Group antigens in slow-growing rhizobia

Summary Internal group antigens of several slow-growing and fast-growing Rhizobium strains were tested by gel-diffusion against antisera to three strains of Rhizobium japonicum. At least one, generally two common antigens were found in 13 strains of R. japonicum, 4 strains of R. lupini, 4 strains isolated from cowpea and two slow-growing strains isolated from Lotus. Forty-six fast-growing rhizobia (including two from Lotus and 4 from Leucaena leucocephala) were clearly distinguished from the slow-growing strains in tests with the same antisera. They were wholly negative (9) or gave a much weaker non-identical line with one antiserum (24 strains), two antisera (8) or three antisera (5). The 5 strains of agrobacteria grouped with the fast-growing rhizobia.     

Genetic Diversity and Pathogenicity of <Emphasis Type="Italic">Xanthomonas axonopodis</Emphasis> Strains Inducing Citrus Canker Disease in Iran and South Korea

For the first time in 1989 citrus bacterial canker disease has seen on Citrus aurantiifolia in southern Iran. A total of 43 strains from affected citrus trees, ten strains from South Korea and representative from all known five pathotypes of Xanthomonas axonopodis pathogenic on citrus trees were used in this study. Isolated strains from Iran were indistinguishable by phenotypic, FAMEs, and SDS-PAGE analyses but showed different host range. First group were pathogenic on all tested citrus seedlings including C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, Poncirus trifoliata X C. paradisi, C. aurantium, C. paradise, C. medica, P. trifoliate, C. grandis, C. sinensis, C. reticulate and C. sinensis X P. trifoliate. Pathogenicity of the second group were limited to C. aurantiifolia, C. limettioides, C. limon, C. jambhiri, P. trifoliata X C. paradis, and C. aurantium. Among the strains studied by AFLP fingerprinting six clusters were found. These clusters were: (1) strains of pathotype C; (2) strains of pathotypes B and D; (3) strains of pathotype A together with the main group of the Iranian strains; (4) strains isolated from Korea; (5) strains of pathotype E; and (6) seven strains from Iran which made a completely separate cluster. Strains from pathotypes B and D could not be differentiated by AFLP. The tested Iranian strains belongs to the two different groups and strains from Korea grouped as a subcluster from main cluster of Iranian strains belong to the pathotype A.     

Agglutination,Toxigenicity and Sorbitol Fermentation of Clostridium difficile

A total of 79 Clostridium difficile strains from different sources (50 strains from the fecal specimens of healthy adults, 13 from patients receiving antibiotics without gastrointestinal complications, 13 from antibiotic-associated pseudomembranous colitis (PMC) or diarrhea patients, and three strains from ATCC) were investigated for agglutinability, using formol-treated cells as antigen, in relation to toxigenicity. C. difficile strains tested were divided into four serovars, I, II, III, and IV, by the cross-agglutination test. The agglutinin absorption test revealed that strains of serovar I, agglutinable with high titers (5,120–10,240) to antiserum prepared against a highly toxigenic C. difficile strain, ATCC 17859, possessed the serovar-specific antigen. All of the strains of serovar I were highly toxigenic and all 13 strains isolated from the fecal specimens of antibiotic-associated PMC or diarrhea patients belonged to this serovar, whereas 19 (38%) out of 50 strains from healthy adults and four (30.8%) out of 13 strains from patients receiving antibiotics without gastrointestinal complications possessed this antigen. None of the strains of other clostridial species than C. difficile were agglutinated by the three reference antisera used. Further study on the sugar fermentation test disclosed that the sorbitol-fermenting property of C. difficile is very closely related to the toxigenicity and agglutinability.     

Yersinia pseudotuberculosis in China

Thirty strains of Yersinia pseudotuberculosis were isolated from rabbits (17 strains), wild rats (9 strains) and house rats (4 strains) in China between 1990 and 1993. The biochemical properties of these isolates were identical with those of Y. pseudotuberculosis and no special characteristics were found in these strains. Serologically, serogroups 4b and 5b were identical to isolates found in Japan, and a new serogroup 1c and unclassified strains have also been detected. The existence of virulence-associated properties were different among strains. The pYV plasmid was detected from 6 strains of 30 isolates. This report documents the presence of Y. pseudotuberculosis in China, providing important epidemiological information.     

