Surface-enhanced Raman scattering as a discrimination method of Streptococcus spp. and alternative approach for identifying capsular types of S. pneumoniae isolates.

The surface-enhanced Raman spectroscopy (SERS) is a method known for its effectiveness in detecting and identifying microorganisms, that was employed to differentiate various bacterial strains both at genus and species level. In this work, we have examined five species belonging to Streptococcus genus, namely S. pneumoniae, S. suis, S. pseudopneumoniae, S. oralis, and S. mitis. Additionally, we conducted SERS experiments on ten S. pneumoniae strains, representing different capsular types. In all of cases we obtained unique SERS signals being spectroscopic fingerprints of bacterial strains tested. Moreover, the principal component analysis (PCA) was performed in order to prove that the spectra of all studied strains can be well separated into five (in case of streptococcal strains) or ten (in case of pneumococcal serotypes) groups. In both investigated situations, the separation at the level of 95% was achieved, proving that SERS-PCA-based method can be used for reliable and fast identification of different strains belonging to the Streptococcus genus, including encapsulated pneumococcal isolates.

Linezolid-resistant enterococci in Polish hospitals: species, clonality and determinants of linezolid resistance.

The significant increase of the linezolid-resistant enterococci (LRE) has been observed in Polish hospitals since 2012 and our study aimed at elucidating the possible reasons for this phenomenon. Polish LRE isolates were analysed by multilocus-sequence typing (MLST) and multiple locus variable-number tandem repeat (VNTR) analysis (MLVA), polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (PCR-RFLP) to establish clonal relatedness and mechanism of linezolid resistance, respectively. Fifty analysed LRE (2008-2015) included mostly Enterococcus faecium (82%) and Enterococcus faecalis (16%). Enterococcus faecium belonged to the hospital-adapted lineages 17/18 and 78, while E. faecalis isolates represented ST6, a hospital-associated type, and ST116, found in both humans and food-production animals. The G2576T 23S rRNA mutation was the most frequent (94%) mechanism of linezolid/tedizolid resistance of LRE. None of the isolates carried the plasmid-associated gene of Cfr methyltransferase, whereas optrA, encoding the ABC-type drug transporter, was identified in two E. faecalis isolates. In these isolates, optrA was located on a plasmid, transferable to both E. faecium and E. faecalis, whose partial (36.3 kb) sequence was 100% identical to the pE394 plasmid, identified previously in China in both clinical and farm animal isolates. The optrA-E. faecium transconjugant displayed a significant growth deficiency, in contrast to the optrA-E. faecalis. Our study indicates the role of mutation acquisition by hospital-adapted clones of enterococci as a major driver of increasing resistance to linezolid and tedizolid. Transferability and apparent lack of a biological cost of resistance suggest that E. faecalis may be a natural reservoir of optrA, an emerging mechanism of oxazolidinone resistance.

Diversity of plasmids and Tn1546-type transposons among VanA Enterococcus faecium in Poland.

The objective of this study was to investigate the antimicrobial resistance, Tn1546 transposon variability and plasmid diversity among Polish vancomycin-resistant Enterococcus faecium (VREfm) isolates of VanA phenotype in the context of their clonal structure. Two hundred sixteen clinical VREfm isolates collected between 1997 and 2010 were studied by antimicrobial susceptibility testing, MLST, MLVA and detection of IS16, esp Efm, pilA, intA and plasmid-specific genes by PCR. Tn1546 structure was revealed by overlapping PCR and sequencing. Selected isolates were subjected to PFGE-S1 and Southern hybridization analyses. The vast majority of the isolates (95.8 %) belonged to lineages 17/18 (during the whole study period 1997-2010) and 78 (mostly in 2006-2010) of hospital-adapted meroclone of E. faecium. All isolates displayed a multi-drug resistance phenotype. Twenty-eight Tn1546 types (including 26 novel ones) were associated with eight different ISs (IS1216, IS1251, ISEfa4, ISEfa5, ISEfm2, ISEf1, IS3-like, ISEfm1-like). The vanA-determinant was typically located on plasmids, which most commonly carried rep2pRE25, rep17pRUM, rep18pEF418, rep1pIP501, ω-ε-ζ and axe-txe genes. VanA isolates from 1997-2005 to 2006-2010 differed in clonal composition, prevalence of gentamicin- and tetracycline-resistance and plasmidome. Our analysis revealed high complexity of Tn1546-type transposons and vanA-plasmids, and suggested that diverse genetic events, such as conjugation transfer, recombination, chromosomal integration and DNA mutations shaped the structure of these elements among Polish VREfm.

