Genetic subgroups of Neisseria meningitidis serogroup A isolated from patients with disseminated forms of meningococcal infection in Moscow, 1969-2006

Results of microbiological monitoring for serogroup A Neisseria meningitidis circulated in Moscow from 2002 to 2006 are presented. Using multilocus sequence-typing, molecular and epidemiologic characteristics of 32 cultures isolated from cerebro-spinal fluid of patients with generalized forms of meningococcal infection. Typed isolates belonged to 4 sequence types: CT-3349 (detected in 24 cultures), CT-2 (detected in 5 cultures), CT-75 (detected in 2 cultures), and CT-5803 (detected in 1 culture). All sequence types (except CT-5803) were detected in Moscow in previous years. Using Internet database (http://pubmlst.org/neisseria) they were genetically characterized and compared with data on serogroup A meningococci circulated in Moscow before 2002., meningococci belonging to epidemically dangerous genetic subgroup III were not detected between characterized strains. Typed isolates were distributed between subgroups VI and X, which are typical for the area under surveillance. Genetic changes in Moscow population of Neisseria meningitidis serogroup A, which manifested by shift of dominating genetic subgroup after 2002-2003, were analyzed.     

Streptokinase alleles and disease association in group A streptocci

Abstract Allele-specific oligonucleotides were used for PCR-based typing of the streptokinase locus of group A streptococcal strains, including well characterized type strains, isolates from patients with acute poststreptococcal glomerulonephritis and strains from Aboriginal communities in the Northern Territory of Australia. The streptokinase SKN allele, previously thought to be associated with glomerulonephritis, was no more frequent in nephritogenic than in non-nephritogenic streptococcal strains in this collection.     

Pulse-electrotypes of Listeria monocytogenes strains, isolated in Moscow

The characterization of the pulse-electrotypes of L. monocytogenes, isolated in 2003-2004 in Moscow from different sources, is presented. Among the cultures, isolated from humans, one outbreak pulse electrotype was detected and from different objects in buildings where a wide variety of food products was produced several probably related and unrelated pulse-electrotypes were obtained. The conclusion was made that several independent L. monocytogenes clones existed on the territory of Moscow, and many products supplied to retail trade and public catering enterprises were contaminated with these clones. Pulse electrophoresis was shown to be the most effective method for intraspecific typing and the study of the molecular epidemiology of listeriosis. Grounds for the necessity to improve the microbiological diagnostics of L. monocytogenes infection are given.     

Epidemiological evaluation of Neisseria meningitidis serogroup B by pulsed-field gel electrophoresis

Abstract Genomic DNA from 25 strains of serogroup B Neisseria meningitidis was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Spe I. N. meningitidis genomic DNA displayed considerable diversity. The diversity we observed among these strains was stable and included isolates from an outbreak that were phenotypically identical. This confirms the value of macrorestriction profiling and PFGE in providing epidemiologically stable strain markers for typing meningococci.     

Polysaccharide-protein complexes isolated from different strains of Neisseria meningitidis serogroup B

Fractionation of the biomass of 3 serogroup B N. meningitidis strains, obtained from solid serum-free and liquid synthetic media, by increasing concentrations of cetavlone revealed that the formation of natural polysaccharide-protein complexes with the ratio of their components approaching 1:1 was possible under the conditions ensuring the intensive synthesis of capsular polysaccharide. Two strains, 125 and 1642, grown on a solid amino peptide-containing medium regularly produced two polysaccharide-protein complexes with the protein/polysaccharide ratio approaching 1:1. One of these complexes passed easily into the supernatant fluid and could be precipitated with 0.1% cetavlone. The second complex was more firmly bound to the outer membrane of the cell and could be precipitated with 1% cetavlone. In most experiments an additional fraction with high protein content in relation to sialic acid was isolated from the biomass.     

Genotyping of Legionella pneumophila serogroup 1 strains isolated in Northern Sicily, Italy

During a three-year period, from April 2002 to May 2005, one hundred-forty-seven samples, taken from technical systems of water distribution at point of use, were repeatedly collected at six different sites in Northern Sicily and assayed for the presence of Legionella pneumophila serogroup 1 and serogroups 2 to 14. At the first samplings, the water distribution systems of all the sites were heavily contaminated, and disinfection treatments by the superheat and flush method were therefore performed. Treatments were always successful against L. pneumophila sg.1, but only in a few cases against all other serogroups. Eighty-six strains of L. pneumophila sg. 1, isolated from 26 of these samples, were characterized by amplified fragment length polymorphism (AFLP) analysis and sequence-based typing (SBT) procedure. Perfectly overlapping results were obtained by both the procedures and four genotypes were identified, accounting for all the isolates. The easy transferability of the SBT data through a web-based database made it possible to identify the presence in Northern Sicily of the two SBT types most commonly circulating in Europe.     

