Differential epithelial cell response upon stimulation with the Aggregatibacter actinomycetemcomitans strains VT 1169, VT 1560 DAM- and ATCC 4318

Aggregatibacter actinomycetemcomitans is known to play a role in human inflammatory oral diseases. The present study aimed to test whether GECs from different individuals show an altered immune response when exposed to wild-type A. actinomycetemcomitans biofilms (VT 1169 and ATCC43718) versus VT 1560 lacking a DNA-adenine-methyltransferase (DAM-). GECs were cultured from biopsies derived from three healthy subjects (A, B, and C). To obtain A. actinomycetemcomitans biofilms, each strain (VT 1169, ATCC 43718, and VT 1560 DAM-) was separately cultured on polymer disks. The mRNA expression of hBD-2, Rnase7, IL-8, and glycerylaldehyd-3-phosphodehydrogenase was analyzed using semi-quantitative RT-PCR. In GECs, the DAM- strain VT 1560 led to a reduced gene expression of hBD-2 and IL-8 compared to VT 1169, but not ATCC 43718. In GECs from subject C, the mRNA expression of hBD-2 and IL-8 was significantly up regulated in response to A. actinomycetemcomitans VT 1169 compared to VT 1560 DAM- and ATCC 43718 (p≤0,05), whereas GECs from subject A and B did not show significant changes for hBD-2 or IL-8 gene expression upon stimulation with either of the strains tested. The present study indicates strain-dependent and subject-specific immune responses in GECs after exposure to DAM+ or DAM- A. actinomycetemcomitans.     

Isolation and characterization of hemolytic genes from Actinobacillus actinomycetemcomitans

Abstract Periodontopathic Actinobacillus actinomycetemcomitans produces hemolysin and other leukotoxins. In the present study, two distinct clones which lysed horse erythrocytes were isolated by screening genomic DNA libraries of A. actinomycetemcomitans ATCC 43718 on blood agar plates. DNA hybridization analysis indicates that there were two distinct hemolytic genes present. Sonicated extracts from both Escherichia coli clones possessed hemolytic activities on horse, sheep and human erythrocytes, but not those of rabbit. Rabbit antiserum to A. actinomycetemcomitans ATCC 43718 whole cells inhibited the hemolytic activities of these clones.     

Heterogeneity in the immune response to serotype b LPS of Actinobacillus actinomycetemcomitans in inbred strains of mice

We investigated the heterogeneity of the humoral immune responses to whole cells and lipopolysaccharide (LPS) of Actinobacillus actinomycetemcomitans serotype b and production of cytokines in inbred strains of mice. Nine such strains were tested: A/J (H-2(a)), C57BL/6 (H-2(b)), BALB/c (H-2(d)), DBA/2 (H-2(d)), B10.BR (H-2(k)), C3H/He (H-2(k)), C3H/HeJ (H-2(k)), DBA/1 (H-2(q)) and B10.S (H-2(s)). Mice were immunized intraperitoneally with whole cells of A. actinomycetemcomitans ATCC 43718 (serotype b) in phosphate buffered saline (PBS; pH 7.2) emulsified with an equal volume of Freund's incomplete adjuvant. Serum immunoglobulin G (IgG), immunoglobulin A (IgA) and immunoglobulin M (IgM) levels against A. actinomycetemcomitans were measured by an ELISA system. ELISA analysis, using LPS fractions from serotype a, b or c strains of A. actinomycetemcomitans as the coating antigens, revealed that mice strains C3H/He, C3H/HeJ, B10.BR and B10.S had an extremely high-IgM response against serotype b LPS. High-IgM titer sera contain also elevated levels of IgA antibodies to the antigen. To compare the cytokine production among inbred mice, the amounts of interleukin-4 (IL-4), interleukin-5 (IL-5), and interleukin-6 (IL-6) released from mouse splenocytes were measured using ELISA systems specific for these cytokines. A. actinomycetemcomitans serotype b LPS stimulation induced IL-6 release from murine splenocytes of all tested strains. However, IL-4 and IL-5 were detected only in high-IgM/IgA responders to A. actinomycetemcomitans serotype b LPS, not in low-IgM/IgA responders. Thus, we found a relationship between the humoral immune response to LPS of A. actinomycetemcomitans serotype b and production of type 2 cytokines by splenocytes.     