Streptococcus suis in invasive human infections in Poland: clonality and determinants of virulence and antimicrobial resistance.

The purpose of this study was to perform an analysis of Streptococcus suis human invasive isolates, collected in Poland by the National Reference Centre for Bacterial Meningitis. Isolates obtained from 21 patients during 2000-2013 were investigated by phenotypic tests, multilocus sequence typing (MLST), analysis of the TR9 locus from the multilocus variable number tandem repeat (VNTR) analysis (MLVA) scheme and pulsed-field gel electrophoresis (PFGE) of SmaI-digested DNA. Determinants of virulence and antimicrobial resistance were detected by polymerase chain reaction (PCR) and analysed by sequencing. All isolates represented sequence type 1 (ST1) and were suggested to be serotype 2. PFGE and analysis of the TR9 locus allowed the discrimination of four and 17 types, respectively. Most of the isolates were haemolysis- and DNase-positive, and around half of them formed biofilm. Genes encoding suilysin, extracellular protein factor, fibronectin-binding protein, muramidase-released protein, surface antigen one, enolase, serum opacity factor and pili were ubiquitous in the studied group, while none of the isolates carried sequences characteristic for the 89K pathogenicity island. All isolates were susceptible to penicillin, cefotaxime, imipenem, moxifloxacin, chloramphenicol, rifampicin, gentamicin, linezolid, vancomycin and daptomycin. Five isolates (24 %) were concomitantly non-susceptible to erythromycin, clindamycin and tetracycline, and harboured the tet(O) and erm(B) genes; for one isolate, lsa(E) and lnu(B) were additionally detected. Streptococcus suis isolated in Poland from human invasive infections belongs to a globally distributed clonal complex of this pathogen, enriched in virulence markers. This is the first report of the lsa(E) and lnu(B) resistance genes in S. suis.

Invasive enterococcal infections in Poland: the current epidemiological situation.

The aim of this study was to investigate human invasive isolates of enterococci, obtained through prospective surveillance in Poland. The consecutive enterococcal isolates were collected in 30 hospitals between May 2010 and June 2011, and studied by species identification, antimicrobial susceptibility testing and, for Enterococcus faecium by detection of markers specific for the hospital meroclone, multilocus VNTR analysis (MLVA) and multilocus sequence typing (MLST). Additionally, the genomic difference regions (GDRs) characteristic for lineage 78 were searched by PCR. Among 259 isolates, a nearly equal number of Enterococcus faecalis (n = 140; 54.1 %) and E. faecium (n = 112; 43.2 %) was found. The observed 14-day mortality rate of infected patients reached 18.1 %. All isolates were susceptible to linezolid and daptomycin. High-level aminoglycoside resistance occurred in over 50 % of isolates. Vancomycin resistance mediated by vanA or vanB was detected in 7.1 % of E. faecium; 71.4 % of isolates were multidrug resistant. E. faecium isolates ubiquitously carried molecular markers of hospital-associated meroclone (IS16, esp(Efm), intA of ICEEfm1) and multilocus sequence typing showed the domination of representatives of lineages 78 and 17/18 (52.7 % and 46.4 %, respectively). Isolates of lineage 78 were significantly enriched in all the GDRs studied. The recent spread of E. faecium from this lineage contributed to the observed increase of E. faecium in enterococcal invasive infections in hospitals in Poland.

Recent trends in epidemiology of invasive pneumococcal disease in Poland.

The objectives of this study were to assess the current incidence of invasive pneumococcal disease (IPD) in Poland (2011-2013), where mass vaccination has not been implemented, and to characterize the Streptococcus pneumoniae isolates responsible for invasive infections by determining their serotype distribution and antimicrobial resistance patterns. For all isolates identification, serotyping and antimicrobial minimal inhibitory concentrations determination were performed based on routine techniques. The highest incidence rates were observed among adults older than 85 years old (4.62/100,000) and children under 1 year of age (4.28/100,000). The general case fatality ratio (CFR) was 25.4%, with the highest CFR in the age group ≥85 years old (59.7%). The most common serotypes were 3, 14, 19A, 4, 9V, 19F, 1, and 23 F (61.3% of all isolates). The 10- and 13-valent pneumococcal conjugated vaccines (PCV) covered 46.0 and 71.8% of all IPD cases, 61.4 and 79.5% of cases in children under two years, and 60.4 and 78.6% of cases involving children under five years of age, respectively. The PCV13 and 23-valent polysaccharide vaccine covered 68.7 and 86.0% of cases in adults >65 years old, respectively. Decreased susceptibility was noted for penicillin (24.8%), cefotaxime (10.0%), meropenem (5.0%), rifampicin (0.8%), chloramphenicol (4.3%), erythromycin (29.7%) and clindamycin (25.6%). Multi-drug resistance characterized 21.6% of the pneumococci tested. Despite deficiencies in the Polish surveillance system and strong underestimation of IPD cases, results of the study showed good theoretical coverage of PCV, which should encourage inclusion of anti-pneumococcal conjugate vaccine into the national immunization program.

