Staphylococcus simulans Recombinant Lysostaphin: Production, Purification, and Determination of Antistaphylococcal Activity.

Staphylococcus simulans lysostaphin is an endopeptidase lysing staphylococcus cell walls by cleaving pentaglycine cross-bridges in their peptidoglycan. A synthetic gene encoding S. simulans lysostaphin was cloned in Escherichia coli cells, and producer strains were designed. The level of produced biologically active lysostaphin comprised 6-30% of total E. coli cell protein (depending on E. coli M15 or BL21 producer) under batch cultivation conditions. New methods were developed for purification of lysostaphin without affinity domains and for testing its enzymatic activity. As judged by PAGE, the purified recombinant lysostaphin is of >97% purity. The produced lysostaphin lysed cells of Staphylococcus aureus and Staphylococcus haemolyticus clinical isolates. In vitro activity and general biochemical properties of purified recombinant lysostaphin produced by M15 or BL21 E. coli strains were identical to those of recombinant lysostaphin supplied by Sigma-Aldrich (USA) and used as reference in other known studies. The prepared recombinant lysostaphin represents a potential product for development of enzymatic preparation for medicine and veterinary due to the simple purification scheme enabling production of the enzyme of high purity and antistaphylococcal activity.

[The express diagnostic of microorganisms affecting respiratory tract of patients with mucoviscidosis].

The shared bacteria Burkholderia capacia complex and Achromobacter sp. infect the respiratory tract of patients with mucoviscidosis brining on disorders of respiratory patency. Burkholderia capacia complex is characterized by transmissivity and higher lethality of patients infected by Burkholderia. Hence, the importance of differentiation of these phenotypically similar microorganisms is obvious. The developed express technique of diagnostic includes the separation of DNA from phlegm amplification and sequenation was fragments of genes recA, gltB, gyrB, 16S rDNA. The evaluation of products of amplification of genes recA, gltB makes it possible to differentiate Burkholderia capacia complex and Achromobacter sp. The analysis of successions of recA, gltB, gyrB makes it possible to identify genotype of Burkholderia capacia complex on the basis of data of allele profiles of strains of Burkholderia capacia complex circulating in Russia. The succession of gene 16S rDNA makes it possible to determine the taxonomic position of microorganism dominating in phlegm and not belonging to Burkholderia capacia complex or Achromobacter sp. The real time polymerase chain reaction in presence of intercalating dye Sybr Green I, DMSO and D(+)-trehalose makes it possible to differentiate Burkholderia capacia complex from other microorganisms infecting respiratory tract of patients with mucoviscidosis. This approach provides additional reduction of diagnostic duration and decrease possibility of contamination.

[Burkholderia cepacia persistence in patients with mucoviscidosis].

AIM: Study features of persistence of Burkholderia cepacia in mucoviscidosis patients. MATERIALS AND METHODS: In the period from 2008 to 2009, 56 B. cepacia strains isolated from children with mucoviscidosis were obtained. 114 medical histories of children with mucoviscidosis from various age groups were analyzed. The developed algorithm for identification and typing including phenotype and molecular biology methods was used to identify B. cepacia bacteria. Strain genotyping was carried out by RAPD-PCR with random oligonucleotide primer as well as pulse-electrophoresis. RESULTS: Persistence of associations ofmicroogranisms in 59.4% of cases was established to be the feature of persistent infection in mucoviscidosis. The feature of persistence of B. cepacia strains in patients with diagnosis ofmuco-viscidosis mixed form, severe course is persistence in association with Pseudomonas aeruginosa. B. cepacia bacteria that can persist in mucoviscidosis patients are characterized by resistance to many antibiotics. A prolonged (up to 1 year and 5 months) persistence of B. cepacia strains isolated from 1 patient was proven by using microflora monitoring of lower respiratory tract. CONCLUSION: B. cepacia bacteria may colonize lower respiratory tract of mucoviscidosis patients, persist for a long time and be transmitted between patients.

