Biovar Strain Research Articles

Phenotypic Characteristics and Cluster Analysis of Japanese and Reference Strains of Ralstonia solanacearum.

Of 54 phenotypic characteristics of 83 strains of Ralstonia solanacearum from Japan and overseas, the strains differed in 20 characters. Japanese strains were divided into four biovars (1-4), of which biovars 3 and 4 were common. Japanese biovar 2 strains were different from foreign biovars 2 and N2 strains for the utilization of trehalose, D-ribose, L-tartrate and/or m-tartrate. Eleven strains of Japanese biovar 3 from tobacco and kalanchoe (Kalanchoe sp.) atypically did not utilize inositol. Cluster analysis based on 20 phenotypic characters using the unweighted average pair-group method (UPGMA) on the simple matching coefficient showed seven clusters at 78% similarity. Biovars 3 and 4 were phenotypically homogeneous, and each grouped into a single cluster. On the other hand, strains in both biovars 1 and 2 were each divided into two clusters, one with the Japanese strains and the other with the overseas strains. The host of the strains in each cluster and biovar was different, and the strains isolated from plants other than solanaceous crops belonged to biovars 3 and 4.

Identification and Natural Antibiotic Susceptibility of Morganella morganii

Our data should elucidate whether or not natural antibiotic susceptibility can be used as an aid for subspecies or biovar discrimination of Morganella morganii (II). Furthermore, our goal was to create a database of the natural susceptibility of M. morganii (III) and we were interested in the relative frequency of the recently described subspecific taxa (I). On the basis of trehalose fermentation (TRE), ornithine decarboxylase (ODC), and lysine decarboxylase (LDC) activities, we determined the biovar for 90 clinical isolates of M. morganii. Within these strains we examined the natural antibiotic susceptibility of 53 morganellae to 70 antibiotics by determination of the MICs with a microdilution procedure. (I): 80 strains (89%) of all morganellae belonged to M. morganii ssp. morganii (TRE−), with biovar A (LDC−, ODC+) predominating (67 strains). The remaining strains of this subspecies were identified as biovar B (LDC+, ODC+; 12 strains) and biovar C (LDC−, ODC−, one strain). Ten strains of M. morganii ssp. sibonii (TRE+) were found: four strains belonged to biovar F (LDC variable, ODC−) and six strains to biovar G (LDC−, ODC). (II): With one exception we found no significant differences in antibiotic susceptibility between different biovars. M. morganii ssp. morganii strains are more susceptible to tetracycline than strains of M. morganii ssp. sibonii, but there is no evidence that this parameter could be useful to differentiate biovars within a subspecies. It could be shown that 8 of 30 strains of biovar A and 2 of 12 strains of biovar B were tetracycline resistant. However, one M. morganii ssp. sibonii strain was clinically susceptible to tetracycline according to French and American standards. (III): The natural population of M. morganii is primarily (naturally) resistant to certain penicillins like benzylpenicillin, oxacillin, and amoxicillin, first and second generation cephalosporins (excluding cefoxitin), cefpodoxime, all antibiotics of the ML group (macrolides and lincosamides), sulfamethoxazole, glycopeptides, fosfomycin, and fusidic acid, naturally sensitive to aminoglycosides, piperacillin, mezlocillin, ticarcillin, third and fourth generation cephalosporins, carbapenems, aztreonam, quinolones, trimethoprim, cotrimoxazole, and chloramphenicol. M. morganii is naturally resistant to a wide range of antibiotics. The natural resistance pattern is useful for validation of sensitivity tests. Susceptibility to antibiotics is an unsuitable parameter for the descrimination of the described subspecific taxa of M. morganii. M. morganii strains that do not belong to biovar A are rare.

Improved diagnostic PCR assay for Actinobacillus pleuropneumoniae based on the nucleotide sequence of an outer membrane lipoprotein.

