Study of the methylation process and DNA methylase specificity in Shigella]

The nature and content of minor bases in DNA of 3 Shigella strains are investigated. DNAs from Shigella stutzeri 2, Sh. sonnei 1188 and Sh. sonnei 311 are found to contain 0.43, 0.56 and 0.45 mol.% of N6-methyladenine respectively. 5-methylcytosine (0.16 mol.%) is discovered in Sh. sonnei 311. Substrate specificity of adenine methylase from Sh. sonnei 1188 with respect to phage DNAs of different host modification is investigated. Recognition sites for guanine methylase of DDVI phage and for adenine methylase of Sh. sonnei 1188 turned to be different. DNA of DDII phage grown in Sh. stutzeri 2 cells does not accept methyl groups under the treatment with Sh. sonnei 1188 extracts, but it is methylated by Escherichia coli extract. Adenine methylases of Sh. sonnei 1188 and Sh. stutzeri 2 are suggested to be either the same enzyme, or enzymes, which recognition sites are partially overlapped.     

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.     

Differential characteristics of the I and II phases and R-form of Sh. sonnei]

Characteristics of various phases of Sh. sonnei were determined on the standard strains of Sh. sonnei of phases I and II and R-form used for the industrial production of agglutinating monoreceptor sera. Bacteria of phase I displayed a distinct morphological, cultural and serological differences. For the differentiation of Sh. sonnei of the II phase and R-form, representing the greatest difficulty in this respect, it is recommended to use phages RFfm and 6-SR, and also indirect immunofluorescence method with the application of agglutinating monophasic serum against the II phase of Sh. sonnei. In addition, a study was made of over 20 various Sh. sonnei strains at different dissociative process phases. Verification data completely confirmed the specificity of phages RFfm and 6-SR for Sh. sonnei in the II phase. The efficacy of the immunofluorescence method was confirmed on 6 strains.     

The effect of Shigella sonnei strains opposite in their virulence on different links in the immune system]

For the first time different action of S. sonnei strains, opposite in their virulence, on hematopoiesis and the functional activity of T- and B-lymphocytes has been shown. The hematopoiesis-disturbing action of virulent shigellae is manifested by their capacity, more pronounced than similar capacity of an avirulent (vaccine) strain, for stimulating the processes of endo- and exocolony formation, the proliferation of hematopoietic stem cells and their migration to the blood. The effect produced by shigellae on T-cell-mediated immune response is manifested by the suppression of macrophage migration and its subsequent activation, whose manifestations and duration depend on the virulence of S. sonnei strains under study. The modulating effect of S. sonnei on B-cell-mediated immune reactions is manifested by the inhibiting action of S. sonnei virulent strain and the stimulating action of S. sonnei vaccine strain on the formation of antibody-producing cells synthesizing S. sonnei lipopolysaccharide antibodies shortly after the injection of shigellae. The results of this study indicate that S. sonnei virulent and avirulent (vaccine) produce multifunctional and differing effects on cell-mediated immune reactions, these processes being dependent on the virulence of shigellae and their individual specific antigens.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Antimicrobial susceptibility and pulsed-field gel electrophoresis of Shigella sonnei strains in Malaysia (1997-2000)

AIMS: Antimicrobial resistance of Shigella sonnei from Malaysia was determined and subtyping was carried by pulsed-field gel electrophoresis (PFGE) to assess the extent of genetic diversity of these strains. METHODS AND RESULTS: A total of 62 isolates of S. sonnei from sporadic cases of shigellosis in different parts of Malaysia were studied by antimicrobial susceptibility test and PFGE. Approximately 35.5% of the strains showed resistance to three or more antimicrobial agents. Eight resistant phenotypes, i.e. RI to RVIII, was defined. Resistant phenotype RV and RVIII only appeared in year 2000. PFGE analysis with NotI and XbaI restriction showed that a great heterogeneity existed at the DNA level among Malaysian S. sonnei isolates. Fifty-eight NotI and 61 XbaI-PFGE profiles were observed in 63 S. sonnei isolates, including ATCC 11060 isolate. Drug sensitive isolates displayed very different profiles from drug-resistant isolates, with a few exceptions. Isolates of resistant phenotype RVI (SXTr.TETr.STRr) showed a greater similarity among each other compared with isolates of resistant phenotype RI and drug-sensitive isolates. CONCLUSION: Multi-drug-resistant S. sonnei were circulated in different parts of Malaysia and the emergence of new resistant phenotype was observed. Wide genetic variations among Malaysian S. sonnei were observed and the drug-sensitive strains could be differentiated from drug-resistant strains by PFGE. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study verifies the usefulness of PFGE in characterizing and comparing strains of S. sonnei. Minor variations among S. sonnei isolates could be detected by PFGE.     

