Preparation of immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horseradish peroxidase with human antibodies to HIV-1]

By using three different linkage methods with carbodiimide, glutaraldehyde and periodite, immunoenzyme conjugates of beta-lactamase from Bacillus licheniformis 749/c and horse radish peroxidase with human antibodies to HIV-1 were prepared. The human antibodies were purified by the affinity procedure on Protein-A-Sepharose 6B. The conjugates were tested in a solid phase immunoenzymatic system for the HIV-1 antigen. It was shown that the conjugates prepared by the carbodiimide linkage method had the highest titer, the beta-lactamase conjugate being superior by its titer to the respective peroxidase conjugate. In the lyophilized state the conjugates prepared with the carbodiimide linkage method were stable.     

Immunoelectrophoretic analysis of the antigenic structure of Sh. sonnei]

The authors studied the antigenic composition of 105 Sh. sonnei strains freshly isolated from patients suffering from acute dysentery and carriers. Immunophoregrams of pure S-and R-forms species were obtained. Up to 13 antigens differing by electrophoretic and diffusion mobility and immunological specificity were revealed among soluble Sh. sonnei antigens The position of common and specific antigens was determined on the immunophoregram. Along with the thermostable somatic O-antigen detected at the I phase of the S-forms, and two thermolabile O-antigen components at the II phase, and the R-forms, there was revealed a surface, relatively thermolabile, K-antigen of A-type capable of agglutinating live bacteria in the O-antiserum; position of the latter on the immunophoregram was also determined.     

An immunoenzyme method of isolation of foot-and-mouth disease virus by using beta-lactamase conjugate with virus-specific antibodies

It was shown that in was feasible to use conjugates of virus-specific antibodies and beta-lactamase from Bacillus licheniformis 749/c to identify aphthosa virus antigens. The antigen titers determined by enzyme immunoassay (EIA) using a beta-lactamase conjugate were 5-64 times higher than the analogous indices of the complement fixation test. Unlike EIA, that by using the antibody conjugates with peroxidase or alkaline phosphatase there were observed no "background" responses.     

Characteristics and trends in the development of an epidemic process of Sonne and Flexner dysentery among preschool children

The results of studies indicate that the morbidity rates of dysentery among children attending preschool institutions and children brought up at home converged in recent years. This phenomenon was most pronounced among children of the kindergarten age group. At the same time dysentery caused by Sh. sonnei and Sh. flexnery produced a higher morbidity rate among children attending nursery in comparison with that among children of the same age group brought up at home. Group infections in preschool institutions were caused by Sh. sonnei in 89.1% of cases and by Sh. flexneri in 10.9% of cases. Outbreaks due to the transfer of infection through everyday contacts were observed only in dysentery caused by Sh. sonnei, constituting 71.4% of the total number of dysentery outbreaks.     

Nature of the epidemic process in Sonne and Flexner dysenteries and the causes of its induction

A comparative study of the epidemic process in Sh. sonnei and Sh. flexneri dysentery in different regions of the USSR revealed that the morbidity level of Sh. sonnei dysentery changed simultaneously in the regions under study at intervals of 2-3 years. Sh. flexneri dysentery showed morbidity rises occurring at intervals 6-8 years, and their occurrence did not coincide with the periods of elevated morbidity in Sh. sonnei dysentery. The data obtained in the cohort analysis and in the study of recurrent morbidity suggest that Sh. flexneri dysentery produces more pronounced postinfection immunity than Sh. sonnei dysentery, and the immunological factor probably affects the dynamics of the epidemic of these Shigella infections.     

Use of data on Shigella contamination and immunity indices for studying the activity of the epidemic process in dysentery

The comparative study of the monthly distribution of characteristics indicating the levels of contamination with Sh. sonnei and Sh. flexneri separately, as well as the seasonal dynamics of the corresponding antibodies, in the years with high and low morbidity levels has been made with the use the indirect hemagglutination test. The possibility of using these characteristics for the evaluation of the activity of the epidemic process in dysentery caused by Sh. sonnei is shown.     