[Persistence of Pseudomonas aeruginosa strains in patients of Federal Scientific Center of Transplantology and Artificial Organs].

AIM: Study genetic diversity of P. aeruginosa strains persisting in patients of Federal Scientific Center of Transplantology and Artificial Organs, and main factors facilitating persistence of strains in the hospital. MATERIALS AND METHODS: 136 P. aeruginosa strains isolated from patients of the center for 3 years 6 months were genotyped by RAPD-PCR and MLST methods and studied for antibiotics resistance and presence of integrons. RESULTS: Genetic diversity of strains persisting in hospital was established. Strains of main genotypes ST235, ST446, ST598 were isolated from patients of various surgical departments. Patients were shown to be colonized by these strains during stay in reanimation and intensive therapy department (RITD) of the hospital. Strains of dominant genotype 235 were isolated from 47% of examined patients during more than 3 years. Only genotype 235 strains contained integron with cassettes of antibiotics resistance genes blaGES5 and aadA6 in the genome. CONCLUSION: The data obtained show that over the period of observation in the center 1 clone of P. aeruginosa that belonged to genotype 235 dominated. This clone was endemic for this hospital and in the process of prolonged persistence became more resistant to antibiotics. Colonization of patients with these strains occurs in RITD. This confirms the necessity of constant monitoring of hospital microflora for advance detection of potentially dangerous epidemic hospital strains able to cause hospital infections.

[Mutants of Burkholderia cenocepacia with a change in synthesis of N-acyl-homoserine lactones--signal molecules of Quorum Sensing regulation].

By means of plasposon mutagenesis, mutants of Burkholderia cenocepacia 370 with the change in production of N-acyl-homoserine lactones (AHL), signal molecules of the Quorum Sensing system of regulation, were obtained. To localize plasposon insertions in mutant strains, fragments of chromosomal DNA containing plasposons were cloned, adjacent DNA regions sequenced, and a search for homologous nucleotide sequences in the GeneBank was initiated. It has been shown that the insertion of plasposon into gene lon encoding lon proteinase drastically decreases AHL synthesis. Upon insertion of plasposon into gene pps encoding phosphoenolpyruvate-synthase, enhancement of AHL production is observed. In mutant carrying inactivated gene lon, a strong decline of extracellular protease activity, hemolytic, and chitinolytic activities was observed in comparison with the original strain; lipase activity was not changed in this mutant. Mutation in gene pps did not affect these properties of B. cenocepacia 370. Mutations in genes lon and pps reduced the virulence of bacteria upon infection of mice.

[Genetic diversity of Staphylococcus aureus strains that colonize nose mucous membrane of children living in a large industrial city].

AIM: Study of genetic diversity of Staphylococcus aureus strains that colonize nose mucous membrane of children living in an ecologically unfavorable district of Krasnoyarsk. MATERIALS AND METHODS: 20 S.aureus strains isolated from mucous membrane of anterior part of nose of children were genotyped by RAPD-PCR (random ampliphied polymorphic DNA) with an oligonucleotide primer (10 Sh1 nucleotide). RESULTS: Presence of 6 genotypes, with 3 of those including 3 and more strains, was detected among the studied strains. The extent of genetic similarity between genotype 1 and 2 was established at 85%. CONCLUSION: 20 of 63 (31.7%) of examined students were resident carriers of S. aureus. 3 main genotypes of S. aureus that circulate in students were detected by RAPD-PCR. This confirms the necessity to use RAPD-PCR method for timely evaluation of epidemic situation in organized groups of children.

[Microbial population of lower respiratory tract in children from different age groups with cystic fibrosis].