Assessment of strains of Pseudomonas syringae pv. tomato from Tanzania for resistance to copper and streptomycin

Abstract

Fifty-six strains of Pseudomonas syringae pv. tomato (P.s. pv. tomato) were collected from tomato-producing areas in Tanzania and assessed for resistance to copper and antibiotics. The collection was done from three tomato-producing regions (Morogoro, Arusha and Iringa), representing three different ecological conditions in the country. After isolation and identification, the P. s. pv. tomato strains were grown on King's medium B (KB) amended with 20% copper sulphate (w/v). The strains were also assessed for resistance to antibiotics. Results indicated that there was widespread resistance of the P. s. pv. tomato strains to copper sulphate. The highest level of resistance was recorded from the Arusha region (Northern Tanzania), 83.3% of the P. s. pv. tomato strains from that region showed resistance to copper sulphate. This was followed by Iringa region (Southern Tanzania), from where strains of the pathogen were moderately resistant to copper sulphate, such that 54.0% of them were able to grow on the KB medium amended with 20% (w/v) of the copper compound.

Out of seven strains of P. s. pv. tomato from Morogoro region (Central Tanzania) included in the study, five (71.5%) were resistant to copper sulphate. The only strain of P. s. pv. tomato from the Dodoma region (Central Tanzania, but with a different ecological condition from the Morogoro region) included in the study was unable to grow on the medium containing 20% copper sulphate. None of the P. s. pv. tomato strains in the four regions included in the study were resistant to streptomycin sulphate. These results suggest that in the Arusha and Iringa regions of Tanzania, there might be possibilities of excessive use of copper compounds in tomato production, such that strains of P. s. pv. tomato strains in the areas have become resistant to the compounds.     

A Possible Role of R Plasmids in Bacterial Permeability for β-Lactam Antibiotics

Four strains of clinical isolates of Serratia marcescens (13039, 13090, 13093, 14093) harboring R plasmids were highly resistant to ampicillin (ABPC) and cephaloridine (CER). With elimination of R plasmids from these strains by acriflavine treatment, ABPC-resistance levels of these strains were markedly reduced. Reduction of CER-resistance levels was also demonstrated in strains 13039 and 13093, but not in strains 13090 and 14093. The permeability of the former strains for CER was also decreased, but not in the latter strains. At the same time, β-lactamase activity of these strains also almost completely disappeared when the R plasmids were eliminated. By broth matings with these strains, the recipient strains of S. marcescens 13031 (rif), Escherichia coli K-12 (rif), and E. coli 15046 (rif) all acquired a high permeability barrier against CER with inheritance of the R plasmids from strains 13039 and 13093, but not from strains 13090 and 14093. The transconjugant of strain 13031 that inherited R plasmid 13093 was resistant not only to CER but also to cefazolin, cephalothin, and cephalexin. Its permeability to these antibiotics was significantly lower than that of the original strain. This fact suggests the possibility that the R plasmid from strain 13093 may be involved not only in production of β-lactamases, but also in regulation of bacterial permeability for cephalosporins.     

Adherence Patterns and DNA Probe Types of Escherichia coli Isolated from Diarrheal Patients in China

