Application of fluorescence internal transcribed spacer-PCR (f-ITS) for the cluster analysis of bacteria isolated from air and deteriorated fresco surfaces

The aim of this work was the evaluation of fluorescence ITS-PCR (f-ITS) as a molecular tool to analyze the microbial community involved in the biodeterioration of cultural heritage surfaces. As a case study we analyzed by f-ITS ninety-two bacterial strains isolated from a medieval fresco and the surrounding air environment. The internal transcribed spacer between the 16S and 23S rRNA genes was amplified, and then the fluorescently labeled PCR products were separated by capillary electrophoresis. Bacterial strains were identified by 16S rDNA sequencing. The f-ITS electropherograms showed different profiles coherent with the affiliation of the strains at the genus and species levels. Among the isolates obtained from the fresco surface, those belonging to the genus Bacillus were the most prevailing exhibiting 8 different f-ITS profiles. The airborne bacilli exhibited only 2 of these 8 profiles. Staphylococcus were mostly isolated from air and produced 4 different profiles. Pseudomonas isolates presented 3 different profiles, and one of them was typical of Pseudomonas putida. Members of the other genera produced their distinctive profiles. Our results show that f-ITS is a promising molecular tool for the rapid selection and clustering of strains isolated from different sources.     

怀地黄活性内生菌的分离鉴定及抗菌抗肿瘤活性

利用平板分离法从怀地黄(Rehmannia glutinosa Libosch)中分离出内生菌共130株,包括67株细菌、50株真菌和13株放线菌。利用大肠杆菌、金黄色葡萄球菌和黑曲霉作为供试菌株,通过对峙试验和平板点接法筛选出8株对3种供试菌株均具有较强拮抗作用的内生菌。经菌种形态观察、生理生化实验以及16S rRNA序列测定,这8株活性内生菌均为假单胞菌属的细菌,并分别与Pseudomonas fluorescens、Pseudomonas thivervalensis、Pseudomonas chlororaphis、Pseudomonas koreensis和一个未定种具有最高相似性。对这8株活性内生菌进行液体发酵、不同有机溶剂抽提和抗菌抗肿瘤研究,结果表明它们的乙醇抽提物和乙酸乙酯抽提物均不同程度地对3种供试菌株和食管癌细胞系Ec9706具有抑制作用。其中2-2号菌株(Pseudomonas chlororaphis)抗菌抗肿瘤作用最强,具有重要的开发价值。     

Characterization of CMR5c and CMR12a, novel fluorescent Pseudomonas strains from the cocoyam rhizosphere with biocontrol activity

AIM: To screen for novel antagonistic Pseudomonas strains producing both phenazines and biosurfactants that are as effective as Pseudomonas aeruginosa PNA1 in the biocontrol of cocoyam root rot caused by Pythium myriotylum. MATERIAL AND RESULTS: Forty pseudomonads were isolated from the rhizosphere of healthy white and red cocoyam plants appearing in natural, heavily infested fields in Cameroon. In vitro tests demonstrated that Py. myriotylum antagonists could be retrieved from the red cocoyam rhizosphere. Except for one isolate, all antagonistic isolates produced phenazines. Results from whole-cell protein profiling showed that the antagonistic isolates are different from other isolated pseudomonads, while BOX-PCR revealed high genomic similarity among them. 16S rDNA sequencing of two representative strains within this group of antagonists confirmed their relatively low similarity with validly described Pseudomonas species. These antagonists are thus provisionally labelled as unidentified Pseudomonas strains. Among the antagonists, Pseudomonas CMR5c and CMR12a were selected because of their combined production of phenazines and biosurfactants. For strain CMR5c also, production of pyrrolnitrin and pyoluteorin was demonstrated. Both CMR5c and CMR12a showed excellent in vivo biocontrol activity against Py. myriotylum to a similar level as Ps. aeruginosa PNA1. CONCLUSION: Pseudomonas CMR5c and CMR12a were identified as novel and promising biocontrol agents of Py. myriotylum on cocoyam, producing an arsenal of antagonistic metabolites. SIGNIFICANCE AND IMPACT OF THE STUDY: Present study reports the identification of two newly isolated fluorescent Pseudomonas strains that can replace the opportunistic human pathogen Ps. aeruginosa PNA1 in the biocontrol of cocoyam root rot and could be taken into account for the suppression of many plant pathogens.     

