[The possibilities for the implementation of whole genome sequencing (WGS) of Neisseria meningitidis in molecular surveillance of invasive meningococcal disease in the Czech Republic]. / Moznosti implementace sekvenace celého genomu (WGS) Neisseria meningitidis do molekulární surveillance invazivního meningokokového onemocnení v Ceské republice.

The implementation of the surveillance of invasive meningococcal disease is recommended worldwide. The Whole Genome Sequencing (WGS) method increasingly comes to the fore, which provides the possibilities for further detailed characterization of Neisseria meningitidis and makes it possible to integrate all conventional sequencing approaches into one method. Six N. meningitidis isolates from 2013 and 2015, characterized previously by Sanger amplicon sequencing, were selected to be studied by the novel WGS method. WGS data analysis has confirmed the accuracy of this method in determining epidemiological markers. The aim of this communication is to point out the possibility for the implementation of WGS into molecular surveillance of invasive meningococcal disease in the Czech Republic. The National Reference Laboratory for Meningococcal Infections (NRL/MEN) will continue to use WGS for molecular characterization of selected isolates of N. meningitidis and for the improvement of molecular surveillance of invasive meningococcal disease in the country.

[Multiple-locus variable number tandem repeat analysis of Bordetella pertussis strains collected in the Czech Republic in 1967-2015: spread of a variant adapted to the population with a high vaccination coverage]. / Multiple-locus variable number tandem repeat analýza souboru kmenu Bordetella pertussis v CR z období 1967-2015: rozsírení varianty adaptované na proockovanou populaci.

AIM: To perform multiple-locus variable number tandem repeat analysis (MLVA) of B. pertussis strains from the collection of the National Reference Laboratory for Diphtheria and Pertussis (NRL/DIPE), National Institute of Public Health (NIPH), Prague. The study strains were isolated from clinical specimens collected mostly in the Czech Republic over a nearly 50-year period from 1967 to 2015 (June). The isolates from three periods characterized by different vaccination strategies and trends in pertussis are compared for genetic diversity and distribution of MLVA types (MT). Based on the results obtained, the suitability for use of MLVA in the analysis of epidemic outbreaks of B. pertussis in the Czech Republic is considered. MATERIAL AND METHODS: DNA samples extracted from B. pertussis strains included in the present study were examined by MLVA using the standard protocol. Data were processed by means of the eBURST algorithm and the calculation of the Simpson diversity index (DI) was used for the statistical analysis. Data were analyzed as a whole and also separately for strains from the three periods: 1967-1980, 1990-2007, and 2008-2015 (June). RESULTS: Fourteen different MT were detected in the study strains, with three of them not being reported before. The most common MTs were MT27 and MT29. MT29 was predominant in 1967-1980 while MT27 was the most prevalent in 1990-2007 and 2008-2015 (June). The DI was the lowest (0.49) in 2008-2015 (June), and comparably higher DIs were calculated for the two previous periods (i.e. 0.667 for 1967-1980 and 0.654 for 1990-2007). CONCLUSION: MLVA revealed a decrease in genetic diversity and shifts in MT distribution of B. pertussis strains isolated from clinical specimens in the Czech Republic from 1967 to 2015 (June). These shifts in the Czech Republic can be characterized as a progressive increase in global MTs at the expense of the locally unique ones. The most common MT, similarly to other geographical areas with long-term high vaccination coverage, is MT27. The results of MLVA of 136 B. pertussis strains can provide a background for using this method in molecular epidemiological analysis of smaller groups of strains.

[Clonal analysis of the meningococcal populations from invasive disease and healthy carriers isolated in the Czech Republic in 1971-2014 (May)]. / Klonální analýza populace meningokoku z invazivního onemocnení a od zdravých nosicu izolovaných v Ceské republice v období 1971--2014 (kveten).

