage and disease, through genomic analyses of a global sample of 301 serotype 3 isolates of the Netherlands3-31 (PMEN31) clone CC180, combined with associated patient data and PCV utilization among countries of isolate collection. We assessed phenotypic variation between dominant clades in capsule charge (zeta potential), capsular polysaccharide shedding, and susceptibility to opsonophagocytic killing, which have previously been associated with carriage duration, invasiveness, and vaccine escape. We identified a recent shift in the CC180 population attributed to a lineage termed Clade II, which was estimated by Bayesian coalescent analysis to have first appeared in 1968 [95% HPD: 1939-1989] and increased in prevalence and effective population size thereafter. Clade II isolates are divergent from the pre-PCV13 serotype 3 population in non-capsular antigenic composition, competence, and antibiotic susceptibility, the last of which resulting from the acquisition of a Tn916-like conjugative transposon. Differences in recombination rates among clades correlated with variations in the ATP-binding subunit of Clp protease, as well as amino acid substitutions in the comCDE operon. Opsonophagocytic killing assays elucidated the low observed efficacy of PCV13 against serotype 3. Variation in PCV13 use among sampled countries was not independently correlated with the CC180 population shift; therefore, genotypic and phenotypic differences in protein antigens and, in particular, antibiotic resistance may have contributed to the increase of Clade II. Our analysis emphasizes the need for routine, representative sampling of isolates from disperse geographic regions, including historically under-sampled areas. We also highlight the value of genomics in resolving antigenic and epidemiological variations within a serotype, which may have implications for future vaccine development.

Identification of Streptococcus pneumoniae and other Mitis streptococci: importance of molecular methods.

The Mitis group of streptococci includes an important human pathogen, Streptococcus pneumoniae (pneumococcus) and about 20 other related species with much lower pathogenicity. In clinical practice, some representatives of these species, especially Streptococcus pseudopneumoniae and Streptococcus mitis, are sometimes mistaken for S. pneumoniae based on the results of classical microbiological methods, such as optochin susceptibility and bile solubility. Several various molecular approaches that address the issue of correct identification of pneumococci and other Mitis streptococci have been proposed and are discussed in this review, including PCR- and gene sequencing-based tests as well as new developments in the genomic field that represents an important advance in our understanding of relationships within the Mitis group.

Emergence of linezolid-resistant Staphylococcus epidermidis in the tertiary children's hospital in Cracow, Poland.

Coagulase-negative staphylococci, ubiquitous commensals of human skin, and mucous membranes represent important pathogens for immunocompromised patients and neonates. The increasing antibiotic resistance among Staphylococcus epidermidis is an emerging problem worldwide. In particular, the linezolid-resistant S. epidermidis (LRSE) strains are observed in Europe since 2014. The aim of our study was to genetically characterize 11 LRSE isolates, recovered mostly from blood in the University Children's Hospital in Krakow, Poland, between 2015 and 2017. For identification of the isolates at the species level, we used 16S rRNA sequencing and RFLP of the saoC gene. Isolates were characterized phenotypically by determining their antimicrobial resistance patterns and using molecular methods such as PFGE, MLST, SCCmec typing, detection of the ica operon, and analysis of antimicrobial resistance determinants. All isolates were multidrug-resistant, including resistance to methicillin, and exhibited so-called PhLOPSA phenotype. In PFGE, all isolates (excluding one from a catheter) represented identical patterns, were identified as ST2, and harbored the ica operon, responsible for biofilm formation. Linezolid resistance was associated with acquisition of A157R mutation in the ribosomal protein L3 and the presence of cfr gene. All isolates revealed new SCCmec cassette element composition. Recently, pediatric patients with serious staphylococcal infections are often treated with linezolid. The increasing linezolid resistance in bacterial strains becomes a real threat for patients, and monitoring such infections combined with surveillance and infection prevention programs is very important to decrease number of linezolid-resistant staphylococcal strains.

Relationships among streptococci from the mitis group, misidentified as Streptococcus pneumoniae.