The gene (omlA) coding for an outer membrane protein of Actinobacillus pleuropneumoniae serotypes 1 and 5 has been described earlier and has formed the basis for development of a specific PCR assay. The corresponding regions of all 12 A. pleuropneumoniae reference strains of biovar 1 were sequenced. Alignment of the sequences revealed conserved terminal and variable middle regions, which divided the reference strains into four distinct groups. Primers were selected from the conserved 5' and 3' termini of the gene. A 950-bp amplicon was obtained from each of 102 tested field isolates of A. pleuropneumoniae obtained from lungs. Their identity was verified by sequencing approximately 500 bp of the amplification product from 50 of the A. pleuropneumoniae isolates, which all showed the expected DNA sequence characteristic of the serotype. To test the specificity of the reaction, 23 other bacterial species related to A. pleuropneumoniae or isolated from pigs were assayed. They were all found negative in the PCR, as were tonsil cultures from 50 pigs of an A. pleuropneumoniae-negative herd. The sensitivity assessed by agarose gel analysis of the PCR product was 10(2) CFU/PCR test tube. The specificity and sensitivity of this PCR compared to those of culture suggest the use of this PCR for routine identification of A. pleuropneumoniae.

Major outer membrane proteins of Pasteurella haemolytica serovars 1–15: comparison of separation techniques and surface-exposed proteins on selected serovars

The Sarkosyl method of obtaining outer membrane proteins (OMPs) from Pasteurella haemolytica A1 was more efficient and less laborious than separating membranes by sucrose gradient centrifugation. More OMPs were recovered and major OMPs were present in greater concentrations in the Sarkosyl-derived preparations. Therefore, OMPs of P. haemolytica serovars 1–15 (serovars 3, 4, 10, and 15 being T biotypes and the remainder being A biotypes) were prepared by the Sarkosyl method and compared by SDS-PAGE. Serovars 1, 2, 5, 6, 7, 8, 11, and 12 which are A biovars had similar OMP profiles characterized by major OMPs of 30.5 and 43 kDa. Biovar T strains were characterized by doublet protein bands in the 26–28 kDa region and a major OMP in the 38–40 kDa range. Serovars 9, 13, and 14, which are also A biovars, had profiles similar, although not identical, to the T biovars. A 43 kDa protein was present in all serovars although concentration was greater in the A biovars. Surface-exposed proteins of P. haemolytica A1 determined by 125I-labeling of whole cells were 94, 84, 53.5, 49, 43, 41, 29.5, and 16 kDa. Iodine-labeling of serovars A2 and A6 which have similar OMP profiles by SDS-PAGE resulted in autoradiographs indistinguishable from A1. These studies expand our knowledge of P. haemolytica OMPs especially showing the utility of the Sarkosyl extraction procedure, variations in OMP profiles among some A biovar strains, and the similarities of OMP profiles and surface-labeled proteins among three of the most important serovars (1, 2, and 6).

Competitive interaction among strains of Rhizobium leguminosarum bv. phaseoli in the nodulation of kidney beans (Phaseolus vulgaris L.)

We examined the competitiveness of five effective Rhizobium leguminosarum biovar phaseoli strains in the nodulation of kidney beans (Phaseolus vulgaris L.), either alone or in pairwise combination, against the indigenous strains. The results showed that the introduced Rhizobium sp. strains (B2, B17, B36, T2, or CIAT 652) occupying 64–79% of the total nodules (as single inocula) were more competitive in nodulation than the native rhizobia. However, the competitiveness of the individual Rhizobium sp. strain either increased or decreased when used in a pairwise combination of double-strain inocula. For example, strain B17, although quite competitive against the indigenous population (68% nodule occupancy), became poorly competitive in the presence of strain B2 (reduced from 68 to 2.5%). A similar reduction in nodule occupancy by strain B17 was observed in the presence of B36 or CIAT 652, indicating that two competitive strains may not always be compatible. These results suggest that it is important to co-select competitive as well as compatible rhizobia for multistrain inoculant formulation.

Vibriophage N4 DNA is nonpermuted and terminally redundant

Phage N4 is one of the five newly isolated phages used in a new phage typing scheme for Vibrio cholerae O1 biovar EITor strains and belongs to the Podoviridae family. Electron microscopic studies showed that the phage N4 has a DNA of 40.4 ± 0.1 kb. A partial denaturation map of the N4 DNA has been constructed. It has been shown with the help of this partial denaturation map that phage N4 genome is nonpermuted. Circularization of the phage genome on treatment with exonuclease III, followed by incubation in 50% formamide, indicates a terminal redundancy.Key words: vibriophage N4, DNA, Vibrio cholerae, denaturation mapping.