The determination in saliva of IgA antibodies to Shigella ribosomes for the diagnosis of dysentery

The levels of antiribosomal antibodies to Shigella ribosomes in serum and saliva samples from 38 dysentery patients (15 S. sonnei cases and 23 S. flexneri cases), 14 patients with salmonellosis and 136 healthy adults were determined in ELISA with ribosomes from S. sonnei R-mutant used as solid-phase antigen. High levels of "normal" antiribosomal IgA, IgG and IgM antibodies were revealed in the sera of healthy persons while the level of salivary IgA antibodies was very low. In dysentery infection no increase in the levels of serum IgG and IgM antibodies and only a slight increase in the level of IgA antibodies were revealed. Local immune response was manifested by the early (on days 2-4 from the onset of infection) and significant augmentation (12- to 16-fold) of salivary antiribosomal IgA antibodies. An increase in the level of these antibodies was registered in 95-100% of dysentery patients but not in patients with salmonellosis, which made it possible to recommend the method for diagnosing shigellosis. Immune response to Shigella ribosomal antigens, in contrast to the response induced by Shigella O-antigen, is almost exclusively local.     

Recurrent Sonne dysentery morbidity caused by Shigella of varying enzymatic types]

The proportion of repeated cases of dysentery caused by Sh. sonnei does not exceed 3.59% of the total number of bacteriologically confirmed cases of this infection. The frequency of reinfection depended on the enzymatic type of the causative agent. The rarest cases of reinfection were those occurring after the primary infection with Sh. sonnei, biochemical type 2, whereas the most frequent after the primary infection with Sh. sonnei, type 3. Reinfection with heterologous Sh. sonnei was registered mostly 6-12 months after the primary infection, and with homologous Sh. sonnei after a 1 year and later.     

Manifestations of the cyclic nature of the epidemic process in dysentery

Different forms of dysentery, especially those caused by Sh. sonnei and Sh. flexneri, have been found to differ considerably in their cyclic recurrence. The development cycles of the epidemic processes of dysentery have an objective character, occur in the presence of any tendencies in the morbidity rate, and depend on the natural factors. Thus, the cycles of increase and decrease in morbidity are 3, 6, 9, 12 years for dysentery caused by Sh. sonnei and 6, 7, 8 years for dysentery caused by Sh. flexneri.     

Development and use of solid-phase test-systems for the detection of O-antigen of Shigella sonnei and Shigella flexneri

The possibility of the diagnosis of dysentery caused by S. sonnei and S. flexneri, as well as the determination of the dynamics of the distribution of specific O-antigen in the patient's body, by means of the enzyme-linked immunosorbent assay system developed on the basis of antibody preparations obtained by immunosorption has been studied. The study has shown that for better diagnosis the use of fecal extracts is preferable in assays; when used in combination with bacteriological analysis, these assays make it possible to increase the confirmation of the diagnosis of dysentery by several fold.     

Experience with the diagnosis of dysentery in adults by determining antibodies to Shigella in the saliva]

Concentration of immunoglobulins and titres of antibodies to Sh. sonnei, Sh. flexneri and enteropathogenic E. coli 0111 was determined in mixed saliva and the blood serum of patients suffering from Sonne dysentery, acute intestinal diseases of unknown etiology, and healthy individuals. The sum total immunoglobulin concentration in mixed saliva proved to be 53--81 times less than in the blood serum, but in the first substrate there was 53--75, and in the second--15% of immunoglobulin A. There proved to be distinct changes in the specific IgA-antibodies in the saliva of patients with Sonne dysentery. A preponderant accumulation of IgG-antibodies was noted in the blood serum. Elevation of both types of antibodies was maximal during the second week of the disease. Sonne dysentery was diagnosed in 80% of the patients by recording the intensity of shifts in the specific antibodies in the saliva, and in 63%--in the blood. The expediency of immunological testing of saliva for the diagnosis of dysentery is substantiated.     

Modern methods of intraspecies typing of Sonne shigellae. II. Spread of Sonne shigellae of different biochemical types]

The paper presents the results of studying peculiarities of the Sh. sonnei of different biochemical types spread established by their typing scheme suggested by the authors earlier according to rhamnose, xylose and maltose. The epidemic process in dysentery both during the years of the rise and of the decline of its incidence at various territories of the countries proved to be maintained on account of circulation of Sh. sonnei of various biochemical types. The results of studying their dissociative and virulent properties confirmed the biological separation of individual biochemical types. An interrelationship between the character of the biochemical pattern of Shigellae sonnei at the individual territories and the persisting activity of different ways of dysentery transmission was determined. The results of studying the biochemical pattern could be used as an indicator of the degree of activity of individual ways of dysentery spread at various territories.     

Inhibition of beta-lactamase of Bacillus licheniformis 749/C by compound PS-5, a new beta-lactam antibiotic.