OBJECTIVE: To present the results of clonal analysis of the meningococcal populations isolated from invasive disease and healthy carriers in the Czech Republic over four decades. MATERIAL AND METHODS: A total of 2179 isolates of Neisseria meningitidis from 1971-2014 (May) were studied: 1093 isolates from patients with invasive meningococcal disease (IMD) and 1086 isolates from healthy carriers. All study isolates were analysed by multilocus sequence typing (MLST), one of the major methods used in molecular epidemiology of IMD. RESULTS: More than 94 % of N. meningitidis isolates from IMD were assigned to serogroups B or C. The strains of the leading serogroup B were genetically highly heterogeneous: 1093 isolates were assigned to 25 clonal complexes. Similarly, the strains of the second leading serogroup C appeared genetically heterogeneous and were classified into 19 clonal complexes. The third leading serogroup Y of IMD isolates showed an opposite tendency and appeared highly homogeneous, with only three clonal complexes being detected. Over 75% of the predominant clonal complexes of IMD isolates of both serogroup Y (cc23) and serogroup C (cc11) were classified as hypervirulent and, as such, posed the highest risk to the host population. Over 80% of IMD isolates of serogroup B were assigned to hypervirulent clonal complexes (cc32, cc41/44, cc18, cc269, and cc11). Of 1086 N. meningitidis isolates from healthy carriers, 41.4% were non-serogroupable, i.e. designated N. meningitidis NG. Classification of these isolates into clonal complexes was highly heterogeneous. In total, 28 clonal complexes were identified of which only a minority were hypervirulent. CONCLUSIONS: The analysis of MLST data on strains collected over four decades revealed that the population of N. meningitidis strains involved in IMD differ genetically from N. meningitidis strains isolated from healthy carriers. These results are relevant to both the optimal use of preventive measures in a focus of IMD and to the development of an effective meningococcal vaccine and vaccination strategy guidelines.

Transformation of Streptomyces lincolnensis protoplasts with plasmid vectors.

A method for the preparation and regeneration of protoplasts of Streptomyces lincolnensis is described. Mycelium in the early exponential phase appeared to be most suitable for this purpose and yielded up to 25% regenerated intact cells. Transformation of S. lincolnensis protoplasts was achieved using broad-host-range streptomycete plasmid vectors pIJ622, pMP66, pRS410 and pIJ943 constructed from replicons pIJ101, pSLG33 and SCP2. The efficiency of transformation was 3.10(3) transformants per micrograms plasmid DNA when (2-5).10(7) recipient protoplasts were used. Interspecific transformations showed that there is no efficient restriction system in S. lincolnensis that would limit the transfer of genetic information from S. lividans or E. coli.

Preparation of protoplasts and regeneration of intact cells of Streptomyces cinnamonensis.

Protoplasts were prepared and intact cells were regenerated in Streptomyces cinnamonensis--a monensin producer--to make genetic manipulations with this strain possible. 70-80% of protoplasts were formed and up to 90% of them could regenerate into intact cells.

Selective regeneration of Bacillus subtilis protoplasts transformed to kanamycin resistance.

An HTY medium osmotically stabilized with 0.5 M D-glucitol was used for regeneration of Bacillus subtilis protoplasts. The application of glucitol as osmotic stabilizer allows simultaneous selection of cells resistant to kanamycin to be made since this antibiotic is not inactivated by glucitol when added to the regeneration medium.

Transfer of liposome-encapsulated plasmid DNA to Bacillus subtilis protoplasts and calcium-treated Escherichia coli cells.

Protoplasts of Bacillus subtilis 168 trpC2 str and cells of Escherichia coli SK 1590 after treatment with calcium chloride were transformed to tetracycline resistance with the recombinant plasmid pUN82 entrapped in the reverse phase evaporation liposomes. Frequency of transfer was 4 X 10(-4)% in B. subtilis and 8 X 10(-6)% in E. coli.

Transformation in Bacillus subtilis. Initial stages of the transformation process and their energy dependence.

NaN3 was found to inhibit transformation but not the irreversible binding of donor 3H-DNA in competent cells of the original low-transformable strain Bacillus subtilis 168 trp2. Addition of NaN3 to cells of two mutants Bacillus subtilis HT39 and HT46 with an increased transformability decreased substantially the irreversible binding of the donor DNA to the competent cells. The decreased irreversible binding of DNA is caused by an increased osmotic sensitivity of competent cells of the mutants HT39 and HT46 in the presence of NaN3, leading preferentially to lysis of the competent cells.