AIM: To study microflora of lower respiratory tract of children from different age groups with cystic fibrosis during follow-up for determination of its variability and possible sources of infectious complications. MATERIALS AND METHODS: One hundred forty-one medical histories of patients from different age groups with cystic fibrosis living in various regions of Russian Federation were analyzed. Eighty-four children with cystic fibrosis living in Moscow and Moscow region treated as outpatients and inpatients were prospectively followed. For identification and characterization of microorganisms, microbiological, molecular biological, and statistical methods were used. RESULTS: It was demonstrated that chronic pseudomonas, staphylococcal or mixed infection was already diagnosed in 25% of children aged 1-4 years, and identified in 80% of patients to the age of 18 years. In two-thirds of cases association of microorganisms was identified, and in hospitalized patients these associations were comprised by 3-5 microorganisms in 60% of cases. Aside from main agents in associations (Pseudomonas aeruginosa and Staphylococcus aureus), representatives of Gram-negative nonfermentative microorganisms (Burkholderia cepacia, Stenotrophomonas maltophilia, Acinetobacter baumanii) were often identified that possibly determined by tropism of these species to lung tissue. CONCLUSION: Chronic mixed infection is characteristic for patients with cystic fibrosis. Identification of possible mechanisms of lung infection in patients with cystic fibrosis will allow to develop evidence-based system of prevention of infectious complications in these patients.

[Role of "quorum sensing" regulatory system in formation of biofilms by Burkholderia cepacia and Pseudomonas aeruginosa].

AIM: To study influence of chemically synthesized lactones with different length of carbonic chain on formation of biofilms by Burkholderia cepacia and Pseudomonas aeruginosa. MATERIALS AND METHODS: Fifty-four strains of B. cepacia including reference strains and clinical isolates as well as etalon strain PA103 of P. aeruginosa were used. Lactones C4 [N-(3-hydroxybutanoyl-L-gomoserine lactone], C6 [N-(3-oxoohexanoyl)-L-gomoserine lactone], C8 [N-(3-oxooctanoyl)-L-gomoserine lactone], C0 [L-gomoserine lactone-HBr] were synthesized and purified by column chromatography. Formation of biofilms was studied by determination the ability of B. capacia strains to adhesion on the surface of 96-well polystyrene plate. RESULTS: Ability to biofilms formation was identified in 83.3% studied strains. It was shown that lactones C4+C8 and C6+C8 when added to cultivation medium improve growth of B. cepacia biofilm. Analysis of optical density (OD) values for P. aeruginosa biofilm revealed that lactones C4, C8, C4+C6, C4+C8, C6+C8 inhibit the formation of biofilm. The most prominent decrease of P. aeruginosa biofilm OD was observed during growth of culture in presence of C0. CONCLUSION: Obtained data point to different effects of lactones and their combinations on formation of biofilms by B. cepacia and P. aeruginosa. Suppression of biofilm formation by P. aeruginosa induced by lactone C0 confirms the need for development of new bacteriostatic drugs, which will be able to inhibit function of the "quorum sensing" regulatory system.

[Preclinical tests of Staphylococcus aureus somatic antigen as the basis for drugs for treatment of staphylococcal skin infections].

AIM: To analyze results of conducted preclinical animal tests of ointment and gel preparations based on the somatic antigen ("Somatin") of Staphylococcus aureus. MATERIALS AND METHODS: Contents of ointment and gel as well as method of "Somatin" extraction from cytosomatin ointment are presented. Standard serum was obtained by immunization of rabbits by "Somatin". Tests for acute and chronic toxicity and sensitizing effect of cytosomatin ointment were performed. 1% gentamycin ointment was used for comparison with cytosomatin ointment and gel for treatment of pyodermia. Preparations were patented. RESULTS: Authenticity and specific activity of ointment and gel preparations were demonstrated. Animal tests for acute and chronic toxicity of cytosomatin ointment did not reveal inflammatory reaction of skin and sclera as well as influence on biochemical parameters of the blood or abnormalities of visceral morphological structure. Assessment of sensitizing effect of cytosomatin ointment did not reveal promotion of allergy. Advantages in treatment of staphylococcal skin infections and wounds with cytosomatin ointment and gel as compared with gentamycin ointment were established. CONCLUSION: The main immunogenic mechanism of "Somatin" substance was activation of complete phagocytosis. The character of immune response was specific. Selected optimal ratios of therapeutic doses for ointment and gel bases promote active permeation of the drug into skin that enhances its therapeutic effect. Duration of treatment with cytosomatin ointment was shortened on 5 days as compared with gentamycin ointment. Duration of treatment with cytosomatin gel was shortened on 8 days as compared with prototype.