Distribution of 3-Ketolactose Formation among Sphingomonas spp. and Other Members of the Alpha Subclass of the Proteobacteria

The distribution of 3-ketolactose formation among members of the alpha subclass of the Proteobacteria was investigated by the Benedict reagent test and by a more sensitive quantitative method in which high-performance liquid chromatography was used. 3-Ketolactose formation activity was found in strains of Agrobacterium biovar 1 and in strains of eight species of the genus Sphingomonas, including Sphingomonas paucimobilis (the type species), which belong to the alpha-2 and alpha-4 subclasses of the Proteobacteria, respectively. Weak activity was found in Erythrobacter longus IFO 14126T (T = type strain), a member of the alpha-4 subclass of the Proteobacteria. The ketosugar was not produced by members of the other taxa of the alpha subclass of the Proteobacteria tested. The ketosugar was isolated from culture broths of S. paucimobilis IFO 13935T and Sphingomonas yanoikuyae IFO 15102T and was identified as 3-ketolactose [4-O-(β-d-xylo-hexopyranosyl-3-ulose)-d-glucopyranose] by chromatographic analyses and 1H nuclear magnetic resonance spectroscopy.

Classification and Identification of Campylobacter sputorum using Numerical Analyses of Phenotypic Tests and of One-dimensional Electrophoretic Protein Profiles

Summary The application of numerical analyses of phenotypic tests and sodium-dodecyl sulphate polyacrylamide gel electrophoretic protein profiles in the classification and identification of Campylobacter sputorum was examined. Thirty-eight strains received, or isolated as, representatives of each of the defined biovars of Campylobacter sputorum (i.e. bubulus, fecalis, and sputorum), along with seventeen type or reference strains of other Campylobacter, Arcobacter , and Helicobacter species, were studied. The results from both analyses indicated, (i) that thirty-two strains formed a distinct cluster which included the type strain of C. sputorum , confirming their identity as members of this species, (ii) that catalase-positive strains of C. sputorum are associated with faeces, (iii) that the normal subdivision of catalase-negative C. sputorum strains into two source-specific biovars is invalid. Thus, catalase-positive strains of C. sputorum should remain as biovar fecalis and catalase-negative strains (i.e. biovars sputorum and bubulus) should be unified as a single biovar sputorum and the biovar bubulus discarded. Five oral isolates initially believed to be C. sputorum biovar sputorum were identified as potentially novel campylobacters. Numerical analysis of both types of data used were shown to be effective methods of classifying and identifying campylobacters and allied taxa.

RTX toxin genotypes and phenotypes in Actinobacillus pleuropneumoniae field strains

Actinobacillus pleuropneumoniae serotype reference strains and 204 A. pleuropneumoniae field strains representing all 12 serotypes and both biovars 1 and 2, obtained from laboratories from various countries worldwide, were analyzed for the presence of the toxin genes apxIC, apxIA, apxIB, apxID, apxIIC, apxIIA, apxIIIC, apxIIIA, apxIIIB, and apxIIID by DNA-DNA hybridization with specific gene probes. Expression of the toxins ApxI, ApxII, and ApxIII was assessed by immunoblot analysis with monoclonal antibodies. The results show that the patterns of apx genes and those of the expressed Apx toxins in biovar 1 field strains are the same as those of the genes and toxins of corresponding serotype reference strain. We found only three strains which had certain apx genes missing compared with the genes in their serotype reference strains. Analysis of the expression of the three toxins showed that nearly all strains expressed their apx genes and produced the same Apx toxins as their serotype reference strain. We found only one strain that did not produce ApxI, although it contained the apxICABD genes, and one strain which did not express ApxII but which contained apxIICA. Several field strains which initially showed that their serotype did not correspond to the apx gene profile of the reference strain and which had an unexpected virulence for the given serotype revealed that their initial serotyping was erroneous. We show that the apx gene profiles are inherent to a given serotype. The method cannot differentiate between all 12 serotypes. However, it allowed us to distinguish five groups of toxin gene patterns which showed pathological, toxicological, and epidemiological significance. None of the biovar 2 strains contained apxIII genes. The apxI and apxII genes in the biovar 2 strains, however, were the same as those found in the serotype reference strains of biovar 1.