烧伤病区感染常见病原菌及抗生素的选择

目的探讨内江市第一人民医院烧伤病区感染的常见病原菌及其对常用抗生素的耐药性．以指导临床抗生素的选择,提出防治措施。方法对1999年1月～2003年12月该院烧伤病区患者因感染而分离出的658株病原菌进行回顾性分析。结果分离出的658株病原菌中．革兰阴性菌425株(64．6％),革兰阳性菌209株(31．8％)．真菌24株(3．6％)。主要病原菌分别为铜绿假单胞菌、金黄色葡萄球菌、大肠埃希菌、凝固酶阴性葡萄球菌和阴沟肠杆菌。药敏提示除真菌外的大多数病原菌出现多重耐药性。革兰阴性菌对亚胺培南、头孢哌酮-舒巴坦、头孢西丁和妥布霉素较为敏感。革兰阳性球菌对替考拉丁和万古霉素高度敏感。结论烧伤感染中以铜绿假单胞菌为主．其次为金黄色葡萄球菌、大肠埃希菌和凝固酶阴性葡萄球菌。了解病原菌的结构及其变化趋势．对于加强抗感染治疗的针对性和主动性,取得最佳的感染防治效果具有重要意义。     

Occurrence of Chloramphenicol-acetylating Enzymes in Various Gram-negative Bacilli

The occurrence of a chloramphenicol-acetylating enzyme, similar to that found in Escherichia coli, carrying an R factor was investigated in various gram-negative bacilli. The acetylated products of chloramphenicol were identified by chromatography and quantitatively assayed after benzene extraction. The investigated strains were of the Salmonella-Arizona group, the Klebsiella-Aerobacter group, Serratia marcescens, the Proteus group, and Pseudomonas aeruginosa, most of which were isolated from 1947 to 1957. Both chloramphenicol-sensitive and -resistant strains were included, but none of them was able to transfer chloramphenicol resistance by conjugation. In the Proteus group, a significant level of a chloramphenicol-acetylating enzyme was found in most strains, whether they were sensitive or resistant to chloramphenicol; the resistant strains showed higher levels of the enzyme. Some chloramphenicol-sensitive strains lacked this enzyme. Only the sensitive strains containing the enzyme could easily produce chloramphenicol-resistant mutants with higher enzyme activity. Thus, the chloramphenicol resistance of this group can be reasonably explained on the basis of the chloramphenicol-acetylating enzyme. All of the Pseudomonas aeruginosa strains were resistant to chloramphenicol, and most strains showed low levels of the enzyme (which, however, did not appear sufficient to explain their resistance). All of the strains of the other groups (except one strain of Enterobacter cloacae) lacked the enzyme, although most strains of the Klebsiella-Aerobacter group and of S. marcescens were resistant to chloramphenicol. With respect to the origin of the resistance gene of the R factor, it is noteworthy that the strains of Proteus mirabilis isolated in 1947 possessed this enzyme before the discovery of chloramphenicol.     

Molecular Identification and Biofilm-Forming Ability of Culturable Aquatic Bacteria In Microbial Biofilms Formed in Drinking Water Distribution Networks