Possible mechanisms underlying the slow lactose fermentation phenotype in Shigella spp.

A Southern hybridization analysis revealed that the region homologous to Escherichia coli lacZ was present on the chromosomal DNAs of beta-galactosidase-positive Shigella strains, such as Shigella dysenteriae serovar 1 and Shigella sonnei strains, whereas this region was absent from chromosomal DNAs of beta-galactosidase-negative strains of Shigella flexneri and Shigella boydii. We found that the lacY-A region was deficient in S. dysenteriae serovar 1 and believe that this is the reason for the slow fermentation of lactose by this strain. S. sonnei strains possessed the region which hybridized with E. coli lacY-A despite their slow hydrolysis of lactose. The whole lactose-fermenting region was cloned from S. sonnei and compared with the cloned lac operon of E. coli K-12. Both clones directed the synthesis of beta-galactosidase in an E. coli K-12 strain lacking indigenous beta-galactosidase activity (strain JM109-1), and we observed no difference in the expression of beta-galactosidase activity in S. sonnei and E. coli. However, E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei exhibited the slow lactose fermentation phenotype like the parental strain. S. sonnei strains had no detectable lactose permease activities. E. coli JM109-1 harboring the lactose-fermenting genes of S. sonnei had a detectable permease activity, possibly because of the multicopy nature of the cloned genes, but this permease activity was much lower than that of strain JM109-1 harboring the lac operon of E. coli K-12. From these results we concluded that slow lactose fermentation by S. sonnei is due to weak lactose permease activity.     

Comparative study of the immunogenicity of neurotoxin preparations and their separate fractions obtained from Shigella sonnei, phases I and II

The immunogenicity of the preparations of phase I S. sonnei neurotoxin, determined by the keratoconjunctival test on guinea pigs, is linked mainly with the presence of a high-molecular component other than endotoxin in these preparations. The preparations of phase II S. sonnei neurotoxin do not contain the endotoxin and high-molecular antigens of S. sonnei, phase I, which protect guinea pigs from the development of experimental keratoconjunctivitis; these preparations are toxic for mice and possess low immunogenicity.     

驱除痢疾杆菌侵袭大质粒的新方法

用质粒不相容性原理驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒 ,先从福氏2a侵袭大质粒分别扩增ori和inc基因 ,将它们克隆至 pMD18 T载体 ,得重组质粒pMDori和 pMDinc ,然后转化 2 4 5 7T和S7,不管是pMDori还是 pMDinc都能竞争驱除痢疾杆菌福氏 2a 2 4 5 7T和宋内S7的侵袭大质粒。     

Acanthamoeba: could it be an environmental host of Shigella?

Shigellosis is a serious public health problem in Korea, because large outbreaks of Shigella sonnei infections were recorded in many parts of the country during the period 1998-2000. However, the epidemiological features of shigellosis are not well known. In this study, we devised conditions suitable for the growth and replication of Shigella in an amoebic intracellular environment, and investigate whether medium conditions affect the survival and replication of Shigella within Acanthamoeba. We evaluated the uptake rates of invasive and non invasive S. sonnei strains by three Acanthamoeba species, namely, A. castellanii Neff, A. astronyxis Ray & Hayes, and A. healyi OC-3A. When A. castellanii Neff was infected with S. sonnei 99OBS1 or 80DH248, shigellae was maintained for a longer time in cytoplasms than in other Acanthamoeba species. S. sonnei 99OBS1 strain (a virulent strain) was recovered in higher numbers than the non-virulent S. sonnei 80DH248 strain in all experiments. Moreover, S. sonnei was more easily engulfed by Acanthamoeba at 18 degrees C. The shigellae uptake rates of Neff strain, which was cultured in free-media (less nutrition), were higher (>10-fold) than those observed in original amoeba culture media (PYG medium) in all time points. S. sonnei 99OBS1 was localized, with an intact membrane, to the vacuoles of Acanthamoeba. We conclude that free-living amoebae more likely act as environmental hosts for shigellae, and thus, may have contributed to outbreaks of shigellosis in Korea.     