Pathogenicity ofAgrobacteriumSpecies from the Noxious Rangeland WeedsEuphorbia esulaandCentaurea repens

Disease surveys were made of Russian knapweed (Centaurea repens) and leafy spurge (Euphorbia esula), two noxious weeds that severely infest large areas of rangelands in the northern Great Plains. Strains of Agrobacterium tumefaciens were isolated from Russian knapweed exhibiting crown galls in New Mexico in 1991. Strains pathogenic to one or more of Helianthus annuus, Lycopersicon esculentum, Nicotiana tabacum, Datura stramonium, or Russian knapweed and known strains of biovars 1 and 2 of A. tumefaciens and A. vitis were inoculated on Russian knapweed and two other knapweed species, diffuse (C. diffusa) and spotted (C. maculosa). Some strains from New Mexico were strongly pathogenic to diffuse knapweed, causing rapidly developing galls that typically girdled, stunted, and caused death of the host. The biovar 1 strains were pathogenic to all three knapweed species, strains of biovar 2 were pathogenic to diffuse and spotted knapweed, and the single A. vitis strain was pathogenic only to diffuse knapweed, forming small galls. Stunted and chlorotic plants of leafy spurge with crown galls, collected in Glacier County, Montana, and plants with root galls collected in eastern North Dakota were infected with A. tumefaciens, which was identified as biovar 1. Leafy spurge plants exhibiting galls on roots collected in North Dakota were infected with strains identified as biovars 1 and 2. Host ranges among nine pathogenic strains from Russian knapweed, leafy spurge, and known strains representing biovars 1 and 2 of A. tumefaciens and A. vitis varied greatly, with six of the nine strains being pathogenic to no more than one additional species besides the original host. These findings indicate that A. tumefaciens may be effective as a biological control of these important rangeland weeds and especially of diffuse knapweed.

Differential Resistance of Tomato Cultigens to Biovars I and III ofPseudomonas solanacearum

The reaction of six tomato cultigens (Yoshimatsu 4-11, Rotam-4, Rodade, Hawaii 7998, CL 1131-0-0-13-0-6, and Irat L3) to Pseudomonas solanacearum biovars I and III was evaluated in a greenhouse trial. Seedlings were root-inoculated with 11 strains of biovar I and nine strains of biovar III. The plants were individually scored 15 days after inoculation for wilt severity on a scale from 1 (no symptoms) to 5 (dead plant). Cluster analysis grouped strains of biovar III in a lower virulent cluster than those of biovar I. Yoshimatsu 4-11, Rotam-4, and Rodade had specific resistance to biovar III but not to biovar 1

Agrobacterium radiobacterstrains K84, K1026 and K84 Agr‐produce an antibiotic‐like substance, activein vitroagainstA. tumefaciensand phytopathogenicErwiniaandPseudomonasspp.

Agrobacterium radiobacter strains K84, K1026 and K84 Agr‐ produced in vitro an antibiotic‐like substance (ALS 84), different from agrocin 84 and observed in mannitol‐glutamate medium. Twenty five out of 39 A. tumefaciens strains of biovars 1, 2 and 3 were sensitive to ALS 84 regardless of their sensitivity to agrocin 84. Sensitivity in A. tumefaciens strain C58 was not encoded by the Ti‐plasmid. Most isolates tested of Erwinia carotovora subsp. carotovora E. carotovora subsp. atroseptica, Pseudomonas corrugata P. cichorii and unidentified isolates from galls were also sensitive to this substance. ALS 84 was not affected by the proteases studied, nor by treatment at 62°C for 30 min and had a bacteriostatic effect. The production of ALS 84 might play a role in the complex mechanism of biological control of crown gall, especially in strains resistant to agrocin 84 and sensitive to ALS 84, and by the creation of an ecological niche favourable to A. radiobacter strains K84, K1026 or K84 Agr‐.