The aim of our study was to investigate phenotypic and genotypic features of streptococci misidentified (misID) as Streptococcus pneumoniae, obtained over 20 years from hospital patients in Poland. Sixty-three isolates demonstrating microbiological features typical for pneumococci (optochin susceptibility and/or bile solubility) were investigated by phenotypic tests, lytA and 16S rRNA gene polymorphism and whole-genome sequencing (WGS). All isolates had a 6-bp deletion in the lytA 3' terminus, characteristic for Mitis streptococc and all but two isolates lacked the pneumococcal signature cytosine at nucleotide position 203 in the 16S rRNA genes. The counterparts of psaA and ply were present in 100% and 81.0% of isolates, respectively; the spn9802 and spn9828 loci were characteristic for 49.2% and 38.1% of isolates, respectively. Phylogenetic trees and networks, based on the multilocus sequence analysis (MLSA) scheme, ribosomal multilocus sequence typing (rMLST) scheme and core-genome analysis, clearly separated investigated isolates from S. pneumoniae and demonstrated the polyclonal character of misID streptococci, associated with the Streptococcus pseudopneumoniae and Streptococcus mitis groups. While the S. pseudopneumoniae clade was relatively well defined in all three analyses, only the core-genome analysis revealed the presence of another cluster comprising a fraction of misID streptococci and a strain proposed elsewhere as a representative of a novel species in the Mitis group. Our findings point to complex phylogenetic and taxonomic relationships among S. mitis-like bacteria and support the notion that this group may in fact consist of several distinct species.

Update on prevalence and mechanisms of resistance to linezolid, tigecycline and daptomycin in enterococci in Europe: Towards a common nomenclature.

Vancomycin-resistant enterococci (VRE) are important nosocomial pathogens. Invasive VRE infections are difficult to treat since common therapeutic options including ampicillin and glycopeptides often fail. In vitro, most VRE remain susceptible to last-resort antibiotics such as linezolid, tigecycline and daptomycin. However, neither tigecycline nor linezolid act in a bactericidal manner, and daptomycin has proven activity only at high dosages licensed for treating enterococcal endocarditis. Despite these pharmacological and therapeutic limitations, reports on resistance to these last-resort drugs in VRE, and enterococci in general, have increased in recent years. In this review, we briefly recapitulate the current knowledge on the mode of action as well as the known and novel mechanisms of resistance and describe surveillance data on resistance to linezolid, tigecycline and daptomycin in enterococci. In addition, we also suggest a common nomenclature for designating enterococci and VRE with resistances to these important last-resort antibiotics.

Linezolid resistance genes and genetic elements enhancing their dissemination in enterococci and streptococci.

Linezolid is considered a last resort drug in treatment of severe infections caused by Gram-positive pathogens, resistant to other antibiotics, such as vancomycin-resistant enterococci (VRE), methicillin-resistant staphylococci and multidrug resistant pneumococci. Although the vast majority of Gram-positive pathogenic bacteria remain susceptible to linezolid, resistant isolates of enterococci, staphylococci and streptococci have been reported worldwide. In these bacteria, apart from mutations, affecting mostly the 23S rRNA genes, acquisition of such genes as cfr, cfr(B), optrA and poxtA, often associated with mobile genetic elements (MGE), plays an important role for resistance. The purpose of this paper is to provide an overview on diversity and epidemiology of MGE carrying linezolid-resistance genes among clinically-relevant Gram-positive pathogens such as enterococci and streptococci.

The changing epidemiology of VanB Enterococcus faecium in Poland.

Increasing prevalence of VanB Enterococcus faecium in Polish hospitals reported to National Reference Centre for Susceptibility Testing (NRCST) prompted us to investigate the basis of this phenomenon. Two-hundred seventy-eight E. faecium isolates of VanB phenotype from the period 1999 to 2010 obtained by NRCST were investigated by multilocus sequence typing (MLST) and multilocus VNTR analysis (MLVA). Localization, transferability, and partial structure of the vanB-carrying Tn1549 transposon were studied by hybridization, PCR mapping, sequencing, and conjugation. VanB isolates almost exclusively represented hospital-associated E. faecium, with a significant shift from representatives of 17/18 lineage to 78 lineage after 2005. The vanB determinant, initially located mostly on transferable plasmids of the pRUM-, pLG1-, and pRE25-replicon types, later on was found almost exclusively on the host chromosome. Fifteen different plasmid and chromosomal insertion sites were identified, typically associated with single transposon coupling sequences, mostly not observed before. Our study demonstrates the significant change in the epidemiology of VanB-E. faecium in Poland, associated with the introduction and spread of the lineage 78 of the hospital-adapted E. faecium. These data point to the importance of the lineage 78 for the spread of vancomycin-resistance, determined by the vanB gene cluster, resulting in an increasing VRE prevalence in hospitals. This study also supports the scenario, in which representatives of the hospital-associated E. faecium independently acquire the vanB determinant de novo and spread within and among hospitals, concomitantly undergoing differentiation.

Multilevel population genetic analysis of vanA and vanB Enterococcus faecium causing nosocomial outbreaks in 27 countries (1986-2012).