Drinking water distribution networks are known to harbor microbial biofilms. The aim of the present work is to (i) identify the culturable bacteria presented in the drinking-water distribution network, (ii) investigate the ability of isolated bacteria to form biofilm under some environmental stress conditions and some eliminating or removing treatments. To achieve it, 57 strains were isolated from biofilm (43 isolates) and water samples (14 isolates) collected from five stations in drinking-water distribution network in Taif city, Kingdom of Saudi Arabia (KSA). Partial sequences of 16S rRNA gene in the 57 isolates ensured the presence of only 22 different strains in biofilm samples. Among these strains, only 14 strains were also detected in water samples. Gram-negative Aeromonas hydrophila was the most occurred bacterium in the microbial biofilm obtained from the purified-water storage tanks followed by Gram-negative Pseudomonas sp. Gram-positive Bacillus subtilis was the most occurred bacterium in the microbial biofilm collected from the ends of the distribution pipes. Among the 22 isolated strains, 13 strains were strong biofilm producers at 30 and 37°C. The effects of environmental stresses including nutrient starvation (diluted TSB, 20:1), heating (100°C for 10 min), UV-treatment (240 nm for 10 min) and dynamic incubation (150 rpm min^?1) on the formation of biofilm were also investigated. These conditions affected the biofilm formation ability of the isolated strains at different levels. Nutrient starvation enhanced biofilm formation by most of the isolates. Among some biofilm deforming treatments, SDS and trypsin had considerable effects on preventing biofilm formation by most of the isolated strains. In conclusion, the results of the present work indicated that not all biofilm strains released from biofilm to the drinking water. Also, not all biofilm strains were able to form biofilm. Most of isolated bacteria had ability to form biofilm at suboptimum temperature of growth. These results may provide basic information on formation of microbial biofilms and overcome the problem of deteriorating of water quality in the drinking-water distribution networks.     

Comparative analysis of antibiotic sensitivity of Streptococcus pneumoniae strains isolated from patients and carriers

The data on antibiotic sensitivity of 38 strains of S. pneumoniae isolated from children and 46 strains isolated from carriers are presented. The isolates from the carriers had significantly higher sensitivity to benzylpenicillin, ampicillin, methicillin, oxacillin, cefazolin, erythromycin, oleandomycin and lincomycin. Resistance to gentamicin was more frequent in the strains isolated from the carriers. Among the strains of S. pneumoniae isolated from the patients and carriers representatives of serovar K19 were more frequent. There were no statistically reliable difference in them by sensitivity to benzylpenicillin, ampicillin, cefazolin, lincomycin and rifampicin. Still, the isolates from the carriers were much more sensitive to methicillin, oxacillin, oleandomycin and erythromycin.     

Screening and selection of stress resistant Lactobacillus spp. isolated from the marine oyster (Crassostrea gigas)

We attempted to isolate Lactobacillus spp. from the marine oyster (Crassostrea gigas) and select stress resistant strains for development of a future marine aquaculture feed adjuvant. A total of 83 lactobacilli strains were isolated from oyster. They were all Gram-positive, rod-shaped and catalase-negative. By performing a stress resistance assay, we selected eighteen isolates. Based on 16S rRNA gene sequencing, Lactobacillus paracasei was the most prevalent species among the selected isolates. The in vitro antagonistic effect of the selected strains against fish pathogens was assayed by measurement of inhibition diameters. Except for MH44, MH51, MH53 and MH62, most of the isolates showed inhibition of Vibrio alginolyticus and Vibrio proteolyticus (diameters over 15 mm). Lactobacillus rhamnosus MH22 was selected as the most stress resistant strain showing the MICs of 1.8 M NaCl, 14% ethanol and 0.014% hydrogen peroxide. L. rhamnosus MH22 isolated from oyster has a potential to be applied as a microbial feed adjuvant for marine aquaculture.     

水霉拮抗放线菌的分离、筛选与鉴定

目的:以珍珠健康养殖水体底泥为材料分离放线菌,筛选对水产动物水霉病病原菌有抗菌活性的放线菌。方法:采用稀释涂布法,选用萘啶酮酸和放线菌酮双抗平板分离获得放线菌;以黄颡鱼和湘云鲫鱼卵水霉病原菌为靶标菌,采用琼脂块法测试所分离菌株抗水霉菌活性及其稳定性;对拮抗活性强的放线菌采用形态观察和16S r DNA序列分析进行分类鉴定。结果:从分离获得的27株放线菌中筛选出3株对水霉病原菌有拮抗活性的菌株QF1、DNC17和QHV2,其中QHV2抗菌活性与稳定性最好;形态学观察与16S r DNA序列分析结果表明QF1、DNC17和QHV2均属于链霉菌属(Streptomycete sp.),分别鉴定为Streptomyces diastatochromogenes、Streptomyces variabilis和Streptomyces collinus。结论:3株放线菌对水霉病原菌具有较好的拮抗活性,具有开发成抗水霉药物的潜在价值。     