Replacement of lysine 269 by arginine in Escherichia coli tryptophan indole-lyase affects the formation and breakdown of quinonoid complexes

Lysine 269 in Escherichia coli tryptophan indole-lyase (tryptophanase) has been changed to arginine by site-directed mutagenesis. The resultant K269R mutant enzyme exhibits kcat values about 10% those of the wild-type enzyme with S-(o-nitrophenyl)-L-cysteine, L-tryptophan, and S-benzyl-L-cysteine, while kcat/Km values are reduced to 2% or less. The pH profile of kcat/Km for S-benzyl-L-cysteine for the mutant enzyme exhibits two pK alpha values which are too close to separate, with an average value of 7.6, while the wild-type enzyme exhibits pK alpha values of 6.0 and 7.8. The pK alpha for the interconversion of the 335 and 412 nm forms of the K269R enzyme is 8.3, while the wild-type enzyme exhibits a pK alpha of 7.4. Steady-state kinetic isotope effects on the reaction of [alpha-2H]S-benzyl-L-cysteine with the K269R mutant enzyme (Dkcat = 2.0; D(kcat/Km) = 3.9) are larger than those of the wild-type enzyme (Dkcat = 1.4; D(kcat/Km) = 2.9). Rapid scanning stopped-flow kinetic studies demonstrate that the K269R mutant enzyme does not accumulate quinonoid intermediates with L-alanine, L-tryptophan, or S-methyl-L-cysteine, but does form quinonoid absorption peaks in complexes with S-benzyl-L-cysteine and oxidolyl-L-alanine, whereas wild-type enzyme forms prominent quinonoid bands with all these amino acids. Single wavelength stopped-flow kinetic studies demonstrate that the alpha-deprotonation of S-benzyl-L-cysteine is 6-fold slower in the K269R mutant enzyme, while the intrinsic deuterium kinetic isotope effect is less for the K269R enzyme (Dk = 4.2) than for the wild-type (Dk = 7.9). The decay of the K269R quinonoid intermediate in the presence of benzimidazole is 7.1-fold slower than that of the wild-type enzyme. These results demonstrate that Lys-269 plays a significant role in the conformational changes or electrostatic effects obligatory to the formation and decomposition of the quinonoid intermediate, although it is not an essential basic residue.     

Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from periodontal disease patients

Leukotoxic activity in Actinobacillus (Haemophilus) actinomycetemcomitans isolated from patients with rapidly progressive periodontitis (RP), gingivitis (G), and juvenile periodontitis (JP), and several oral bacteria, was determined by observation of morphological changes in polymorphonuclear leukocytes (PMNs). Many A. actinomycetemcomitans isolates yielded both rough-surfaced and umbonate-shaped colonies (A-type), and smooth-surfaced and convex-shaped colonies (B-type), when stock cultures were streaked on agar medium. Both types of cells were identical in terms of Gram stain, cell morphology, sugar fermentation profile, nitrate reduction and cellular fatty acid composition. Sonic extracts were prepared from 32 A. actinomycetemcomitans strains isolated from patients and from 3 American Type Culture Collection (ATCC) strains. Sonic extracts from 8 isolates and 2 ATCC strains induced sphering of PMNs during a 45-50 min period of incubation at 37 C. Extracts from the other oral bacteria had no effects on PMN morphology. The sphered PMNs were found by their fluorochromatic-negative reactions to be damaged cells. The leukotoxic substance was heat-sensitive (56 C, 30 min), trypsin-sensitive and did not induce sphering of PMNs at 4 C. There was no clear correlation between colony type and leukotoxicity. Among 8 leukotoxic strains, 5 were isolates from an RP patient.     

Chemical composition and immunobiological activities of sodium dodecyl sulphate extracts from the cell envelopes of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis and Fusobacterium nucleatum