One hundred and seventy-two strains of Escherichia coli isolated from diarrheal patients in Beijing, P. R. China, were analyzed for plasmid DNA profile, HEp-2 cell adherence ability and reactivity to 10 previously described DNA probes. They had not been recognized as pathogenic E. coli in China. Of the 110 strains tested, 76 (69%) contained one or multiple large plasmids. Of the 71 strains with the large plasmids 64 could adhere to HEp-2 cells. Of the 172 strains, 102 (59.3%) were hybridized with at least one of the 10 probes. Of those, seven strains hybridized with enteroaggregative E. coli (EAggEC) probe. Their serotypes were O128 (two strains), O6 (one strain), and O111 (one strain). Three strains were untypable. Six and three strains were hybridized with enteropathogenic E. coli (EPEC) attaching and effacing genes (eae) or EPEC adherence factor (EAF) probe, respectively. Two non-O157: H7 strains hybridized with enterohemorrhagic E. coli (EHEC) probe. Seventy-two strains (41.9%) hybridized with shiga-like toxin 2 or 1 (SLT2 or SLT1) probes. Among the SLT1 or SLT2 probe-positive strains, 54 hybridized with invasive (INV) plasmid probe developed for identification of enteroinvasive E. coli (EIEC) and Shigella species. The INV and SLT probe-positive strains might represent a new variety of verotoxin-producing E. coli (VTEC).     

Phenotypic and Genetic Diversity of Erwinia carotovora ssp. carotovora Strains from Asia

A collection of 87 strains of the soft rot pathogen Erwinia carotovora ssp. carotovora (Ecc) isolated from various host plants in Japan, Korea and Thailand was characterized by bacteriological, pathological and genetic properties. On the basis of pathogenicity on the potato, tomato, onion and cucumber, strains were divided into four groups. They were also characterized by PCR‐restriction fragment length polymorphisms (RFLP) of 16S ribosomal DNA (rDNA), 16S‐23S rDNA intergenic spacer regions (ISRs) and a pel gene encoding pectate lyase. By analysis of 16S rDNA RFLP generated by Hinf I, Ecc strains were differentiated into two groups where it was discovered that most strains from Korea and Japan belonged to the same group. In the analysis of ISRs RFLP with MboI, two patterns were found. All Thai strains showed the same pattern. In the analysis of the pel gene RFLP with Sau3AI, all strains were separated into two independent patterns except for one strain. The strain (MAFF 301937) isolated from the mulberry showed a unique RFLP pattern of the pel gene. In cluster analysis based on 26 phenotypic characters, Ecc strains were composed of two groups, A and B. Group A contained typical Ecc strains which provided negative reactions in testing the production of reducing substances from sucrose and acids from α‐methyl glucoside. All Thai strains and most of the Korean strains belonged to group A, whereas group B contained atypical Ecc strains, which were isolated in Japan and Korea; the properties of this group were similar to those of E. carotovora ssp. atroseptica. The research reported here was undertaken to provide information on the strains of E. carotovora ssp. carotovora in Asia.     

Enterovirulent <Emphasis Type="Italic">E. coli</Emphasis> in inflammatory and noninflammatory bowel diseases

We determined the incidence of enterovirulent E. coli (EVEC; which can to cause gastrointestinal infections) in strains isolated from patients with both of the major inflammatory bowel diseases (IBD), Crohn’s disease (CD) and ulcerative colitis (UC) and from patients with noninflammatory bowel diseases (nonIBD). Cell detachment E. coli (CDEC) were detected in 14 % of all strains. A significant difference in the presence of CDEC was found between the groups of strains isolated from UC (24.1 %), nonIBD (11.9 %) and CD (4.7 %). Enteroaggregative E. coli (EAggEC) were detected in 2.5 %, typical enteropathogenic strains (EPEC) in 1.3 % and enterotoxigenic ones (ETEC) in 1.5 %. Enteroinvasive (EIEC) and shigatoxin producing E. coli (STEC) were not detected. Some strains showed a high invasion level in gentamicin-protection assay. These strains could therefore belong to adherent-invasive E. coli (AIEC) because they are free of genes encoding invasins (ipaH, ial) and are equipped with fimA gene. However, complete characterization of these strains and their classification as AIEC will require further tests.     