High abundance and diversity of antimicrobial resistance determinants among early vancomycin-resistant Enterococcus faecium in Poland.

The purpose of this study was to investigate the clonal structure, antimicrobial resistance phenotypes and their determinants among early vancomycin-resistant Enterococcus faecium (VREm) isolates in Poland. Two hundred and eighty-one VREm isolates collected between 1997 and 2005 were studied. VREm isolates were characterised by multilocus sequence typing (MLST). The presence of antimicrobial resistance determinants, transposon-specific genes, IS16 and esp Efm was checked by polymerase chain reaction (PCR). Ciprofloxacin and ampicillin resistance determinants were investigated by sequencing. Two hundred and twenty-two (79 %) and 59 (21 %) VREm isolates were vanA- and vanB-positive, respectively. Among 135 representative isolates, MLST yielded 33 different sequence types (STs), of which 29 were characteristic of hospital-associated E. faecium; 128 (94.8 %) and 123 (91.1 %) isolates harboured the IS16 and esp Efm genes, and all 135 isolates were resistant to ciprofloxacin and ampicillin. Resistance to tetracycline (71.1 % isolates) was mostly associated with tetM (75.0 %) and the concomitant presence of the Tn916 integrase gene. High-level resistance to streptomycin (93.3 % of isolates) and high-level resistance to gentamicin (94.1 % of isolates) were due to ant(6')-Ia and aac(6')-Ie-aph(2″) genes, respectively, the latter of which is known to be located on various Tn4001-type transposons. Fifteen combinations of mutations in the quinolone-determining regions of GyrA and ParC were identified, including changes not previously reported, such as S83F and A84P in GyrA. Twenty-three variants of the penicillin-binding protein PBP5 occurred in the studied group, and novel insertions at amino acid positions 433 and 568 were identified. This analysis revealed the predominance of hospital-associated strains of E. faecium, carrying an abundant and divergent range of resistance determinants among early VREm isolates in Poland.

Comparative population analysis of Klebsiella pneumoniae strains with extended-spectrum ß-lactamases colonizing patients in rehabilitation centers in four countries.

The international project MOSAR was conducted in five rehabilitation centers; patients were screened for rectal carriage of extended-spectrum ß-lactamase (ESBL)-producing members of the Enterobacteriaceae. Among 229 Klebsiella pneumoniae isolates, four clonal groups (CG) or complexes (CC) prevailed: CG17 in France, CG101 in Italy, CG15 in Spain, and CC147 in Israel. ESBLs, mainly CTX-Ms, were produced by 226 isolates; three isolates expressed AmpC-like cephalosporinases. High genetic diversity of K. pneumoniae populations was observed, with specific characteristics at each center.

Evolution and spread of a multidrug-resistant Proteus mirabilis clone with chromosomal AmpC-type cephalosporinases in Europe.

Proteus mirabilis isolates obtained in 1999 to 2008 from three European countries were analyzed; all carried chromosomal AmpC-type cephalosporinase bla(CMY) genes from a Citrobacter freundii origin (bla(CMY-2)-like genes). Isolates from Poland harbored several bla(CMY) genes (bla(CMY-4), bla(CMY-12), bla(CMY-14), bla(CMY-15), and bla(CMY-38) and the new gene bla(CMY-45)), while isolates from Italy and Greece harbored bla(CMY-16) only. Earlier isolates with bla(CMY-4) or bla(CMY-12), recovered in France from Greek and Algerian patients, were also studied. All isolates showed striking similarities. Their bla(CMY) genes resided within ISEcp1 transposition modules, named Tn6093, characterized by a 110-bp distance between ISEcp1 and bla(CMY), and identical fragments of both C. freundii DNA and a ColE1-type plasmid backbone. Moreover, these modules were inserted into the same chromosomal site, within the pepQ gene. Since ColE1 plasmids carrying ISEcp1 with similar C. freundii DNA fragments (Tn6114) had been identified earlier, it is likely that a similar molecule had mediated at some stage this DNA transfer between C. freundii and P. mirabilis. In addition, isolates with bla(CMY-12), bla(CMY-15), and bla(CMY-38) genes harbored a second bla(CMY) copy within a shorter ISEcp1 module (Tn6113), always inserted downstream of the ppiD gene. Sequence analysis of all mobile bla(CMY-2)-like genes indicated that those integrated in the P. mirabilis chromosome form a distinct cluster that may have evolved by the stepwise accumulation of mutations. All of these observations, coupled to strain typing data, suggest that the bla(CMY) genes studied here may have originated from a single ISEcp1-mediated mobilization-transfer-integration process, followed by the spread and evolution of a P. mirabilis clone over time and a large geographic area.

