Identification of cinically relevant Streptococcus and Enterococcus species based on biochemical methods and 16S rRNA, sodA, tuf, rpoB, and recA gene sequencing

Streptococci and enterococci are significant opportunistic pathogens in epidemiology and infectious medicine. High genetic and taxonomic similarities and several reclassifications within genera are the most challenging in species identification. The aim of this study was to identify Streptococcus and Enterococcus species using genetic and phenotypic methods and to determine the most discriminatory identification method. Thirty strains recovered from clinical samples representing 15 streptococcal species, five enterococcal species, and four nonstreptococcal species were subjected to bacterial identification by the Vitek® 2 system and Sanger-based sequencing methods targeting the 16S rRNA, sodA, tuf, rpoB, and recA genes. Phenotypic methods allowed the identification of 10 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains (Leuconostoc, Granulicatella, and Globicatella genera). The combination of sequencing methods allowed the identification of 21 streptococcal strains, five enterococcal strains, and four nonstreptococcal strains. The 16S rRNA and rpoB genes had the highest identification potential. Only a combination of several molecular methods was sufficient for unambiguous confirmation of species identity. This study will be useful for comparison of several identification methods, both those used as a first choice in routine microbiology and those used for final confirmation

Development and validation of a reference data set for assigning Staphylococcus species based on next-generation sequencing of the 16S-23S rRNA region

Many members of the Staphylococcus genus are clinically relevant opportunistic pathogens that warrant accurate and rapid identification for targeted therapy. The aim of this study was to develop a careful assignment scheme for staphylococcal species based on next-generation sequencing (NGS) of the 16S-23S rRNA region. All reference staphylococcal strains were identified at the species level using Sanger sequencing of the 16S rRNA, sodA, tuf, and rpoB genes and NGS of the 16S-23S rRNA region. To broaden the database, an additional 100 staphylococcal strains, including 29 species, were identified by routine diagnostic methods, 16S rRNA Sanger sequencing and NGS of the 16S-23S rRNA region. The results enabled development of reference sequences encompassing the 16S-23S rRNA region for 50 species (including one newly proposed species) and 6 subspecies of the Staphylococcus genus. This study showed sodA and rpoB targets were the most discriminative but NGS of the 16S-23S rRNA region was more discriminative than tuf gene sequencing and much more discriminative than 16S rRNA gene sequencing. Almost all Staphylococcus species could be distinguished when the max score was 99.0% or higher and the sequence similarity between the best and second best species was equal to or >0.2% (min. 9 nucleotides). This study allowed development of reference sequences for 21 staphylococcal species and enrichment for 29 species for which sequences were publicly available. We confirmed the usefulness of NGS of the 16S-23S rRNA region by identifying the whole species content in 45 clinical samples and comparing the results to those obtained using routine diagnostic methods. Based on the developed reference database, all staphylococcal species can be reliably detected based on the 16S-23S rRNA sequences in samples composed of both single species and more complex polymicrobial communities. This study will be useful for introduction of a novel diagnostic tool, which undoubtedly is an improvement for reliable species identification in polymicrobial samples. The introduction of this new method is hindered by a lack of reference sequences for the 16S-23S rRNA region for many bacterial species. The results will allow identification of all Staphylococcus species, which are clinically relevant pathogens

Preliminary evaluation of application of a 3-dimensional network structure of siloxanes Dergall preparation on chick embryo development and microbiological status of eggshells