Identification of some Bacteria from Paddy Antagonistic to Several Rice Fungal Pathogens

The effect of 23 bacterial strains from ricefields in the tropics on rice seed germination and on radicle and hypocotyl development of four rice cultivars was determined. There was a varietal difference in response to seed bacterization with the different bacterial strains. Germination of cv. IR58 increased from 78 to 93 %, that of cv. IR64, from 89 to 97 %. Less effects on germination of cvs IR42 and IR36 were observed. All strains inhibited the mycelial growth of Rhizoctonia solani in vitro. The three strains, identified as Bacillus subtilis, inhibited the mycelial growth of eight fungal pathogens whereas the other strains were pathogen-specific. Seed bacterization with these bacterial strains provided a sheath blight protection of 4. 5 to 73 % in the glasshouse trial. These 23 bacterial strains were identified by phenotypic tests using the API systems, morphological and biochemical features, and by comparison of electrophoretic patterns after sodium dodecyl sulphate polyacrylamide gel electrophoresis. Bacterial strains were identified (number of strains in brackets) as: Bacillus subtilis (3), Bacillus laterosporus (1), Bacillus pumilus (1), Pseudomonas aeruginosa (7), Pseudomonas belonging to section 1 (5), Erwina herbicola-like (1), and Serratia marcescens (1). The features of the other four strains were similar to Serratia except for the DNAase and lipase activities.     

Drug Resistance and R Plasmids in Vibrio anguillarum Isolated in Cultured Ayu (Plecoglossus altivelis)

Two hundred twenty-six strains of Vibrio anguillarum collected from cultured ayu (Plecoglossus altivelis) between 1978 and 1980 were studied for their sensitivities to 10 chemotherapeutics. In order to determine whether the drug-resistant strains possessed transferable R plasmids, they were conjugated with Escherichia coli. Almost all the strains isolated during the 3 years showed resistance to nalidixic acid (NA) and/or furazolidone (NF). NA and NF resistance were not transferred to Escherichia coli from any of the strains. Chloramphenicol-resistant strains were isolated in every year and almost all of them carried transferable R plasmids. Only one strain with tetracycline resistance was found among the strains tested. Strains resistant to sulfonamides, streptomycin, ampicillin (ABP), and trimethoprim (TMP) increased rapidly in 1980, and a large number of them carried transferable R plasmids. Transferable R plasmids encoded with resistance to ABP and TMP were detected for the first time in V. anguillarum strains. The R plasmids detected in the strains isolated in 1980 were classified into incompatibility groups E, A, and an untypable group. The R plasmid DNAs were cleaved by EcoRI to yield 11 to 13 fragments. The estimated molecular weights of the R plasmids from the five strains ranged from 97 to 104 M daltons.     

Phenotypic and genotypic characterization of tetracycline and minocycline resistance in Clostridium perfringens

The aim of this study was to determine the incidence of tetracycline resistance and the prevalence of tetracycline-resistance genes in strains of Clostridium perfringens isolated from different sources between 1994 and 2005. Susceptibility to tetracycline and minocycline in strains from humans (35 isolates), chickens (15 isolates), food (21 isolates), soil (16 isolates) and veterinary sources (6 isolates) was determined, and tetracycline-resistance genes were detected. Resistance was most common in strains isolated from chickens, followed by those from soils, clinical samples and foods. The most highly resistant strains were found among clinical and food isolates. tetA(P) was the most common resistance gene, and along with tetB(P) was found in all resistant strains and some sensitive strains. One tetracycline-resistant food isolate had an intact tet(M) gene. However, PCR fragments of 0.4 or 0.8 kb with high degrees of identity to parts of the tet(M) sequences of other bacteria were found, mainly in clinical isolates, and often in isolates with tetB(P). No correlation between level of sensitivity to tetracycline or minocycline and the presence of tetA(P), tetB(P) or part of tet(M) was found. The presence of part of tet(M) in some strains of C. perfringens containing tetB(P) may have occurred by recent gene transfer.     