R plasmid distribution in S. typhi isolated on the territory of the left coast of the Ukraine in 1970-1982

A total of 373 strains of S. typhi isolated in 1970-1982 were tested with respect to their sensitivity to 9 antibiotics active against gram-negative bacteria. It was shown that about 1/3 of the isolates were resistant to 1-3 antibiotics. Among the resistant isolates the number of strains resistant to 4-5 and more antibiotics amounted to 12.5 per cent. The plasmid nature of the antibiotic resistance in the isolated strains of S. typhi was shown. Transmissive R plasmids were detected in 13 per cent of the strains studied. Within the last 5 years there was an increase in the proportion of strains with transmissive R plasmids in patients with sporadic typhoid fever or especially in groups of patients with the disease.     

土霉素暴露对小麦根际抗生素抗性细菌及土壤酶活性的影响

通过培养的方法研究了土霉素暴露和小麦根际抗性细菌的数量、种类、分布特征及土壤酶活性之间的剂量效应关系。结果表明,土霉素暴露下小麦根际单一抗生素抗性细菌数量和抗土霉素—链霉素双重抗性细菌数都明显增加,且与暴露剂量呈正效应关系;同时,土壤磷酸酶、脱氢酶活性下降,但与土霉素的剂量效应关系不明显。从土霉素暴露的土壤中分离到50株抗性细菌,经形态观察、RFLP分组和16S rDNA序列测定与分析,将它们聚集在Actinobacteria、Bacilli、Alphaproteobacteria、Gammaproteobacteria 和Sphingobacteria类群。其中放线菌最多（15株）,占抗性菌总数的30 %;其次是Bacillus属细菌（9株）和Pseudomonas属细菌（8株）,分别占18 %和16 %。同时,具有抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌在土霉素暴露的样品中均被分离到,分别占抗性菌株总数的16 %、8 %和4 %。值得注意的是,随着土霉素暴露剂量的增加,小麦根际优势促生菌Bacillus属细菌的抗性检出率逐步降低;但具有抗生素抗性的人类机会致病菌Pseudomonas、Sphingomonas和Stenotrophomonas属细菌的检出率却明显增加,提示可能会进一步增大其机会致病性。     

相似穿孔线虫拮抗细菌的筛选及分子鉴定

【背景】相似穿孔线虫是一种重要的检疫性有害生物,其寄主范围广,危害具有毁灭性,已给我国农业生产造成潜在威胁。由于目前大多化学杀线剂毒性较高,筛选获得对相似穿孔线虫具有拮抗活性的微生物菌株对线虫防控具有重要意义。【方法】广泛从染病香蕉植株根际采集土壤样本,运用等比稀释涂布平板法分离菌株;采用摇瓶发酵法制备拮抗菌株上清液和发酵液。通过室内生物测定和盆栽试验,筛选出对相似穿孔线虫具有拮抗作用的细菌菌株,并通过16S rDNA的PCR扩增和序列分析,对拮抗菌株进行种类鉴定。【结果】获得5株对相似穿孔线虫具有较理想拮抗活性的细菌菌株。其中,菌株HD-86的作用效果最好,其发酵上清液处理24 h后的相似穿孔线虫死亡率达100%。盆栽试验表明,菌株HD-86发酵液处理42和70 d后对相似穿孔线虫的相对防效分别为77.34%和90.51%,均优于对照药剂阿维菌素。【结论与意义】菌株HD-86对相似穿孔线虫具有很强的拮抗作用,将其鉴定为洋葱伯克氏菌Burkholderia cepacia。本研究可为相似穿孔线虫生防制剂的研发和病害的防控提供依据。     

Resistance of Shigella to antibiotics

Antibiotic sensitivity of 104 Shigella clinical strains and 104 Escherichia coli strains isolated from patients with acute dysentery not treated with antibiotics in 1986-1987 was studied. It was shown that 100 per cent of the dysentery pathogens and colon bacilli were antibiotic resistant. Strains resistant simultaneously to chloramphenicol, ampicillin, streptomycin, tetracycline, monomycin and kanamycin were the most frequent among the dysentery pathogens. Colon bacilli and dysentery pathogens isolated from the same patient had specific sets of antibiotic resistance markers. Pathogenetic therapy of dysentery and exclusion of antibiotic use for several years did not result in lower numbers of Shigella antibiotic resistant strains.     