By use of a new computer-assisted u.v.-spectrophotometric assay method, the kinetic parameters of the reaction catalysed by Bacillus licheniformis 749/C beta-lactamase were re-examined and the mode of inhibition of the enzyme by compound PS-5, a novel beta-lactam antibiotic, was studied with benzylpenicillin as substrate. (1) The fundamental assay conditions for the determination of Km and V were examined in detail with benzylpenicillin as substrate. In 0.1 M-sodium/potassium phosphate buffer, pH 6.8, at 30 degrees C, initial substrate concentrations of benzylpenicillin above 0.7 mM were very likely to lead to substrate inhibition. The Km value of the enzyme for benzylpenicillin at initial concentrations from 1.96 to 0.07 mM was calculated to be 97-108 microM. (2) The Km values of the enzyme for 6-aminopenicillanic acid, ampicillin and cephaloridine were found to be 25, 154-161 and 144-161 microM respectively. (3) Compound PS-5 was virtually unattacked by Bacillus licheniformis 749/C beta-lactamase. (4) The activity of the enzyme was diminished by compound PS-5, to extents depending on the duration of incubation and the concentration of the inhibitor. The rate of inactivation of the enzyme by compound PS-5 followed first-order kinetics. (5) In an Appendix, a new computer-assisted u.v.-spectrophotometric enzyme assay method, in which a single reaction progress curve of time-absorbance was analysed by the integrated Michaelis-Menten equation, was devised for the accurate and precise determination of the kinetic constants of beta-lactamase. For conversion of absorbance readings into molar substrate concentrations, the initial or final absorbance reading that was independent of the reaction time was used as the basis of calculation. In calculation of Km and V three systematic methods of data combination were employed for finer analysis of the reaction progress curve. A list of the computer program named YF6TAIM is obtainable from the author on request or as Supplementary Publication SUP 50100 (12 pages) from the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., on the terms indicated in Biochem. J. (1978) 169, 5.     

Tendencies in the epidemic process of dysentery in Ryazan

In shigellosis caused by Sh. sonnei and Sh. flexneri the epidemic process was found to have considerable difference in the tendencies and pace of its development. Shigellosis which dominated in the etiological structure at the period following 1966 was dysentery caused by Sh. sonnei; it showed the tendency towards a decrease in morbidity rate. Dysentery caused by Sh. flexneri was second in respect to the frequency of its occurrence after 1966 and showed the tendency towards increase. The simultaneous circulation of Sh. sonnei and Sh. flexneri, the differences in the epidemiology of these types of shigellosis make it imperative that they be studied separately, taking into ccount their etiological selectivity to the main routes of transmitting the infection.     

Data to substantiate the early immunological diagnosis of dysentery]

The interaction of the whole blood from patients with dysentery and gastrointestinal diseases of non-dysenteric etiology, with the causative agents of dysentery, Sh. sonnei and Sh. flexneri, and saprophytic, staphylococci labeled with radioactive isotopes was studied in vitro. In dysentery an increase in the capacity of the blood for Shigella fixation was observed from the beginning of the disease. During the 1st week of the disease this reaction was strictly specific and accompanied by a decrease in the fixation of staphylococci, but later the reaction became relatively specific. An increase in Shigella fixation occurred considerably earlier than immune antibody formation, as revealed by the indirect hemagglutination test. This research substantiates the possibility of an earlier immunological diagnosis of dysentery as compared with the serological methods.     

Serological method of detecting the antigens of the causative agent of Sonne dysentery in lysates of the fecal inoculates from patients]

Serological method of detection of Sh. sonnei antigens in the lysates of the patients, fecal cultures is suggested and approved. In the majority of cases of the results of bacteriological and serological methods of study of the feces coincided. Data confirming the specificity of the antibody neutralization test (ANT) in Sonne dysentery are presented. In connection with detection of the screening action of the Vi-antigen of typhoid bacilli there were elaborated additional methods for verifying the specificity of the ANT results. It is recommended to keep agar plates after selection of suspicious colonies during the bacteriological test; the lysate of the microbial crop should be additionally subjected to the ANT, this considerably increasing the percentage of laboratory confirmations of dysentery caused by Sh. sonnei.     