Isolation and Identification of Black Yeasts by Enrichment on Atmospheres of Monoaromatic Hydrocarbons

Black yeast members of the Herpotrichiellaceae present a complex ecological behavior: They are often isolated from rather extreme environments polluted with aromatic hydrocarbons, while they are also regularly involved in human opportunistic infections. A selective technique to promote the in vitro growth of herpotrichiellaceous fungi was applied to investigate their ecophysiology. Samples from natural ecological niches and man-made environments that might contain black yeasts were enriched on an inert solid support at low humidity and under a controlled atmosphere rich in volatile aromatic hydrocarbons. Benzene, toluene, and xylene were provided separately as the sole carbon and energy source via the gas phase. The assayed isolation protocol was highly specific toward mesophilic Exophiala species (70 strains of this genus out of 71 isolates). Those were obtained predominantly from creosote-treated railway ties (53 strains), but isolates were also found on wild berries (11 strains) and in guano-rich soil samples (six strains). Most of the isolates were obtained on toluene (43 strains), but enrichments on xylene and benzene also yielded herpotrichiellaceous fungi (17 and 10 isolates, respectively). Based upon morphological characterizations and DNA sequences of the full internal transcriber spacers (ITS) and the 8.5S rRNA genes, the majority of the obtained isolates were affiliated to the recently described species Exophiala xenobiotica (32 strains) and Exophiala bergeri (nine strains). Members of two other phylogenetic groups (24 and two strains, respectively) somewhat related to E. bergeri were also found, and a last group (three strains) corresponded to an undescribed Exophiala species.     

Diversity of Rhizobia isolated from various Hedysarum species

Cultural and physiological properties, serology, plasmid profiles and infective traits were determined for 23 strains of rhizobia isolated from various Hedysarum species: H. coronarium (common name: sulla) (16), H. carnosum (1), H. alpinum (3), H. mackenzii (2) and H. pallens (1) from Portugal, Spain, Tunisia, Alaska and Israel. Strains isolated from H. alpinum, H. mackenzii and H. pallens have slow growth rates on yeast-extract mannitol medium and were unable to nodulate H. coronarium plants, whereas the latter were effectively nodulated by all sixteen fast growing strains from sulla. Regardless of the country of origin all H. coronarium strains fell into one serogroup and were not serologically related with strains of other Hedysarum species. The RAPD (random amplified polymorphic DNA) fingerprinting method which was carried out on five H. coronarium and three H. alpinum strains allowed distinction to be made among serologically related rhizobia. No particular plasmid profile pattern was observed in relation to the host or geographical origin of the strains.     

The Incidence of R Factors in Bordetella bronchiseptica Isolated from Pigs

Bordetella bronchiseptica strains isolated from the nasal cavities of young pigs in Japan from 1969 to 1972 were surveyed for drug resistance and distribution of R factors. Of 304 strains examined, 71 (23%) were resistant to either one or more of following three drugs, streptomycin (SM), sulfadimethoxine (SA), and aminobenzyl penicillin (APC). Triple (SM.SA.APC)-resistance was most frequent among these resistant strains. Strains of double (SM. SA)- or single (SM)- and (SA)-resistance were also isolated, but were very few in numbers. Of the 71 drug-resistant strains, 61 (86%) were found to carry R factors which were capable of conjugal transfer. All of these R factors had the triple (SM.SA.APC)-resistant markers and were identified as fi^– (no fertility inhibition) type. The (SM.SA.APC)-resistant strains carrying R factors had been isolated from pigs reared on various farms in different districts, and consequently the prevalence of B. bronchiseptica strains carrying R factors was considered to be relatively wide-spread in young pigs.     

Antibacterial Activity of Marine Culturable Bacteria Collected from a Global Sampling of Ocean Surface Waters and Surface Swabs of Marine Organisms