[Genotypic features of Pseudomonas aeruginosa strains circulating in surgical hospital].

AIM: To study genetic diversity of Pseudomonas aeruginosa strains circulating in intensive care unit (ICU), to determine the source of these strains and duration of circulation of epidemically-significant clone in the hospital. MATERIALS AND METHODS: Genotyping of 106 P. aeruginosa strains isolated from patients, clinical specimens and fomites was performed by random amplified polymorphic DNA analysis with oligonucleotide primer Sh1 of 10 bp long. RESULTS: Out of 106 P. aeruginosa isolates, 72.6% belonged to the same genotype, which was dominated in ICU during whole study period. It was established that 58.3% of examined patients were colonized by identical strains belonged to prevalent genotype that indicates the intrahospital transmission of epidemic strain. CONCLUSION: Obtained data show that during the period of observation (15 months) one clone of P. aeruginosa dominated in ICU, which was characterized by multiple resistance to antibiotics and caused nosocomial infection in 58.3% of patients. This confirms the need of continuous molecular-microbiological monitoring of hospital microflora in order to early detect potentially dangerous epidemic hospital strains, which are able to cause nosocomial infections.

[Biofilm formation by strains of Burkholderia cepacia complex in dependence of their phenotypic and genotypic characteristics].

Biofilm formation was studied in 54 strains of Burkholderia cepacia complex isolated in 7 Moscow hospitals. 80% of strains (biofilm groups I and II) had the capacity to biofilm formation and only 16.7% of strains (group III) were not capable to biofilm formation. Molecular genetic methods allowed to identify one of the epidemic markers (CBL, IS hybrid sequence, Burkholderia Cepacia Epidemic Strain Marker - BCESM) in 46.7, 23.3, and 33.3% of strains from group I, II, and III respectively. Gene cepR from the Quorum Sensing system was identified in three biofilm groups in nearly equal frequency (92.3, 96.2 and 100% for group I, II, and III respectively), whereas cepl gene was found more often in group I (76.9%) and II (65.4%). Strains from all three groups had protease and lipase activity and 13.3% of group I strains had chitinolytic activity. B. cepacia strains from group I produced hemolysin in 33.3% of cases, from group II--in 26.6%, and from group III--in 11.1% of cases. The majority of Moscow hospital strains of B. cepacia complex were identified as B. cenocepacia (genomovar III, group A). RAPD-PCR method enabled to differentiate isolated strains into several genotypic variants. 13.3% of strains from group I were susceptible to imipenem/ciprofloxacin, as well as 33.3% of isolates from group II and 44.4% of isolates from group III.

[The role of "quorum sensing" regulation system in symbiotic interaction of bacteria Burkholderia cepacia and Pseudomonas aeruginosa during mixed infection].

Data concerning mechanisms of strengthening virulence of bacteria B.cepacia and P.aeruginosa during experimental mixed-infection are submitted. Results of in vitro studies of acylhomoserine lactones (AHL) with various length of a carbon lateral circuit have shown that butanoyl-homoser-ine lactone (C4), hexanoyl-homoserine lactone (C6) and octanoyl-homoserine lactone (C8) separately and in various combinations display different effects on growth of bacteria, formation of colonies and production of pathogenicity factors. Experiments on animals have shown strengthening virulence of bacteria B.cepacia by C4, C6 and C8 lactones when introduced simultaneously. Strengthening of virulence of the P.aeruginosa strain under the action of lactone C6 and simultaneous action of C4, C6 and C8 lactones was observed. The data obtained give grounds for assumption, that bacteria may use exogenous lactones, including those produced by closely related bacteria, during their life cycle.

[Antibiotic resistance and putative origin of Staphylococcus aureus and Klebsiella strains isolated from the children with intestinal dysbacteriosis].