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom

R.J. BIRTLES, N. DOSHI, N.A. SAUNDERS AND T.G. HARRISON. 1991. A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

Mapping phosphoproteins in Neisseria meningitidis serogroup A

To investigate the phosphorylation capability of serogroup A Neisseria meningitidis (MenA) and to implement our knowledge in meningococcal biology and in bacterial post-translational modifications, cell extracts were separated by 2-DE and 51 novel phosphoproteins were revealed by the use of the highly specific Ser/Thr/Tyr-phosphorylated proteins staining by Pro-Q Diamond and identified by MALDI-ToF/MS. Our results indicate that phosphorylation in MenA is comparable to that of other bacterial species. A first functional characterization of the identified modified proteins was also given, in order to understand their role in meningococcal physiopathology.     

Haemophilus influenzae antibiotic resistant strains isolated in Moscow in 2002-2004]

The results of 3-year observation on emergence of H. influenzae antibiotic resistant strains in Moscow are summarized. The study included 566 strains isolated from patients in 2002-2004. The susceptibility was determined by the 2-fold microdilution method on the Haemophilus test medium. The percentage of the resistant strains isolated in 2002, 2003 and 2004 was the following: ampicillin --4.9, 3.2 and 3.6%, tetracycline--3.3, 3.2 and 1.8% and co-trimoxazole--10.9, 20.9 and 20% respectively. The strains isolated in 2003 and 2004 were resistant to azithromycin in 0.6 and 1.8% of the isolates and to clarithromycin in 1.3 and 3.2% of the isolates respectively. Five isolates differed by the minimum resistance to ampicillin whereas producing no beta-lactamase (BLNAR strains). The drugs of choice for the treatment of respiratory tract infections mainly due to H. influenzae, i.e. acute otitis and sinusitis, chronic bronchitis exacerbation and sometimes pneumonia remain betalactam antibiotics. From the microbiological viewpoint the inhibitor-protected aminopenicillins, cefuroxime and cefotaxime have no significant advantages vs. amoxycillin. The use of cotrimoxazole and chloramphenicol should be considered inexpedient.     

A clonal analysis of Neisseria meningitidis serogroup A

A typing scheme has recently been developed for Neisseria meningitidis serogroup A based on the clonal population structure of these bacteria. An international strain collection consisting of 423 group A strains isolated from 23 epidemics or outbreaks since 1963, as well as from older epidemics and numerous non-epidemic situations was used in the analysis. Strains were first segregated into electrophoretic types, depending on the combined score for the electrophoretic mobilities of 7 cytoplasmic isoenzymes resolved by starch gel electrophoresis and of 2 outer membrane proteins resolved by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The bacteria were subsequently assigned to one of 21 clones after numerical analysis of their electrophoretic types.The epidemiological value of the typing scheme was assessed by examining case and carrier strains isolated during (1982–83) and subsequent to (1984–85) an epidemic in the Gambia, West Africa. The case isolates, all of which were serogroup A, were of a single clonal type. All serogroup A carrier isolates were also of this clone, while carrier strains of other serogroups showed greater clonal diversity. These results indicate that case strains during an epidemic show little clonal diversity and thus that the typing scheme is of value in distinguishing the etiology of epidemics.A retrospective epidemiological analysis of the strains in the international collection showed that most serogroup A epidemics were associated with a single or predominant clone, although some epidemics were of mixed etiology. The survey included 256 isolates from 15 African epidemics since 1963, a period which covers 3 major epidemic waves (1960–63; 1967–73 and 1981–83), thus permitting a detailed epidemiological analysis of serogroup A epidemics in this continent.Epidemiological records indicate that seven clones have been responsible for sets of epidemics throughout the world since 1915 and that at least two of these sets can be considered to represent mutually exclusive pandemics, linking numerous epidemics between 1967–75 and 1973–83, respectively.     

Second serogroup of Legionella quinlivanii isolated from two unrelated sources in the United Kingdom.

A series of strains, presumptively identified as legionellas on the basis of their nutritional requirements and biochemical reactivity, were isolated from two unrelated environmental sources in the UK. Representatives of each of these series had a restriction endonuclease digest pattern indistinguishable from that of the Legionella quinlivanii type strain (1442-AUS-E) and the identity of these strains was confirmed by DNA homology studies. Serological examination of the two strains showed that they were distinct from the type strain 1442-AUS-E but indistinguishable from each other. A second serogroup, L. quinlivanii serogroup 2 (type strain LC870; NCTC 12434), is proposed to accommodate these strains.     