The spatial network structure of Dergall is based on substances nontoxic to humans and the environment which, when applied on solid surfaces, creates a coating that reduces bacterial cell adhesion. The bacteriostatic properties of siloxanes are based on a purely physical action mechanism which excludes development of drug-resistant microorganisms. The aims of the present study were to 1) evaluate a Dergall layer formed on the eggshell surface regarding the potential harmful effects on the chick embryo; 2) evaluate antimicrobial activity and estimate the prolongation time of Dergall's potential antimicrobial activity. Dergall at a concentration of 0.6% formed a layer on the eggshell surface. In vitro testing of the potential harmful effects of Dergall by means of a hen embryo test of the chorioallantoic membrane showed no irritation reaction at a concentration of 3% and lower. The hatchability of the groups sprayed with a Dergall water solution with a concentration of 0 to 5% was 89.1 to 93.8% for fertilized eggs (P > 0.05) but decreased to 63.7% (P < 0.05) in the group sprayed with a 6% concentration of the solution. This phenomenon was caused by embryo mortality in the first week of incubation. At the concentration of 0.6%, Dergall exhibited strong antibacterial properties against bacteria such as Staphylococcus aureus, Escherichia coli, Shigella dysenteriae, Shigella flexneri, and Salmonella typhimurium. For Streptococcus pyogenes, the highest antibacterial activity of Dergall was reported in the concentrations of 100 and 50%. For Pseudomonas aeruginosa, no antibacterial activity of Dergall was generally observed, but in vivo testing showed a strong decrease of all gram-negative bacteria growth. Moreover, a prolonged antimicrobial effect lasting until 3 D after disinfection was observed, which makes Dergall a safe and efficient disinfectant

Clustering of Staphylococcus aureus bovine mastitis strains from regions of Central-Eastern Poland based on their biochemical and genetic characteristics

Staphylococcus aureus strains were isolated from mastitic milk of cows with infected mammary glands. The animals were living in 12 different farms near Lublin, in Central-Eastern Poland. A biochemical identification method based on enzymatic assay was performed, followed by haemolytic and proteolytic tests. PCR-RFLP targeted on the gap gene allowed the genetic identification of strains at the species level and verified phenotypic identification results. A molecular typing method using triplex PCR was performed to recognize the genetic similarity of the analyzed strains. DNA microarray hybridization (StaphyType, Alere Technologies) was used for detection of antibiotic resistance and virulence associated markers. The results obtained indicate high genetic similarity in strains isolated from the same sites. High genetic similarities were also detected between strains isolated from cows from different farms of the same region. A slightly lower similarity was noted however, in strains from various regions indicating that the strains are herd specific and that the cow's infections caused by S. aureus were of a clonal character. In 21 representative isolates selected for DNA-microarray testing, only fosfomycin (fosB) and penicillin resistance markers (blaZ, blaI, blaR) were detected. The presence of genes coding for haemolysins (lukF, lukS, hlgA, hla, hld, hlb), proteases (aur, sspA, sspB, sspP), enterotoxins (entA, entD, entG, entI, entJ, entM, entN, entO, entR, entU, egc-cluster), adhesins (icaA, icaC, icaD, bbp, clfA, clfB, fib, fnbA, map, vwb) or immune evasion proteins (scn, chp, sak) was common and, with exceptions, matched triplex PCR-defined clusters

Development of a reference data set for assigning Streptococcus and Enterococcus species based on next generation sequencing of the 16S-23S rRNA region