Plant growth-promoting potential of endophytic bacteria isolated from roots of coastal sand dune plants

Endophytic bacteria associated with the roots of coastal sand dune plants were isolated, taxonomically characterized, and tested for their plant growth-promoting activities. Ninety-one endophytic bacterial isolates were collected and assigned to 17 different genera of 6 major bacterial phyla based on partial 16S rDNA sequence analyses. Gammaproteobacteria represented the majority of the isolates (65.9%), and members of Pseudomonas constituted 49.5% of the total isolates. When testing for antagonism towards plant pathogenic fungi, 25 strains were antagonistic towards Rhizoctonia solani, 57 strains were antagonistic towards Pythium ultimum, 53 strains were antagonistic towards Fusarium oxysporum, and 41 strains were antagonistic towards Botrytis cinerea. Seven strains were shown to produce indole acetic acid (IAA), 33 to produce siderophores, 23 to produce protease, 37 to produce pectinase, and 38 to produce chitinase. The broadest spectra of activities were observed among the Pseudomonas strains, indicating outstanding plant growth-promoting potential. The isolates from C. kobomugi and M. sibirica also exhibited good plant growth-promoting potential. The correlations among individual plant growth-promoting activities were examined using phi coefficients, and the resulting data indicated that the production of protease, pectinase, chitinase, and siderophores was highly related.     

Distribution and characteristics of Bacillus bacteria associated with hydrobionts and the waters of the Peter the Great Bay, Sea of Japan

Bacilli of the species Bacillus subtilis, B. pumilus, B. mycoides, B. marinus and B. licheniformis (a total of 53 strains) were isolated from 15 invertebrate species and the water of the Vostok Bay, Peter the Great Bay, Sea of Japan. Bacilli were most often isolated from bivalves (22.7%) and sea cucumbers (18.9%); they occurred less frequently in sea urchins and starfish (13.2 and 7.5%, respectively). Most of bacilli strains were isolated from invertebrates inhabiting silted sediments. No Bacillus spp. strains were isolated from invertebrates inhabiting stony and sandy environments. The species diversity of bacilli isolated from marine objects under study was low. Almost all bacterial isolates were resistant to lincomycin. Unlike B. pumilus, B. subtilis isolates were mostly resistant to benzylpenicillin and ampicillin. Antibiotic sensitivity of B. licheniformis strains was variable (two strains were resistant to benzylpenicillin and oxacillin, while one was sensitive). A significant fraction of isolated bacilli contained pigments. Pigmented strains were more often isolated from seawater samples, while colorless ones predominated within hydrobionts. B. subtilis colonies had the broadest range of colors. In the Bacillus strains obtained, DNase, RNase, phosphatase, elastolytic, chitinase, and agarolytic activity was detected. Bacilli strains with hydrolytic activity occurred in invertebrates more often than in seawater.     

Involvement of secondary metabolites and extracellular lytic enzymes produced by Pseudomonas fluorescens in inhibition of Rhizoctonia solani, the rice sheath blight pathogen

Fourteen strains of Pseudomonas fluorescens isolated from rhizosphere soil of rice were tested for their antagonistic effect towards Rhizoctonia solani, the rice sheath blight fungus. Among them, PfMDU2 was the most effective in inhibiting mycelial growth of R. solani in vitro. Production of chitinase, beta-1,3-glucanase, siderophores, salicylic acid (SA) and hydrogen cyanide (HCN) by P. fluorescens strains was evaluated. The highest beta-1,3-glucanase activity, siderophore production, SA production and HCN production were recorded with PfMDU2. A significant relationship between the antagonistic potential of P. fluorescens against R. solani and its level of beta-1,3-glucanase, SA and HCN was observed.     