Effects of caffeic acid, chlorogenic acid and ferulic acid on growth and arylamine N-acetyltransferase activity in Shigella sonnei (group D)

Arylamine N-acetyltransferase (NAT) activities with 2-aminofluorene (2-AF) as substrates were determined in Shigella sonnei (group D) collected from patients with diarrhoeal disease. The NAT activity was determined using an acetyl CoA recycling assay and high pressure liquid chromatography. Inhibition of growth studies from S. sonnei (group D) demonstrated that caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) elicited a dose-dependent bactericidal effect in S. sonnei (group D) cultures, i.e. the greater the concentration of CA, CGA and FA, the greater the inhibition of growth of S. sonnei (group D). Cytosols or suspensions of S. sonnei (group D) with and without selected concentrations of CA, CGA and FA co-treatment showed different percentages of 2-AF acetylation. The data indicated that there was reduced NAT activity associated with increased CA, CGA and FA in Shigella dysenteriae (group D) cytosols and intact cells. For the cytosol and intact bacteria examinations, the apparent values of K(m) and Vmax decreased after being co-treated with 400 microM CA, CGA and FA. This report is the first demonstration of plant phenolic inhibition (CA, CGA and FA) of arylamine NAT activity and growth in the bacterium S. sonnei (group D).     

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.     

Production of auxotrophic mutants of Shigella sonnei]

Use of selection method with R. Ffm phage offered a possibility of obtaining R-forms of Sh. sonnei from the population of bacteria of phase II with a low frequency of natural dissociation. Mono- and polyauxotrophic mutant of I, II and R-phases by hir, ilv, pro, and mtl markers were obtained by treatment of Sh. sonnei strains with nitrosoguanidine and ethyl-methanesulfonate. The frequency of mutation in two Sh. sonnei strains studied in this work (UBC and No. 941) by the same genetic regions proved to differ. The change of Sh. sonnei phase I into phase II was observed only in selection by proline marker which could be connected with the existence in this region of a gene controlling the dissociation process.     

A novel marker for the species-specific detection and quantitation of Shigella sonnei by targeting a methylase gene

Shigella sonnei is a causal agent of fever, nausea, stomach cramps, vomiting, and diarrheal disease. The present study describes a quantitative polymerase chain reaction (qPCR) assay for the specific detection of S. sonnei using a primer pair based on the methylase gene for the amplification of a 325 bp DNA fragment. The qPCR primer set for the accurate diagnosis of Shigella sonnei was developed from publically available genome sequences. This quantitative PCR-based method will potentially simplify and facilitate the diagnosis of this pathogen and guide disease management.     

The plasmid heterogeneity of Shigella sonnei phase-I and -II strains

The plasmid composition of S. sonnei standard strains has been studied by the method of electron microscopy of the preparations of plasmid DNA. In S. sonnei cells I-941-HP, phase I, plasmids of 2,500; 5,000; 5,600; 6,100 and 6,800 base pairs, as well as plasmids of 85,000-117,000 and 170,000-235,000 base pairs have been detected. In S. sonnei cells, phase II, plasmids of 2,500; 4,900 and 6,100 base pairs, as well as plasmids of 85,000-109,000 base pairs, have been found. Thus, virulent S. sonnei in phase I contain additional plasmids of 5,600; 6,800; 110,000-117,000 and 170,000-237,000 base pairs. The range of plasmid lengths between 85,000-117,000 and 170,000-237,000 base pairs exceeds the usual background of electron-microscopic studies, which makes it possible to come to the conclusion on the intrastrain heterogeneity of these classes of plasmids. The suggestion has been made that the transition of S. sonnei from phase I to phase II is linked with the loss of fragments of the genetic material, limited by inverted DNA repetitions.     

A multi-prefectural outbreak of Shigella sonnei infections associated with eating oysters in Japan

Among roughly one thousand incidents of shigellosis annually in Japan, approximately 70% of the cases are estimated to be associated with overseas travel. However, at the end of 2001, reports of domestically acquired Shigella sonnei infections suddenly increased. We report here the first multi-prefectural outbreak of Shigella sonnei infections linked to the consumption of imported oysters in Japan at the end of 2001. Isolates of S. sonnei from patients epidemiologically linked to eating contaminated oysters and from the imported oysters themselves showed an indistinguishable pulsed-field gel electrophoresis pattern and drug resistance pattern.     