The purpose of the present study was to isolate marine culturable bacteria with antibacterial activity and hence a potential biotechnological use. Seawater samples (244) and 309 swab samples from biotic or abiotic surfaces were collected on a global Danish marine research expedition (Galathea 3). Total cell counts at the seawater surface were 5 × 10⁵ to 10⁶ cells/ml, of which 0.1–0.2% were culturable on dilute marine agar (20°C). Three percent of the colonies cultured from seawater inhibited Vibrio anguillarum, whereas a significantly higher proportion (13%) of colonies from inert or biotic surfaces was inhibitory. It was not possible to relate a specific kind of eukaryotic surface or a specific geographic location to a general high occurrence of antagonistic bacteria. Five hundred and nineteen strains representing all samples and geographic locations were identified on the basis of partial 16S rRNA gene sequence homology and belonged to three major groups: Vibrionaceae (309 strains), Pseudoalteromonas spp. (128 strains), and the Roseobacter clade (29 strains). Of the latter, 25 strains were identified as Ruegeria mobilis or pelagia. When re-testing against V. anguillarum, only 409 (79%) retained some level of inhibitory activity. Many strains, especially Pseudoalteromonas spp. and Ruegeria spp., also inhibited Staphylococcus aureus. The most pronounced antibacterial strains were pigmented Pseudoalteromonas strains and Ruegeria spp. The inhibitory, pigmented Pseudoalteromonas were predominantly isolated in warmer waters from swabs of live or inert surfaces. Ruegeria strains were isolated from all ocean areas except for Arctic and Antarctic waters and inhibitory activity caused by production of tropodithietic acid.     

Toxicity of chlorpyrifos‐methyl to Sitophilus zeamais collected in Korea and biochemical differences

In this study, lethal concentration (LC₅₀) values of chlorpyrifos‐methyl (CPM) were determined for two Korean strains (CBNU and KNU) of Sitophilus zeamais. The two strains had similar susceptibilities (1.70 and 1.86 μg a.i./cm², respectively) to CPM. Carboxylesterase (CE) activity was twice as high in the CBNU strain as in the KNU strain. Lower acetylcholinesterase (AChE) activity was also noted in the latter; however, the activity of glutathione S‐transferase (GST) was twice as high as in the CBNU strain. Gel electrophoresis of CE of crude extracts from adults of the two strains of S. zeamais showed clearly different band patterns, with molecular weights of 60 kDa and 71 kDa in the CBNU and KNU strains, respectively. MALDI‐TOF MS/MS was used to profile small proteins (less than 10 kDa), with results indicating that 206 proteins are expressed differently in the two strains. The peak of interest of 2247.7 m/z was applied to TOF‐TOF MS and its de novo peptide sequence was identified as a tyrosine phosphatase fragment. Phospholipids from the two strains were analyzed and 34 phospholipids were found to be significantly different between strains. Results suggest that the two strains collected from Korea showed different biochemical results, presumably differences in insecticide selection by different living locations.     

Interactions between Plants and Epiphytic Bacteria Regarding Their Auxin Metabolism

Using hydrocultured pea plants, 109 bacterial strains (42 from shoots) were isolated from shoots, roots, and from the hydroculture medium. 58 different strains (26 from shoots) were able to produce IAA from tryptophan, 15 different strains (7 from shoots) were able lo destroy IAA. (Included are 13 strains possessing both properties.) As far as they could be identified, the IAA-producing and -destroying strains belong to Pseudomonas (by far dominating), Achromobacter, Alcaligenses, Bacillus, and Flavobacterium. The IAA-destroying activity strongly depends on the physiological state of the bacteria and the milieu conditions. Bacterial IAA production (but not IAA-degradation) is supposed to be important for the plant.     

Detection of Shiga toxins,intimin and enterohemolysin in Escherichia coli strains isolated from children in eastern Slovakia

FiftyEscherichia coli strains isolated from stool samples of 51 healthy children, 143 strains isolated from stool samples of 327 children with diarrhea and 24 strains isolated from stool samples of 21 children with suspected hemolytic uremic syndrome were examined for the presence of Shiga toxin-producingE. coli virulence factors (shiga toxin 1 and 2, intimin and enterohemolysin) and their genes. Vero-cell assay and latex agglutination were used for detection of Shiga toxin 1 and 2, TSB agar with washed erythrocytes was used for detection of enterohemolysin; genes encoding shiga toxin 1 and 2, intimin and enterohemolysin were detected using multiplex PCR. The presence ofE. coli strains harboring genes encoding shiga toxin 1 and 2 (12 strains), intimin (34 strains) and enterohemolysin (12 strains) was demonstrated.     

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.