Assessment of ecological diversity of rhizobacterial communities in vermicompost and analysis of their potential to improve plant growth

Present study was designed to determine the microbial diversity from three distinctive sites (amended with vermicompost) of Gujarat, India. A set of 76 strains were screened from total of 438 strains that exhibit plant growthpromoting (PGP) and antagonistic potential isolated from sites PS1 (Mehsana district), BS2 (Dantiwada district) and VS3 (Gandhinagar district). Their diversity indices were studied for determining the species richness and evenness of screened isolates. Results revealed that site BS2 showed the most significant diversity indices in terms of Shannon (H′ 1.525) and Simpson (1/D 5.120) than other two samples. Principal component analysis showed that bacterial diversity (H′) was correlated with the soil characteristics. Chickpea and groundnut plants inoculated with MBCU1 and MBCU3 isolates showed an increase in the vegetative growth parameters that evaluate plant growth when compared to uninoculated controls. Strains MBCU1 and MBCU3 were identified as Pseudomonas stutzeri and Pseudomonas mosselii, respectively, according to sequence analysis of the 16S rRNA gene. These both isolates belong to site BS2 and they showed specific PGP traits suggesting that these isolates can promote plant growth by more than one mechanism with respect to their higher diversity index.     

Inhibitory effect against pathogenic and spoilage bacteria of Pseudomonas strains isolated from spoiled and fresh fish.

The antibacterial effects of 209 Pseudomonas strains isolated from spoiled iced fish and newly caught fish were assessed by screening target organisms in agar diffusion assays. One-third (67 strains) inhibited the growth of one or several of six target organisms (Escherichia coli, Shewanella putrefaciens, Aeromonas sobria, Pseudomonas fluorescens, Listeria monocytogenes, and Staphylococcus aureus), of which S. aureus and A. sobria were the most sensitive. The inhibitory action was most pronounced among the strains producing siderophores, and the presence of iron eliminated the antibacterial effect of two-thirds of the inhibitory strains. Siderophore-mediated competition for iron may explain the inhibitory activity of these strains. All but nine of the inhibiting strains were found to inhibit the growth of 38 psychrotrophic S. putrefaciens strains isolated from spoiling fish and fish products. Siderophore-containing Pseudomonas culture supernatants inhibited growth of S. putrefaciens, as did the addition of iron chelators (ethylenediamine dihydroxyphenylacetic acid [EDDHA]). In particular, Pseudomonas strains isolated from newly caught and spoiled Nile perch (Lates niloticus) inhibited S. putrefaciens. This suggests that microbial interaction (e.g., competition or antagonism) may influence the selection of a microflora for some chilled food products.     

Microbiological control of drug resistance of causative agents in suppurative-septic infections in newborn infants

Resistance of the main causative agents of purulent septic infections such as pneumonia, meningitis, sepsis, etc. to 11 chemotherapeutic drugs was studied. The pathogens were isolated from autopsies of 203 newborns who died within 1981 to 1987. Among 2978 isolates belonging to Enterobacteriaceae (2466 strains) and Pseudomonadaceae (512 strains) which constituted 88% of all the isolates, strains with multiple resistance predominated: 90% of the strains resistant to 4 or more antibiotics, 77% of the strains resistant to 6 or more antibiotics and 48% of the strains resistant to 8 or more antibiotics. The highest number of the isolates were resistant to 8 and 9 chemotherapeutics (19 and 20%, respectively). 84% of all the isolates belonged to 4 genera: Klebsiella (34%), Escherichia (21%), Serratia (14%) and Pseudomonas (15%). They were characterized by the highest resistance spectra. In all the cases massive colonization of the intestine by the strains with multiple resistance which caused purulent septic infections was observed. The most frequent variants of the drug resistance combination were determined. In the total frequency of the isolate with multiple resistance no significant differences were detected in 1981 and thereafter. In 1986-1987 the frequency of S. marcescens strains increased 3-5 times with simultaneous broadening of their drug resistance spectra. Strains of S. marcescens and K. pneumoniae with multiple drug resistance endemic for definite hospitals were detected.     

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.