The chemical composition and immunobiological activities in vivo and in vitro of sodium dodecyl sulphate extracts (SDS-SE) derived from periodontopathic bacteria (three strains of Actinobacillus actinomycetemcomitans, two strains of Bacteroides gingivalis, and one strain of Fusobacterium nucleatum) were investigated. The main components of SDS-SE were protein and lipid, with negligible amounts of peptidoglycan and lipopolysaccharide. Immunopotentiating activity was detected in both delayed-type hypersensitivity and antibody formation against the elicitation of a protein antigen with the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and B. gingivalis 381 and 1021. On the other hand the SDS-SE of A. actinomycetemcomitans ATCC 29522 enhanced only the induction of a delayed-type hypersensitivity response. All the SDS-SE preparations had mitogenic activity to splenocytes from BALB/c nu/nu, C3H/HeN and C3H/HeJ mice. Migration-stimulating activity for human peripheral blood monocytes was detected especially in the SDS-SE preparations of A. actinomycetemcomitans ATCC 29524 and Y4. All of the SDS-SE samples inhibited [3H]thymidine uptake in human gingival fibroblasts and caused degradation of the cells. The results suggest that the cell membrane components extractable with sodium dodecyl sulphate from periodontopathic bacteria are involved in the pathogenesis of periodontal disease.     

Endothelial cells in peripheral blood of healthy subjects and patients with metastatic carcinomas.

BACKGROUND: A lack of standardized assays and consensus of cell definition has lead to a wide variation in the reported range of circulating endothelial cells (CECs). METHODS: An automated rare cell analysis system was used to enumerate nucleated, CD146+/CD105+/CD45- CECs in 4 mL of blood. RESULTS: Recoveries of spiked HUVECs were linear over a range of 0-1,241 cells (R2>or=0.99) with recoveries of >or=70% at each spike level. Correlation coefficient values for interoperator variability and duplicate sample variation were (R2=0.99 and 0.90), respectively. Correlation of CEC counts between tubes 1-2 and 2-3 drawn from the same subject in sequence differed (R2=0.48 and 0.63, respectively). The normal CEC reference range established in 249 healthy donors was 1-20 CECs/mL blood. CEC counts were significantly higher in the 206 metastatic carcinoma patients (P<0.0001). CONCLUSION: CECs can be accurately and reproducibly enumerated in blood and are elevated in metastatic carcinomas compared with healthy donors. Phlebotomy procedures can affect endothelial cell counts.     

Renibacterium salmoninarum: effect of hypochlorite treatment, and survival in water

The effect of different concentrations of sodium hypochlorite on Renibacterium salmoninarum and the survival of the bacterium in autoclaved river water and groundwater were examined. The disinfection trial was performed using R. salmoninarum ATCC 33209. The concentrations of free chlorine were 10, 50, 100 and 200 mg 1(-1), the contact times were 5, 15, and 30 min and 24 h, and the test suspensions were subcultured both on Kidney disease medium (KDM2) agar and in 3 parallel KDM2 broths, which were then subcultured on KDM2 and selective KDM (SKDM) agar. The survival of the bacterium in river water and groundwater was studied using 4 isolates of R. salmoninarum including ATCC 33209. Treatment with sodium hypochlorite effectively reduced the number of culturable cells of R. salmoninarum, but use of the recovery broth showed that small numbers of cells remained viable at all concentrations of free chlorine. The numbers of R. salmoninarum decreased to an undetectable level after 4 wk incubation in the survival trials, but low numbers of colonies were again found in the subculture after 5 wk incubation. Viable cells of R. salmoninarum were still detected in subcultures of all strains after 20 wk of incubation in river water.     

Biosynthesis of enantiopure (S)-3-hydroxybutyric acid in metabolically engineered Escherichia coli

A biosynthetic pathway for the production of (S)-3-hydroxybutyric acid (S3HB) from glucose was established in recombinant Escherichia coli by introducing the beta-ketothiolase gene from Ralstonia eutropha H16, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene from R. eutropha H16, or Clostridium acetobutylicum ATCC824, and the 3-hydroxyisobutyryl-CoA hydrolase gene from Bacillus cereus ATCC14579. Artificial operon consisting of these genes was constructed and was expressed in E. coli BL21 (DE3) codon plus under T7 promoter by isopropyl beta-D: -thiogalactoside (IPTG) induction. Recombinant E. coli BL21 (DE3) codon plus expressing the beta-ketothiolase gene, the (S)-3-hydroxybutyryl-CoA dehydrogenase gene, and the 3-hydroxyisobutyryl-CoA hydrolase gene could synthesize enantiomerically pure S3HB to the concentration of 0.61 g l(-1) from 20 g l(-1) of glucose in Luria-Bertani medium. Fed-batch cultures of recombinant E. coli BL21 (DE3) codon plus were carried out to achieve higher titer of S3HB with varying induction time and glucose concentration during fermentation. Protein expression was induced by addition of 1 mM IPTG when cell concentration reached 10 and 20 g l(-1) (OD(600) = 30 and 60), respectively. When protein expression was induced at 60 of OD(600) and glucose was fed to the concentration of 15 g l(-1), 10.3 g l(-1) of S3HB was obtained in 38 h with the S3HB productivity of 0.21 g l(-1)h(-1). Lowering glucose concentration to 5 g l(-1) and induction of protein expression at 30 of OD(600) significantly reduced final S3HB concentration to 3.7 g l(-1), which also resulted in the decrease of the S3HB productivity to 0.05 g l(-1)h(-1).     