OBJECTIVES: Vancomycin-resistant Enterococcus faecium (VREfm) have been increasingly reported since the 1980s. Despite the high number of published studies about VRE epidemiology, the dynamics and evolvability of these microorganisms are still not fully understood. A multilevel population genetic analysis of VREfm outbreak strains since 1986, representing the first comprehensive characterization of plasmid content in E. faecium, was performed to provide a detailed view of potential transmissible units. METHODS: From a comprehensive MeSH search, we identified VREfm strains causing hospital outbreaks (1986-2012). In total, 53 VanA and 18 VanB isolates (27 countries, 5 continents) were analysed and 82 vancomycin-susceptible E. faecium (VSEfm) were included for comparison. Clonal relatedness was established by PFGE and MLST (goeBURST/Bayesian Analysis of Population Structure, BAPS). Characterization of van transposons (PCR mapping, RFLP, sequencing), plasmids (transfer, ClaI-RFLP, PCR typing of relaxases, replication-initiation proteins and toxin-antitoxin systems, hybridization, sequencing), bacteriocins and virulence determinants (PCR, hybridization, sequencing) was performed. RESULTS: VREfm were mainly associated with major human lineages ST17, ST18 and ST78. VREfm and VSEfm harboured plasmids of different families [RCR, small theta plasmids, RepA_N (pRUM/pLG1) and Inc18] able to yield mosaic elements. Tn1546-vanA was mainly located on pRUM/Axe-Txe (USA) and Inc18-pIP186 (Europe) plasmids. The VanB2 type (Tn5382/Tn1549) was predominant among VanB strains (chromosome and plasmids). CONCLUSIONS: Both strains and plasmids contributed to the spread and persistence of vancomycin resistance among E. faecium. Horizontal gene transfer events among genetic elements from different clonal lineages (same or different species) result in chimeras with different stability and host range, complicating the surveillance of epidemic plasmids.

Investigating the mobilome in clinically important lineages of Enterococcus faecium and Enterococcus faecalis.

BACKGROUND: The success of Enterococcus faecium and E. faecalis evolving as multi-resistant nosocomial pathogens is associated with their ability to acquire and share adaptive traits, including antimicrobial resistance genes encoded by mobile genetic elements (MGEs). Here, we investigate this mobilome in successful hospital associated genetic lineages, E. faecium sequence type (ST)17 (n=10) and ST78 (n=10), E. faecalis ST6 (n=10) and ST40 (n=10) by DNA microarray analyses. RESULTS: The hybridization patterns of 272 representative targets including plasmid backbones (n=85), transposable elements (n=85), resistance determinants (n=67), prophages (n=29) and clustered regularly interspaced short palindromic repeats (CRISPR)-cas sequences (n=6) separated the strains according to species, and for E. faecalis also according to STs. RCR-, Rep_3-, RepA_N- and Inc18-family plasmids were highly prevalent and with the exception of Rep_3, evenly distributed between the species. There was a considerable difference in the replicon profile, with rep 17/pRUM , rep 2/pRE25 , rep 14/EFNP1 and rep 20/pLG1 dominating in E. faecium and rep 9/pCF10 , rep 2/pRE25 and rep 7 in E. faecalis strains. We observed an overall high correlation between the presence and absence of genes coding for resistance towards antibiotics, metals, biocides and their corresponding MGEs as well as their phenotypic antimicrobial susceptibility pattern. Although most IS families were represented in both E. faecalis and E. faecium, specific IS elements within these families were distributed in only one species. The prevalence of IS256-, IS3-, ISL3-, IS200/IS605-, IS110-, IS982- and IS4-transposases was significantly higher in E. faecium than E. faecalis, and that of IS110-, IS982- and IS1182-transposases in E. faecalis ST6 compared to ST40. Notably, the transposases of IS981, ISEfm1 and IS1678 that have only been reported in few enterococcal isolates were well represented in the E. faecium strains. E. faecalis ST40 strains harboured possible functional CRISPR-Cas systems, and still resistance and prophage sequences were generally well represented. CONCLUSIONS: The targeted MGEs were highly prevalent among the selected STs, underlining their potential importance in the evolution of hospital-adapted lineages of enterococci. Although the propensity of inter-species horizontal gene transfer (HGT) must be emphasized, the considerable species-specificity of these MGEs indicates a separate vertical evolution of MGEs within each species, and for E. faecalis within each ST.

Comprehensive molecular, genomic and phenotypic analysis of a major clone of Enterococcus faecalis MLST ST40.