Proposal for Rejection of Agrobacterium tumefaciens and Revised Descriptions for the Genus Agrobacterium and for Agrobacterium radiobacter and Agrobacterium rhizogenes

The 16S rRNA sequences of seven representative Agrobacterium strains, eight representative Rhizobium strains, and the type strains of Azorhizobium caulinodans and Bradyrhizobium japonicum were determined. These strains included the type strains of Agrobacterium tumefaciens, Agrobacterium rhizogenes, Agrobacterium radiobacter, Agrobacterium vitis, Agrobacterium rubi, Rhizobium fredii, Rhizobium galegae, Rhizobium huakuii, Rhizobium leguminosarum, Rhizobium loti, Rhizobium meliloti, and Rhizobium tropici. A phylogenetic analysis showed that the 15 strains of Agrobacterium and Rhizobium species formed a compact phylogenetic cluster clearly separated from the other members of the alpha subclass of the Proteobacteria. However, Agrobacterium species and Rhizobium species are phylogenetically entwined with one another, and the two genera cannot be separated. In the Agrobacterium species, the strains of biovar 1, biovar 2, Agrobacterium rubi, and Agrobacterium vitis were clearly separated. The two biovars exhibited homogeneity in their phenotypic, chemotaxonomic, and phylogenetic characteristics, and two species should be established for the two biovars. We considered the nomenclature of the two biovars, and revised descriptions of Agrobacterium radiobacter (for the biovar 1 strains) and Agrobacterium rhizogenes (for the biovar 2 strains) are proposed. The name Agrobacterium tumefaciens is rejected because the type strain of this species was assigned to Agrobacterium radiobacter, and consequently the description of the genus Agrobacterium is revised.

Genotypic typing and phylogenetic analysis of Salmonella paratyphi B and S. java with IS200.

Salmonella paratyphi B and Salmonella java are biovars of common serotype 1,4,[5],12:b:1,2 which respectively cause human paratyphoid fever and gastroenteritis. In order to define genotypes and phylogenetic relationships in this group, we examined representative strains for restriction fragment length polymorphisms (RFLPs) in and around the 16S ribosomal RNA (rrn) genes, and the five to eleven insertion sites of the Salmonella-specific DNA insertion sequence IS200. One of four 16S rrn profiles was predominant, and was shared by the majority of strains, irrespective of their designation as S. paratyphi B or S. java. On the other hand, thirteen unique IS200 profiles were found and this technique was able to distinguish, for the first time, distinct genotypes for S. paratyphi B and S. java. One of the S. paratyphi B profiles, Spj-IP1.0, represented a globally-distributed clone. Greater diversity was detected within IS200 profiles of S. java than within those of S. paratyphi B. IS200 profiles described a phylogenetic complex in which strains of both biovars could be placed. They constituted reproducible molecular fingerprints, which could be compared in a band-matching database suitable for molecular epidemiological typing.

Taxonomic Significance of the Lipopolysaccharide Composition of the Three Biovars of Agrobacterium tumefaciens

Summary The sugar and fatty acid composition of 9 strains belonging to the 3 biovars of Agrobacterium tumefaciens and to Agrobacterium rubi was investigated. All strains showed a complex composition of the sugar moieties which comprised mostly 6-deoxyhexoses, hexoses and acidic sugars, such as GalA, 3-deoxy-lyxo-heptulosaric acid and Kdo. More striking however, was the different pattern in DOC-PAGE indicating that biovar 3 strains (Agrobacterium vitis), as well as Agrobacterium rubi contained an R-type LPS (or an R-type with only a very truncated O-chain). In contrast, strains of biovars 1 and 2 showed very long O-chains comprising more than 20 O-repeating units. The fatty acid profiles of the lipid A moieties of the LPS of biovars 1, 2 and 3 were characterized by the predominance of hydroxylated fatty acids which comprised 2–5 different 3-OH-fatty acids, as well as (n-2) hydroxylated long chain fatty acids, such as 27-OH-28 : 0. Biovar 2 strains were characterized by a high content of the rare iso-branched 3-OH-15 : 0, which was lacking in the other biovars and may serve as a characteristic chemotaxonomic marker. The presence of the rare 3-OH-18 : 0 and 3-OH-18 : 1 in 7 out of 9, or 5 out of 9 strains, respectively, was conspicuous.