Differences in nitric oxide steady states between arginine, hypoxanthine, uracil auxotrophs (AHU) and non-AHU strains of Neisseria gonorrhoeae during anaerobic respiration in the presence of nitrite

Neisseria gonorrhoeae can grow by anaerobic respiration using nitrite as an alternative electron acceptor. Under these growth conditions, N. gonorrhoeae produces and degrades nitric oxide (NO), an important host defense molecule. Laboratory strain F62 has been shown to establish and maintain a NO steady-state level that is a function of the nitrite reductase/NO reductase ratio and is independent of cell number. The nitrite reductase activities (122-197 nmol NO2 reduced.min-1.OD600-1) and NO reductase activities (88-155 nmol NO reduced.min-1.OD600-1) in a variety of gonococcal clinical isolates were similar to the specific activities seen in F62 (241 nmol NO2 reduced.min-1.OD600-1 and 88 nmol NO reduced.min-1.OD600-1, respectively). In seven gonococcal strains, the NO steady-state levels established in the presence of nitrite were similar to that of F62 (801-2121 nmol.L-1 NO), while six of the strains, identified as arginine, hypoxanthine, and uracil auxotrophs (AHU), that cause asymptomatic infection in men had either two- to threefold (373-579 nmol.L-1 NO) or about 100-fold (13-24 nmol.L-1 NO) lower NO steady-state concentrations. All tested strains in the presence of a NO donor, 2,2'-(hydroxynitrosohydrazono)bis-ethanimine/NO, quickly lowered and maintained NO levels in the noninflammatory range of NO (<300 nmol.L-1). The generation of a NO steady-state concentration was directly affected by alterations in respiratory control in both F62 and an AHU strain, although differences in membrane function are suspected to be responsible for NO steady-state level differences in AHU strains.     

3种农杆菌对茶树发状根诱导的影响

以茶树'福云6号'和'铁观音'成熟种子下胚轴、未成熟种子下胚轴和愈伤组织为材料,以发状根诱导率为指标,探究菌液浓度、农杆菌菌株、外植体类型和预培养时间对发状根诱导的影响.结果表明:(1)菌液浓度OD600在0.4～1.2范围内,'福云6号'成熟种子下胚轴发状根诱导率先升高后降低,ATCC15834在OD 600为0.6...     

Mechanism of additive genetic transformation in Haemophilus influenzae.

Transforming deoxyribonucleic acid (DNA) preparations from Haemophilus influenzae Rd strains carrying a chromosomally integrated, conjugative, antibiotic resistance transfer (R) plasmid were exposed to ultraviolet radiation and then assayed for antibiotic resistance transfer on sensitive wild-type Rd competent suspensions and on similar suspensions of a uvr-1 mutant unable to excise pyrimidine dimers. No host cell reactivation of resistance transfer (DNA repair) was observed. Parallel experiments with ethanol-precipitated, heated, free R plasmid DNA preparations gave much higher survival when assayed on the wild-type strain compared to the survival on the uvr-1 strain. These observations indicate that additive genetic transformation (in this case, the addition of the integrated R plasmid to the recipient genome) involves single-strand insertion.     