BACKGROUND: Enterococcus faecalis is a multifaceted microorganism known to act as a beneficial intestinal commensal bacterium. It is also a dreaded nosocomial pathogen causing life-threatening infections in hospitalised patients. Isolates of a distinct MLST type ST40 represent the most frequent strain type of this species, distributed worldwide and originating from various sources (animal, human, environmental) and different conditions (colonisation/infection). Since enterococci are known to be highly recombinogenic we determined to analyse the microevolution and niche adaptation of this highly distributed clonal type. RESULTS: We compared a set of 42 ST40 isolates by assessing key molecular determinants, performing whole genome sequencing (WGS) and a number of phenotypic assays including resistance profiling, formation of biofilm and utilisation of carbon sources. We generated the first circular closed reference genome of an E. faecalis isolate D32 of animal origin and compared it with the genomes of other reference strains. D32 was used as a template for detailed WGS comparisons of high-quality draft genomes of 14 ST40 isolates. Genomic and phylogenetic analyses suggest a high level of similarity regarding the core genome, also demonstrated by similar carbon utilisation patterns. Distribution of known and putative virulence-associated genes did not differentiate between ST40 strains from a commensal and clinical background or an animal or human source. Further analyses of mobile genetic elements (MGE) revealed genomic diversity owed to: (1) a modularly structured pathogenicity island; (2) a site-specifically integrated and previously unknown genomic island of 138 kb in two strains putatively involved in exopolysaccharide synthesis; and (3) isolate-specific plasmid and phage patterns. Moreover, we used different cell-biological and animal experiments to compare the isolate D32 with a closely related ST40 endocarditis isolate whose draft genome sequence was also generated. D32 generally showed a greater capacity of adherence to human cell lines and an increased pathogenic potential in various animal models in combination with an even faster growth in vivo (not in vitro). CONCLUSION: Molecular, genomic and phenotypic analysis of representative isolates of a major clone of E. faecalis MLST ST40 revealed new insights into the microbiology of a commensal bacterium which can turn into a conditional pathogen.

Drug-resistant and hospital-associated Enterococcus faecium from wastewater, riverine estuary and anthropogenically impacted marine catchment basin.

BACKGROUND: Enterococci, ubiquitous colonizers of humans and other animals, play an increasingly important role in health-care associated infections (HAIs). It is believed that the recent evolution of two clinically relevant species, Enterococcus faecalis and Enterococcus faecium occurred in a big part in a hospital environment, leading to formation of high-risk enterococcal clonal complexes (HiRECCs), which combine multidrug resistance with increased pathogenicity and epidemicity. The aim of this study was to establish the species composition in wastewater, its marine recipient as well as a river estuary and to investigate the antimicrobial susceptibility of collected isolates. Molecular methods were additionally applied to test the presence of HiRRECC-related E. faecium. RESULTS: Two wastewater treatment plants (WWTPs), their marine outfalls and Vistula river that influence significantly the quality of waters in Gulf of Gdansk were sampled to investigate the presence of Enterococcus spp. Four-hundred-twenty-eight isolates were obtained, including E. faecium (244 isolates, 57.0%), E. hirae (113 isolates, 26.4%) and E. faecalis (63 isolates, 14.7%); other species (E. gallinarum/casseliflavus, E. durans and E. avium) accounted for 1.9%. Antimicrobial susceptibility testing revealed the presence of isolates resistant to erythromycin, tetracycline, amipicillin, fluoroquinolones and aminoglycosides (high-level resistance), especially among E. faecium, where such isolates were usually characterized by multilocus sequence types associated with nosocomial lineages 17, 18 and 78 of this species representing HiRECC, formerly called CC17. These isolates not only carried several resistance determinants but were also enriched in genes encoding pathogenicity factors (Esp, pili) and genes associated with mobile genetic elements (MGE), a feature also typical for nosocomial HiRECC. CONCLUSIONS: Our data show that WWTPs constitute an important source of enterococcal strains carrying antimicrobial resistance determinants, often associated with the presence of MGE, for the recipient water environment, thus increasing a pool of such genes for other organisms. The presence of HiRECCs in wastewaters and marine/river environment may indicate that adaptations gained in hospitals may be also beneficial for survival of such clones in other settings. There is an obvious need to monitor the release and spread of such strains in order to elucidate better ways to curb their dissemination.

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.

Comparative genomic analysis of two ST320 Streptococcus pneumoniae isolates, representing serotypes 19A and 19F.