A new leptospiral serovar in the autumnalis serogroup

Strain A6 of leptospire was isolated from blood culture of a case with leptospirosis on July 1, 1962 in Mengla county, Xishuangbanna, Yunnan. It is proved a new serovar belonging to serogroup Autumnalis by cross agglutination and cross agglutinin-absorption tests. The name, Leprospira interrogans serovar nanla with reference strain A6 is proposed.     

Differences in the susceptibility of field mice from the Altai Territory and Moscow Province to Leptospira of serovar mozdok and serogroup Pomona

Adult striped field mice (Apodemus agrarius) caught in the Altai region proved to be insusceptible to experimental infection when inoculated with Leptospira of serovar mozdok, serogroup Pomona. In pregnant females, though infected with this organism, no leptospiruria was observed. At the same time nonpubescent animals became Leptospira carriers, females becoming carriers 4.5 times more frequently than males. The formation of antibodies to Leptospira in the test rodents was poorly pronounced and did not depend on their sex, age, physiological state and the presence of renal leptospirosis. But all adult striped field mice belonging to the population of the Moscow region became Leptospira carriers in such experiments.     

Proteome analysis of Neisseria meningitidis serogroup A

Neisseria meningitidis is an encapsulated Gram-negative bacterium responsible for significant morbidity and mortality worldwide. Meningococci are opportunistic pathogens, carried in the nasopharynx of approximately 10% of asymptomatic adults. Occasionally they enter the bloodstream to cause septicaemia and meningitis. Meningococci are classified into serogroups on the basis of polysaccharide capsule diversity, and serogroup A strains have caused major epidemics mainly in the developing world. Here we describe a two-dimensional gel electrophoresis protein map of the serogroup A strain Z4970, a clinical isolate classified as ancestral to several pandemic waves. To our knowledge this is the first systematically annotated proteomic map for N. meningitidis. Total protein samples from bacteria grown on GC-agar were electrophoretically separated and protein species were identified by matrix-assisted laser desorption/ionization time of flight spectrometry. We identified the products of 273 genes, covering several functional classes, including 94 proteins so far considered as hypothetical. We also describe several protein species encoded by genes reported by DNA microarray studies as being regulated in physiological conditions which are relevant to natural meningococcal pathogenicity. Since menA differs from other serogroups by having a fairly stable clonal population structure (i.e. with a low degree of variability), we envisaged comparative mapping as a useful tool for microevolution studies, in conjunction with established genotyping methods. As a proof of principle, we performed a comparative analysis on the B subunit of the meningococcal transferrin receptor, a vaccine candidate encoded by the tbpB gene, and a known marker of population diversity in meningococci. The results show that TbpB spot pattern variation observed in the maps of nine clinical isolates from diverse epidemic spreads, fits previous analyses based on allelic variations of the tbpB gene.     

Antigenic structure of leptospires of the Canicola serogroup isolated in the north-western regions of the RSFSR

The comparative study of 16 Leptospira cultures, serogroup Canicola, isolated from humans and animals in different years in the North-West of the RSFSR and 2 reference strains of the Canicola serovar, Hond Utrecht IV and Kashirsky, was carried out in the agglutinin cross-adsorption tests. The absence of the antigenic homogeneity of the cultures under test was established: 9 of them proved to be identical to strain Kashirsky and 7, to strain Hond Utrecht IV.     

Clinico-epidemiologic aspects of campylobacteriosis in Moscow and Moscow Province

The clinical course and epidemiological features of campylobacteriosis in Moscow and Moscow Province in 1986-1987 are described. The specific proportion of this infection in the structure of acute enteric infections is, on the average, 4.8%. The level of Campylobacter contamination of animals and birds and their role in the spread of campylobacteriosis have been established. The possible ways and factors of the transfer of this infection are discussed.     

Molecular genetic typing of Legionella pneumophila serogroup 1 isolated in town Verkhnyaya Pyshma using international standards

Strains of Legionella pneumophila Pyshma-1 and Pyshma-2 were typed according to the international standard - STB protocol developed by EGWLI. Allelic profile of the strains was determined. Identity of strains on the locus pilE, which codes protein important for virulence of bacterium, was shown. Close similarity of nucleotide sequences in Pyshma-1 and Pyshma-2 strains (mainly, 98-100%) was noted. Both strains differed more from reference strain Philadelphia-1 ATCC 33152. Maximal differences (5-6%) were observed in fragment of mompS gene.The study revealed considerable need for conducting of systemic analysis of collected and newly isolated strains in order to get information picture on endemic strains of L. pneumophila in Russia.