Emergence and spread of vancomycin resistance among enterococci in Europe.

Nowadays, six types of acquired vancomycin resistance in enterococci are known; however, only VanA and to a lesser extent VanB are widely prevalent. Various genes encode acquired vancomycin resistance and these are typically associated with mobile genetic elements which allow resistance to spread clonally and laterally. The major reservoir of acquired vancomycin resistance is Enterococcus faecium; vancomycin-resistant Enterococcus faecalis are still rare. Population analysis of E. faecium has revealed a distinct subpopulation of hospital-acquired strain types, which can be differentiated by molecular typing methods (MLVA, MLST) from human commensal and animal strains. Hospital-acquired E. faecium have additional genomic content (accessory genome) including several factors known or supposed to be virulence-associated. Acquired ampicillin resistance is a major phenotypic marker of hospital-acquired E. faecium in Europe and experience has shown that it often precedes increasing rates of VRE with a delay of several years. Several factors are known to promote VRE colonisation and transmission; however, despite having populations with similar predispositions and preconditions, rates of VRE vary all over Europe.

Hospital outbreak of vancomycin-resistant enterococci caused by a single clone of Enterococcus raffinosus and several clones of Enterococcus faecium.

A mixed outbreak caused by vancomycin-resistant Enterococcus raffinosus and Enterococcus faecium carrying the vanA gene was analysed. The outbreak occurred in a large hospital in Poland and affected 27 patients, most of whom were colonised, in three wards, including the haematology unit. The E. raffinosus isolates had a high-level multiresistant phenotype and were initially misidentified as Enterococcus avium; their unambiguous identification was provided by multilocus sequence analysis. The molecular investigation demonstrated the clonal character of the E. raffinosus outbreak and the polyclonal structure of the E. faecium isolates. All of the isolates carried the same Tn1546-like element containing an IS1251-like insertion sequence, located on a c. 50-kb conjugative plasmid. One of the E. faecium clones, found previously to be endemic in the hospital, was probably the source of the plasmid. The results of the study suggest that difficulties in identification may have led to an underestimate of the importance of E. raffinosus in vancomycin-resistant enterococci (VRE) control strategies.

Identification of Neisseria meningitidis sequence type 66 in Poland.

Investigation of two cases of invasive meningococcal disease within a single family revealed the presence of isolates of Neisseria meningitidis phenotype C:2b:P1.2,P1.5 belonging to sequence type (ST) 66. The ST66 clone is a single-locus variant of the widely distributed ST8 complex, which has been observed previously in Spain, Belgium, Australia and New Zealand. This hypervariable meningococcal lineage has been responsible for local epidemics worldwide. This is the first report of ST66 meningococcal isolates of this phenotype from Poland.

Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression.

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.

Infectious transcripts from cloned cDNA of potato leafroll luteovirus.

Infectious transcripts play a key role in the research on plant viruses at the molecular level. A number of cDNA clones covering the whole genome of the Polish isolate of potato leafroll virus were constructed. Four overlapping clones were selected and assembled using restriction sites. The full copy was positioned between T7 RNA polymerase promoter and unique ScaI site. The full-length capped transcripts of the sequence of the viral genome synthesised in vitro were able to replicate in protoplasts and to produce the viral coat protein.

Sulfate and thiosulfate transport in Escherichia coli K-12: evidence for a functional overlapping of sulfate- and thiosulfate-binding proteins.

In Escherichia coli, sulfate and thiosulfate ions are transported by an ABC-type transporter consisting of both the membrane components (the products of cysT, cysW, and cysA genes) and the periplasmic binders (the products of cysP and sbp genes). The single cysP and sbp mutants are able to utilize both sulfate and thiosulfate as a sole sulfur source, while the inactivation of both genes leads to cysteine auxotrophy resulting from the block in the transport of both ions.

Nucleotide sequence of RNA of a Polish isolate of potato leafroll luteovirus.

The nucleotide sequence of the genomic RNA of a Polish isolate of a potato leafroll virus was determined. Some variations between the determined sequences were observed. A comparison of the frequency of sequence variants in particular regions of the genome is presented.