Inducible bacteriophages of Actinobacillus actinomycetemcomitans

Doses of 0.1 to 1.0 g/ml of mitomycin C induced cell lysis of six of eight strains of Actinobacillus actinomycetemcomitans tested. Infectious phages were induced from ATCC strains 43717, 29524, 33384, and 43719; non-plaque-forming, possibly defective phages were induced from ATCC strains 29522 and 29523. No phages were detected in strain FDC 651 or ATCC strain 43718. No correlation between lysogeny and leukotoxin production or serotype of the strains could be established. Gel electrophoresis of phage DNAs indicated that the induced phages were of three types, based on size. By electron microscopy, the phages were found to belong to either morphotype A1 or morphotype B1; no other morphotypes were observed. Curing experiments led to the isolation of nonlysogenic derivatives of two strains, which supported plaque formation by the phages they originally carried. On the basis of our results, lysogeny appears to be widespread in A. actinomycetemcomitans.     

Limitations of the enzyme-linked immunosorbent assay for routine determination of legume inoculant quality

Peat inoculants containing strains of either Rhizobium or Bradyrhizobium spp. were used to determine correlations between cell numbers and A₄₀₅ values obtained with double antibody sandwich enzyme-linked immunosorbent assay (DAS ELISA) and indirect ELISA. ELISA values of inoculants containing strains of Rhizobium were weak and non-specific; with Bradyrhizobium spp. strains, readings were higher and cross-reactions negligible when heated inoculant suspensions were allowed to stand for 3 h before ELISA determinations were made. With soybean inoculant, correlation coefficients of r = 0.93 and 0.83 were obtained with DAS and indirect ELISA, respectively. A linear curve relating log cell numbers to A₄₀₅ values was used to determine the reliability of DAS ELISA values obtained over 2 years with tests on commercially produced soybean inoculants. In the range 5 times 10⁸-ca 3 times 10⁹ cells/g inoculant, DAS ELISA estimates closely followed plate counts but no significant correlation was found when inoculants contained >ca 3 times 10⁹ cells/g. With a minimum requirement of 1 times 10⁹ cells/g inoculant, discrepancies between DAS ELISA estimates and plate counts obtained with inoculants produced with gamma-irradiated peat would have resulted in the erroneous rejection or acceptance of 14.5% of all inoculants tested, based on DAS ELISA estimates. With inoculants produced with steam-sterilized peat, which was unfavourable for survival of strain WB1, 70.0% of the inoculants rejected because of low plate counts would have been acceptable on the basis of DAS ELISA estimates.     

Leukotoxic Activity of Actinobacillus actinomycetemcomitans Isolated from Brazilian Periodontal Patients

The leukotoxic activity of 31 Actinobacillus actinomycetemcomitans isolates from Brazilian periodontal patients [nine from Localized Juvenile Periodontitis (LJP) patients, 22 from patients with AIDS-associated Necrotizing Ulcerative Periodontitis (AIDS/NUP)], and from the reference strain A. actinomycetemcomitans ATCC43718, were analysed for their cytotoxicity on human monocytes. A cytotoxicity inhibitory assay of the isolate P35 and the reference strain ATCC 43718 with sera from ten LJP patients and ten healthy subjects was also performed and leukotoxin reactivity was evaluated with serum from rabbits immune to leukotoxin from A. actinomycetemcomitans ATCC 43718. The cytotoxicity results were not statistically different among groups of A. actinomycetemcomitans isolates from LJP and AIDS/NUP patients, but the individual analysis of each isolate showed two isolates (P24 and P35) from LJP patients with high leukotoxic activity (P<0.05). Also, a high leucotoxic inhibitory effect with LJP patients' sera compared with healthy subjects with sonic extract from isolate P35 (P<0.05) and the reactivity of rabbit antiserum to leukotoxin were observed. Both leukotoxic and non-leukotoxic strains of A. actinomycetemcomitans can be isolated from LJP and AIDS/NUP patients, but A. actinomycetemcomitans with high leukotoxic activity is more frequent in PJL than AIDS/NUP patients. Even though A. actinomycetemcomitans exhibits leukotoxic activity, there is an immune response to the leucotoxin in LJP patients.     

Identification of Actinobacillus actinomycetemcomitans using species-specific 16S rDNA primers

The purpose of this study was to develop species-specific PCR primers for use in the identification and detection of Actinobacillus actinomycetemcomitans. These primers target variable regions of the 16S ribosomal RNA coding gene (rDNA). We assessed the specificity of the primers against 9 A. actinomycetemcomitans strains and 11 strains (3 species) of the Haemophilus genus. Primer sensitivity was determined by testing serial dilutions of the purified genomic DNAs of A. actinomycetemcomitans ATCC 33384T. Our obtained data revealed that we had obtained species-specific amplicons for all of the tested A. actinomycetemcomitans strains, and that none of these amplicons occurred in any of the other species. Our PCR protocol proved able to detect as little as 4 fg of A. actinomycetemcomitans chromosomal DNA. Our findings suggest that these PCR primers are incredibly sensitive, and should prove suitable for application in epidemiological studies, as well as the diagnosis and monitoring of periodontal pathogens after treatment for periodontitis.     