The results of the statistical treatment of data on the analyses of 766 children, the residents of Moscow, for dysbacteriosis are presented; of these children, 34 were aged up to 1 month and 732, from 1 month to 1 year. This study revealed that in the fist year of life in children with dysbacteriosis the dominating bacterial species were S. aureus, bacteria of the genus Klebsiella and fungi of the genus Candida. From the intestine of children aged up to 1 month S. aureus and Klebsiella were isolated more often than from children aged up to 1 year. The results of the study of antibioticograms demonstrated that 21.6% of S. aureus strains and 74.4% of Klebsiella strains were multiresistant to antibiotics. Taking into account the fact that multiresistance to antibiotics was characteristic of hospital strains, the suggestion was made that the isolated strains were of hospital origin and such strains could colonize the intestine of children in maternity hospitals.

[Microbiological aspects of enteric dysbacteriosis in patients of different age groups, visiting outpatient clinics of Moscow].

The analysis of the intestinal microflora in 2,378 patients of different age revealed changes in the state of enteric microflora in all examined patients. In the maximum percent of cases a decrease in the amount of bifidobacteria was observed in children of up to 1 month old and in the amount of lactobacilli, in children aged 6 - 14 years. In patients of all age groups the representatives of such facultative microflora as Staphylococcus aureus or fungi of the genus Candida dominated. The highest proportion of isolated staphylococci was characteristic of children in the first year of life. In the highest percent of cases a decrease in the amount of Escherichia coli with typical properties was observed in persons over 65 years old. Other enterobacteria were most often isolated from adults aged 56 - 65, but the percentage of their isolation was 1.5 times lower than that of Candida. The conclusion was made that the treatment of patients with quantitative and qualitative disturbances of normal enteric microflora needed individual approach in each concrete case with due regard to the patient's age.

[Epidemiological aspects of the present-day demographic situation in Russia].

Study of reference rate in healthcare institutions in 1991 - 2001 demonstrated a steady growth of general mortality and disease incidence among the population (by 22.7% and 8.1%, respectively). The number of patients with acute and chronic diseases registered in 2001 was by 16.4% higher than that in 1991. These trends were characteristic of all disease classes, but the most intensive growth was observed in such classes as diseases of blood and hemopoetic organs (2.5 times), endocrinal and urogenital diseases (1.8 times). Disease incidence and general mortality among children aged 0 to 14 had grown by 25% and 33.2%, among adolescents--by 60% and 71.1%, among adults--by 5.2% and 20.3%, respectively. These negative trends in population health were accompanied by a constant growth of incapacitation rate in 1990 - 2000 (2.4 times, and 4.3 times among children and adolescents younger than 18). There is a correlation between mortality and traditionally used demographic parameters, which suggests that mortality should be recognized the key demographic parameter. In order to achieve a satisfactory level of population health and adequate demographic transition, and to develop general sanatory strategy, the State should undertake powerful, goal-seeking, society-oriented measures, including deep clinical and epidemiological study of human pathology.

[The influence of bacteria of the Burkholderia cepacia and Pseudomonas aeruginosa complex on cell-mediated immune reactions in experimental animals].

The evidence has been obtained that various species, as well as individual strains having pathogenicity factors, produced different effect on the functional activity of immunocompetent B and T lymphocytes of mice infected intraperitoneally. The injection of live P. aerruginosa PA 103 and B. cepacia 8240 cells resulted in imunosuppression of antibody-forming cells, synthesizing antibodies to heterologous antigens. On the contrary, in the animals infected with B. cepacia 8236 the functional activity of B lymphocytes increased. An increase in the proliferative activity of spleen cells was noted in the presence of T and B mitogens after the infection of mice with P. aeruginosa PA 103 in comparison with B. cepacia 8236 and B. cepacia 8240 which produced a faintly pronounced modulating effect. The pathogenesis mechanisms of infections induced by these microorganisms as well as the development of chronic, persisting forms of the infectious process are discussed.