Mobilization of phase I plasmid in Shigella sonnei and stability of its inheritance in rough strains of Shigella and Escherichia

The plasmid pSS120, determining the synthesis of species specific I phase antigen of Shigella sonnei is mobilized for genetic transfer into E. coli K12 recipient cells with the frequency 12-41%. The frequency depends on the type of mobilized plasmid and recipient strain. The I phase antigen is normally expressed in II phase recipient cells and in E. coli cells. During mobilization pSS120 forms cointegrates representing a recombinant of mobilizing and mobilized plasmids DNA. The study of pSS120 inheritance stability has shown the plasmid to be unstable during culturing of bacteria and to be partially lost from the parent Shigella sonnei strains as well as from the "hybrid" transconjugants obtained. The 60 Md plasmid present in the donor strains of Shigella sonnei is prone to structural fragmentation particularly expressed in Shigella sonnei/E. coli hybrids.     

Latex test used for diagnosis of dysentery caused by Shigella sonnei]

The aim of this study was to evaluate the usefulness of latex reagent coated with immunoglobulins specific for antigens of phase I and II of S. sonnei for detection of these antigens in primary, mixed bacterial cultures. The study was performed on 919 fecal samples from individuals with clinical symptoms of dysentery, convalescents and from contact individuals. Material used for the test was bacterial suspension collected from McConkey or SS agars and a culture from selenite F broth heated at 100C. The results of the latex test were compared with the results of isolation of S. sonnei from the same cultures. S. sonnei was isolated from feces of 140 individuals (15.2%), while the latex test was positive in 215 cases (23.4%). The highest testing effectiveness , significantly higher than when isolation of pathogen was performed, was obtained only when 18-20 hr culture on Selenite F medium was used for latex test. The correlation between efficacy of testing for S. sonnei and phosphate content of Selenite F and a mode of its preparation was found. The latex test allows to eliminate from further bacteriological studies cultures free of S. sonnei thus it gives measurable economical profits and it shortens significantly time period of bacteriological examination.     

宋内Ⅰ相抗原和霍乱CT-B共表达的免疫保护效果观察

将编码宋内氏痢疾菌(Shigella sonnei)I相O抗原的基因和霍乱弧菌(Vibrio choler-ae)的CT-B基因克隆至带asd基因的质粒PYA248,得重组质粒PMGL105。将该重组质粒转入asd基因缺失的减毒伤寒沙门氏菌X4072,构成了一个不带抗药性基因的载体-宿主平衡致死系统。一系列实验表明,该重组菌X4072(PMGL105)能稳定地表达宋内I相O抗原和霍乱弧菌的CT-B抗原。小鼠免疫保护实验表明,该重组菌对有毒的宋内氏I相痢疾杆菌及霍乱弧菌的攻击均具有良好保护作用。     

Detecting low concentrations of Shigella sonnei in environmental water samples by PCR

Outbreaks of Shigella sonnei associated with contaminated water have been reported and methods for the simultaneous detection of Shigellae and enteroinvasive Escherichia coli in water samples have been developed with detection limits of 10(1)-10(2) CFU mL(-1) of water. Because 10(1)-10(2)Shigellae can cause disease, a more sensitive detection method as an addition to the existing methods for detection of Shigella sonnei in water samples is reported here. Initially, 33 Shigella sonnei and 72 non-Shigella sonnei isolates were tested and one primer pair was found capable of specifically amplifying a 369-bp insertion sequence 1 (IS1) fragment from all 33 Shigella sonnei isolates and one Shigella dysenteriae ATCC isolate by PCR. The detection method was developed, which included filtration of 50 mL of water through a membrane and application of PCR to the membrane using this primer pair. Environmental water samples with total bacterial numbers of 384-2.84 x 10(7) CFU L(-1) were collected and seeded with 13 Shigella sonnei and the Shigella dysenteriae ATCC isolates. Detection limits were determined as 1.7-24.7 and 270-8000 CFU per 50 mL of water, respectively, using this detection method.