重组巴氏毕赤酵母恒化培养动力学及代谢迁移特性研究

通过对甲醇营养型毕赤酵母基因工程菌以碳源甘油为限制性基质进行恒化培养动力学试验 ,结果认为 :(1 )细胞光密度与其干、湿重呈线性关系 ,当细胞光密度 (OD60 0 )为 1 0 0时细胞湿重 (WCW)为 1 2 8 3g L ,细胞干重 (WDW)则为 2 2 9g L ;(2 )基因工程菌P .pastoris的生长与限制性基质甘油残留浓度的关系符合Monod关系式 ,通过 1 μ对 1 S进行线性回归得 μmax=0 .366h- 1,Ks=0 .1 82 3g L ,经参数推导甘油最大菌体得率系数YG =0 .54g g ,菌体维持生长消耗底物系数m =0 .0 0 69g (g·h) ;氧最大菌体系数YX O2 =30 .96g moL ,菌体维持生长时消耗氧系数mO2 =0 .0 0 0 8mol (g·h) ,最适理论稀释速率Dm =0 .341h- 1;(3)从氨水的消耗速率和呼吸商 (RQ)的变化认为随着比生长速率 (μ)的增大 ,甘油代谢流从糖原异生和磷酸戊糖途径线性地向糖酵解和三羧酸循环途径进行代谢迁移 ,即糖酵解和三羧酸循环途径的代谢流量在线性地增大     

Antibacterial effect of protamine assayed by impedimetry

Impedimetric measurements were used to assay the antibacterial effect of protamine. A good linear correlation between the impedance detection time and the initial cell counts was obtained ( r = 0.99, n = 2). As basic peptides may cause clumping of cells, this correlation curve was used when estimating the cell number after protamine treatment, rather than colony counts.
Protamine from salmon killed growing Gram-positive bacteria and significantly inhibited growth of Gram-negative bacteria in Tryptone Soy Broth (TSB) at 25°C. In general Gram-positive bacteria were more sensitive to protamine than Gram-negative bacteria; the minimum inhibitory concentrations (MIC) determined for Gram-positive strains varied from 20 to 1000 μ ml^-1 and for Gram-negative strains from 500 μ ml^-1 to more than 4000 μ ml^-1.
The effect of protamine on non-growing Listeria monocytogenes Scott A suspended in buffer was not lethal as was the effect on growing cells; however, protamine (50–500 μg ml^-1) killed the Gram-negative fish spoilage bacteria Shewanella putrefaciens when the live cells were suspended in buffer.     

Drug Resistance of Enteric Bacteria

Drug resistance of 3,000 Shigella strains isolated in 1965 were investigated. These strains originated from 10 City Hospitals and 4 Prefectural Health Centers, which are located in different parts of Japan. One hundred and seventy strains which were resistant to 4 drugs, chloramphenicol (CM), tetracycline (TC), dihydrostreptomycin (SM), and sulfanilamide (SA), were selected at random from these stock cultures in this laboratory and the distribution of R factors in these isolates was examined. It was found that the strains all harbored R factors which were capable of transferring drug resistance by usual conjugal process. Among the strains carrying R factors, 85 per cent harbored a single type of R factor and 15 per cent carried two types of R factor in a cell. The latter is called the hetero-R state. Among the strains in the hetero-R state, isolation of strains harboring both R (SM.SA) and R (TC.CM.SM.SA) factors was most frequent. It was found that 25 R (SM.SA) factors isolated from strains in hetero-R had the genetic determinant iR^?, while most of the R (TC.CM.SM.SA) factors isolated from natural sources were iR⁺. When two types of R factor, R (SM.SA) and R (TC.CM.SM.SA) derived from the same host cells, were brought together in a host cell by superinfection with both factors, they were found to exist stably in a host bacterium. These results confirmed the stable existence of both factors in Shigella strains isolated from dysenteric patients.