[A study of action of subinhibiting concentrations of antibiotics on the expression of genes regulating production of pathogenicity factors in Burkholderia cepacia and Pseudomonas aeruginosa].

Described in the paper are the results of a study of differential expression of pathogenicity of B. cepacia and P. aeruginosa in subinhibiting concentrations (SIC) of cyprofloxacyne. While identifying genes expressing differentially in the antibiotic SIC, genes cepR B. cepacia and P. aeruginosa expressing without cyprofloxacyne in the cultivation medium and not expressing in the antibiotic SIC were detected. Finally, involvement of cepR B. cepacia in regulating the pathogenicity expression factors according to "quorum sensing" is under discussion.

[Virulent properties of hospital strains of bacteria of the Burkholderia cepacia complex, isolated in hospitals of Moscow].

The results of the study of hospital strains of the B. cepacia complex, isolated in hospitals of Moscow, with the use of phenotypical and molecular-genetic methods are presented. The phenotypical methods made it possible to differentiate Russian strains and classify them with a group of genomovars (I, III, IV). As the result the epidemic importance of the strains with epidemic markers, having specific characteristics for every clinic, was determined. The detection of the collection of genes cepI and cepR in the strains made confirmed the epidemic importance of the stains which had, due to the regulatory "quorum sensing" (QS) system, the potential capacity for inducing infection and persisting in the patient's body. The presence of gene cepR in all strains and the absence of gene cepl in 33% of strains gave evidence to suggest that in some strains the activation of the production of pathogenicity factors required the presence of other bacteria having the fully developed QS system. Thus, the new complex approach with the use of phenotypical and molecular-genetic methods permits more precise identification of the source of hospital infection induced by the bacteria of the B. cepacia complex.

[Development of the controlled model of persisting infection , caused by Pseudomonas aeruginosa and bacteria of the complex Burkholderia cepacia]. / Razrabotka upravliaemoi modeli persistiruiushchei infektsii, vyzvannoi Pseudomonas aeruginosa i bakteriiami kompleksa Burkholderia cepacia.

As the result of testing three different variants, the experimental models of persisting infection for P. aeruginosa and B. cepacia have been developed. These doses differ in the time of administration, doses of antibiotics and the infective doses of the microorganisms. The administration of the sub-inhibiting concentration of antibiotics for 5 days and the subsequent infection of laboratory animals (non-inbred mice) B. cepacia strains in a dose of LD50 leads to a considerable increase in the survival rate of mice and to a longer period (up to 20 days) of obtaining inoculative material from the spleen. The isolated cultures are characterized by a sharply slower growth on artificial culture media (up to 5-7 days as compared with 24-48 hours for the initial culture). The newly developed models have made it possible to control different stages of the infectious process in the induced increase or decrease of the virulent properties of the infective agent and in changes in the immune status of the host. As the result of these studies, in some mice (10%) infected with B. cepacia after the injection of gamma-hydroxybutyric acid lactone the infection has taken the acute form, while in the mice infected with P. aeruginosa no such effect has been observed. On the contrary, in the mice infected with P. aeruginosa and then receiving cyclophosphamide the transition of the infection into the acute form has been observed in 30% of the animals. In the mice infected with B. cepacia no such effect has been noted after the injection of this preparation. Different effects produced by cyclophosphamide and lactone are discussed from the positions of "quorum sensing" in pathogenic bacteria.

[Clinical strains of Burkholderia cepacia: characteristic and detection of the components in the quorum sensing regulatory system]. / Klinicheskie shtammy kompleksa Burkholderia cepacia: kharakteristika i vyiavlenie komponentov reguliatornoi sistemy quorum sensing.

Described in the paper are characteristics of B. cepacia clinical strains isolated from patients at Moscow hospitals. The strains were investigated for the presence of proteolytic, chitinolytic, hemolytic and lipase activities as well as for presence of components of the "Quorum sensing" gene activity regulatory system by using biological test-systems and in the polymerase chain reaction with primers to genes cepI and cepR.