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.

[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.

[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.

[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.

[Study of higher schooling load effect on the health status of schoolchildren]. / Izuchenie vliianiia povyshennoi uchebnoi nagruzki na sostoianie zdorov'ia uchashchikhsia.

Higher schooling load, non-observance of school routine, inactivity, etc. have a negative impact on health. The purpose of the study was to determine the impact of intensive training on the health status of schoolchildren. An intensive training college, a physico-mathematical school, a private school-gymnasia were chosen as objects of studies. A general educational school was matched as a control object. In each educational establishment, 180-320 schoolchildren from the 8th-10th forms were examined. The examination ascertained that the schooling load at new-type schools was greater than the standards established. Before going to new-type schools, schoolchildren had a better health status than did the general educational school children of their age; during schooling, worse health was, however, more noticeable in new-type school children than in the matched schoolchildren. A higher schooling load also affects the performance of schoolchildren unfavorably.