Background: Many members of Streptococcus and Enterococcus genera are clinically relevant opportunistic pathogens warranting accurate and rapid identification for targeted therapy. Currently, the developed method based on next generation sequencing (NGS) of the 16S-23S rRNA region proved to be a rapid, reliable and precise approach for species identification directly from polymicrobial and challenging clinical samples. The introduction of this new method to routine diagnostics is hindered by a lack of the reference sequences for the 16S-23S rRNA region for many bacterial species. The aim of this study was to develop a careful assignment for streptococcal and enterococcal species based on NGS of the 16S-23S rRNA region. Methods: Thirty two strains recovered from clinical samples and 19 reference strains representing 42 streptococcal species and nine enterococcal species were subjected to bacterial identification by four Sanger-based sequencing methods targeting the genes encoding (i) 16S rRNA, (ii) sodA, (iii) tuf and (iv) rpoB; and NGS of the 16S-23S rRNA region. Results: This study allowed obtainment and deposition of reference sequences of the 16S-23S rRNA region for 15 streptococcal and 3 enterococcal species followed by enrichment for 27 and 6 species, respectively, for which reference sequences were available in the databases. For Streptococcus, NGS of the 16S-23S rRNA region was as discriminative as Sanger sequencing of the tuf and rpoB genes allowing for an unambiguous identification of 93% of analyzed species. For Enterococcus, sodA, tuf and rpoB genes sequencing allowed for identification of all species, while the NGS-based method did not allow for identification of only one enterococcal species. For both genera, the sequence analysis of the 16S rRNA gene was endowed with a low identification potential and was inferior to that of other tested identification methods. Moreover, in case of phylogenetically related species the sequence analysis of only the intergenic spacer region was not sufficient enough to precisely identify Streptococcus strains at the species level. Conclusions: Based on the developed reference dataset, clinically relevant streptococcal and enterococcal species can now be reliably identified by 16S-23S rRNA sequences in samples. This study will be useful for introduction of a novel diagnostic tool, NGS of the 16S-23S rRNA region, which undoubtedly is an improvement for reliable culture-independent species identification directly from polymicrobially constituted clinical samples

New Insight into Genotypic and Phenotypic Relatedness of Staphylococcus aureus Strains from Human Infections or Animal Reservoirs

Cilj ovog rada bio je ispitati učinkovitost ekstrakcije bioaktivnih spojeva iz listova koprive (Urtica dioica L.) primjenom ubrzane ekstrakcije otapalima pri povišenom tlaku (ASE). Uz etanol kao ekstrakcijsko otapalo, ispitivani su sljedeći parametri ekstrakcije: (i) statičko vrijeme ekstrakcije (5 i 10 min); (ii) broj ciklusa ekstrakcije (1, 2, 3 i 4 ciklusa); te (iii) temperatura (20 i 50 °C). U dobivenim ekstraktima provedeno je spektrofotometrijsko određivanje ukupnih fenola, karotenoida te klorofila a i b. Rezultati istraživanja su pokazali kako sva tri varirana parametra značano utječu na masene udjele ispitivanih spojeva (p ≤ 0,01). Maseni udjeli ukupnih fenola određeni su u rasponu 106,44 do 739,72 mg 100 g-1, klorofila a od 116,44 do 490,42 mg 100 g-1, klorofila b od 44,88 do 166,85 mg g-1 te karotenoida od 36,62 do 133,99 mg 100 g-1. Najveći prinosi dobiveni su pri sljedećim uvjetima ekstrakcije: ukupni fenoli, karotenoidi i klorofil a pri temperaturi 50 °C i 4 ekstrakcijska ciklusa od 10 min; klorofil b pri temperaturi ekstrakcije od 50 °C, 3 ekstrakcijska ciklusa od 10 min.The aim of this study was to investigate the extraction efficiency of the bioactive compounds from stinging nettle (Urtica dioica L.) leaves by using Accelerated Solvent Extraction (ASE). Ethanol was used as the extraction solvent and the following extraction parameters were investigated: (i) static extraction time (5 and 10 min); (ii) number of extraction cycles (1, 2, 3 and 4 cycles); and (iii) temperature (20 and 50 °C). All extracts were spectrophotometrically evaluated in terms of total phenols, carotenoids and chlorophylls a and b. The results of the research have shown that all extraction parameters significantly affected the concetrations of the investigated compounds (p≤0,01). Mass fractions of total phenols ranged from 106,44 to 739,72 mg GAE 100 g-1, chlorophyll a from 116,44 to 490,42 mg 100 g-1, chlorophyll b from 44,88 to 166,85 mg 100 g-1 and carotenoids from 36,62 to 133,99 mg 100 g-1. The highest yields were obtained under the following extraction conditions: extraction temperature of 50 °C and four 10 minute cycles for total phenols, carotenoids and chlorophyll a and extraction temperature of 50 °C and three 10 minutes cycles for chlorophyll b