BACKGROUND: Streptococcus pneumoniae (pneumococcus) represents an important human pathogen, responsible for respiratory and invasive infections in the community. The efficacy of polysaccharide conjugate vaccines formulated against pneumococci is reduced by the phenomenon of serotype replacement in population of this pathogen. The aim of the current study was to obtain and compare complete genomic sequences of two pneumococcal isolates, both belonging to ST320 but differing by the serotype. RESULTS: Here, we report genomic sequences of two isolates of important human pathogen, S. pneumoniae. Genomic sequencing resulted in complete sequences of chromosomes of both isolates, 2,069,241 bp and 2,103,144 bp in size, and confirmed the presence of cps loci specific for serotypes 19A and 19F. The comparative analysis of these genomes revealed several instances of recombination, which involved not only S. pneumoniae but also presumably other streptococci as donors. CONCLUSIONS: We report the complete genomic sequences of two S. pneumoniae isolates of ST320 and serotypes 19A and 19F. The detailed comparative analysis of these genomes revealed the history of several recombination events, clustered in the region including the cps locus.

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.

Highly Resistant Serotype 19A Streptococcus pneumoniae of the GPSC1/CC320 Clone from Invasive Infections in Poland Prior to Antipneumococcal Vaccination of Children.

INTRODUCTION: The introduction of pneumococcal conjugate vaccines (PCV) into the national immunization programs (NIPs) has significantly reduced the number of pneumococcal infections. However, infections caused by isolates of non-vaccine serotypes (NVT) started spreading shortly thereafter and strains of NVT 19A have become the main cause of invasive pneumococcal disease burden worldwide. The aim of the study was to characterize serotype 19A invasive pneumococci of GPSC1/CC320 circulating in Poland before the introduction of PCV into the Polish NIP in 2017 and to compare them to isolates from other countries where PCVs were implemented much earlier than in Poland. METHODS: All the GPSC1/CC320 isolates were analyzed by serotyping, susceptibility testing, and whole genome sequencing followed by analyses of resistome, virulome, and core genome multilocus sequence typing (cgMLST), including comparative analysis with isolates with publicly accessible genomic sequences (PubMLST). RESULTS: During continuous surveillance the NRCBM collected 4237 invasive Streptococcus pneumoniae isolates between 1997 and 2016, including 200 isolates (4.7%) of serotype 19A. The most prevalent among 19A pneumococci were highly resistant representatives of Global Pneumococcal Sequence Cluster 1/Clonal Complex 320, GPSC1/CC320 (n = 97, 48.5%). Isolates of GPSC1/CC320 belonged to three sequence types (STs): ST320 (75.2%) ST4768 (23.7%), and ST15047 (1.0%), which all represented the 19A-III cps subtype and had complete loci for both PI-1 and PI-2 pili types. On the basis of the cgMLST analysis the majority of Polish GPSC1/CC320 isolates formed a group clearly distinct from pneumococci of this clone observed in other countries. CONCLUSION: Before introduction of PCV in the Polish NIP we noticed an unexpected increase of serotype 19A in invasive pneumococcal infections, with the most common being representatives of highly drug-resistant GPSC1/CC320 clone, rarely identified in Europe both before and even after PCV introduction.

Insight into antimicrobial susceptibility and population structure of contemporary human Enterococcus faecalis isolates from Europe.

OBJECTIVES: To investigate antimicrobial susceptibility and clonal relatedness of Enterococcus faecalis human isolates recovered recently (2006-09) in six European countries. METHODS: Antimicrobial susceptibility of 386 isolates from Denmark, Germany, Norway, Poland, Spain and The Netherlands, from hospital infections (223 isolates), carriage (82 isolates) and from colonization in the community (81 isolates) was determined by the broth microdilution method. Clonal relatedness of isolates was assessed by multilocus sequence typing. RESULTS: All isolates were susceptible to benzylpenicillin, ampicillin, linezolid, tigecycline and daptomycin. Non-susceptibility to tetracycline (77.6%), rifampicin (57.3%), ciprofloxacin (51.2%), aminoglycosides (43.3% high-level gentamicin resistance, 40.0% high-level streptomycin resistance) was frequent among hospital isolates, while non-susceptibility to glycopeptides was rare and associated mostly with vanA. Multidrug resistance was found in 59.7% of hospital isolates and 16.1% of community isolates. Isolates were classified into 105 sequence types (STs), of which 21 STs, representing more than half of the collected isolates (53.9%), grouped with 6 large E. faecalis clonal complexes (CCs; CC2, CC16, CC21, CC30, CC40 and CC87). Two of these, CC2 (frequently recovered in Spain and The Netherlands) and CC87 (prevalent in Poland), were found almost exclusively in hospitals and included the highest proportion of multiresistant isolates. CONCLUSIONS: While hospital-acquired E. faecalis in Europe remains susceptible to ampicillin and glycopeptides, the high prevalence of strains that are highly resistant to aminoglycosides excludes these antibiotics from combination therapies. Genotyping revealed that nosocomial infections by multiresistant E. faecalis are largely caused by only a few hospital-associated clones.