Assessment of the diagnostic effectiveness of antigenic preparations from Sonne shigellae]

The authors studied the diagnostic possibilities of 4 diagnistic agents from Sh. sonnei. A total of 1500 from 1122 persons were investigated. Specificity and sensitivity of the diagnostic agents from Sh. sonnei were determined. Methodical priciples of the objective assessment of the diagnostic efficacy of the antigenic preparations in the controlled epidemiological trial were elaborated. A possibility of establishment of the etiological diagnosis of Sonne dysentery in the passive hemagglutination or blast agglutination tests by the results of testing with the diagnostic agents of the serum obtained from the patient once or by determining the dynamics of the antibody titre rise in coupled serum portions was not great.     

The fate of the BlaI repressor during the induction of the Bacillus licheniformis BlaP beta-lactamase

The induction of the Staphylococcus aureus BlaZ and Bacillus licheniformis 749/I BlaP beta-lactamases by beta-lactam antibiotics occurs according to similar processes. In both bacteria, the products of the blaI and blaRl genes share a high degree of sequence homology and act as repressors and penicillin-sensory transducers respectively. It has been shown in S. aureus that the BlaI repressor, which controls the expression of BlaZ negatively, is degraded after the addition of the inducer. In the present study,we followed the fate of BlaI during beta-lactamase induction in B. licheniformis 749/I and in a recombinant Bacillus subtilis 168 strain harbouring the pDML995 plasmid, which carries the B. licheniformis blaP, blaI and blaRl genes. In contrast to the situation in B. licheniformis 749/I, beta-lactamase induction in B.subtilis 168/pDML995 was not correlated with the proteolysis of BlaI. To exclude molecular variations undetectable by SDS-PAGE, two-dimensional gel electrophoresis was performed with cellular extracts from uninduced or induced B. subtilis 168/pDML995cells. No variation in the Blal isoelectric point was observed in induced cells, whereas the DNA-binding property was lost. Cross-linking experiments with dithiobis(succimidylpropionate) confirmed that, in uninduced recombinant B. subtilis cells, BlaI was present as a homodimer and that this situation was not altered in induced conditions. This latter result is incompatible with a mechanism of inactivation of BlaI by proteolysis and suggests that the inactivation of BlaI results from a non-covalent modification by a co-activator and that the subsequent proteolysis of BlaI might be a secondary phenomenon. In addition to the presence of this co-activator, our results show that the presence of penicillin stress is also required for full induction of beta-lactamase biosynthesis.     

Use of intraspecific differentiation of Sh. sonnei for the purpose of studying patterns in the epidemic process of dysentery]

The use of complex typing of Sh. sonnei by the colicinogenic and enzymatic properties in epidemiological analysis of dysentery caused by Sh. sonnei, apart from establishment of epidemiological connections in the foci, offered a possibility of introducing significant corrections in the determination of the sizes and duration of existence of the epidemic foci, as well as in detection of the sources and factors of transmission of the infection established in epidemiological examination. Typing of Sh. sonnei also aided in ascertaining that, apart from general regularities in the individual territories, the epidemic process in individual groups of the population and in different seasons of the year could also course autonomously.     

A second regulatory gene, blaR1, encoding a potential penicillin-binding protein required for induction of beta-lactamase in Bacillus licheniformis.

A second regulatory locus (blaR1) required for the induction of beta-lactamase synthesis in Bacillus licheniformis 749 was cloned and sequenced. The gene was located on a 5.2-kilobase-pair SphI DNA fragment which also contained the beta-lactamase (blaP) and repressor (blaI) genes. Bacillus subtilis BD224 carrying these three genes synthesized beta-lactamase on exposure to cephalosporin C, whereas Escherichia coli HB101 carrying the genes did not show any detectable induction of the enzyme. An open reading frame of 1,803 bases was identified as the blaR1 gene by subcloning and DNA sequencing. The gene started 2 bases downstream of the termination codon of bla1 and was preceded by a putative Shine-Dalgarno sequence (AAGGA) with a spacing of 5 bases. The deduced blaR1 product (601 amino acids) had a molecular weight of 68,425. Five transmembrane regions were predicted from the hydrophobicity profile. The region around Phe-Ala-Pro-Ala-Ser-Thr-Tyr-Lys (amino acids 398 to 405), which appeared to be located outside the membrane, was homologous to the binding regions of penicillin-binding proteins, including the beta-lactamases. The segment of 22 amino acids from 400 to 421 showed more than 70% homology to the penicillin-binding region of PBP 2 of E. coli. The blaR1 gene encodes a potential penicillin receptor which is required for the induction of beta-lactamase in B. licheniformis 749.