Molecular typing of Chlamydia trachomatis by random amplification of polymorphic DNA

The random amplification of polymorphic DNA (RAPD) was used for epidemiological typing of Chlamydia trachomatis strains. DNA samples from 39 C. trachomatis, 1 C. pneumoniae and 2 C. psittaci strains were screened by the use of 4 single 10-mer primers. Different and reproducible banding profiles were observed on agarose gel electrophoresis. No common profiles were recorded for strains from different Chlamydia species. All C. trachomatis strains of trachoma biovar were distinguished from lymphogranuloma venereum biovar. Moreover, serotypes A to C were separated from serotypes D to K, and some groups of strains sharing the same serotype D to K were further subdivided by RAPD. Conversely, strains of different serotypes could produce identical patterns of amplification, indicating that RAPD did not reflect serotyping. The patterns of amplified products were compared to the restriction fragment length polymorphism of the omp 1 gene after amplification and to DNA fingerprinting by use of ribosomal RNA or randomly cloned DNA probes. RAPD seemed to be an alternative molecular typing procedure for epidemiological study and strain identification in urogenital infections due to serotypes D to K.

Efficient Adsorption of Lysostaphin on Bacterial Cells of Lysostaphin‐Resistant Staphylococcus aureus Mutant

A simple and efficient method for the purification of staphylolytic endopeptidase (lysostaphin) contained in culture supernatant of Staphylococcus simulans biovar staphylolyticus strain by adsorption of the enzyme on bacterial cells of lysostaphin-resistant S. aureus mutant was successfully devised. Lysostaphin was sufficiently absorbed on the heat-killed mutant cells derived from S. aureus Cowan I and efficiently eluted by 3 M KSCN. Enzyme preparation obtained by a single procedure of the affinity purification was pure enough for practical use. The yield of the enzyme was 25 mg from 1 liter culture and recovery rate was 64%.

Survival of south african strains of biovar 2 and biovar 3 ofPseudomonas solanacearum in the roots and stems of weeds

South African strains of biovar 2 and biovar 3 ofPseudomonas solanacearum were compared for their ability to survive in the roots and stems of ten weed species. After artificial inoculation with a biovar 2 strain none of the weed species developed wilt symptoms, but the pathogen was isolated from one species,Physalis angulata L. Using inoculum of a biovar 3 strain, two of the weed species developed wilt symptoms (Datura stramonium L.,Solanum nigrum L.), and the pathogen was isolated from four species,Eragrostis curvula, Amaranthus hybridus L.,Datura stramonium L. andSolanum nigrum.

A rapid and sensitive method for the identification of Brucella species with a monoclonal antibody

A coagglutination test using monoclonal antibody BmE10-5 with specificity for the M antigen of Brucella melitensis 16M has been developed for the rapid identification of the smooth Brucella species. All reference strains of several biovars of B. melitensis, B. abortus, B. suis and B. neotomae tested were positive in this assay. No significant differences in reaction intensity were observed in relation to the different distribution of the A and M antigens among the Brucella serovars analysed. Conversely, rough Brucella species, with the exception of B. abortus 45/20, were negative in the assay. Among the different organisms tested not belonging to the genus Brucella, serovar 09 of Yersinia enterocolitica was the only one that gave a weak positive reaction of coagglutination. Thus, in view of its rapidity, simplicity, specificity and low costs, this technique could be highly useful for rapid identification of smooth Brucella strains in diagnostic laboratories.

Serogroups of Agrobacterium tumefaciens Biovar 3 Determined Using Somatic Antigens.

Serological specificity of Agrobacterium tumefaciens biovar 3 was tested by means of agglutination and Ouchterlony double diffusion tests, using rabbit antisera prepared by injecting heat-treated (100°C, 1hr) cell suspensions of 4 Japanese strains of G-Ag-19, 26, 27 and 60, and strain NCPPB 2562, a representative strain for biovar 3 isolated in Greece. In the agglutination test, 39 strains of biovar 3 except strain G-Ag-19 were separated into 4 distinct groups (A-D) according to the titers against 5 antisera. The same group-specific reactions were observed in the Ouchterlony double diffusion test as those in the agglutination test. Each of the biovar 3 strains except strain G-Ag-19 formed several group-specific precipitin bands only in combination with the antiserum prepared against the member of the same group. These results suggest that the biovar 3 strains used are serologically heterogeneous and can be separated into 4 serogroups (A-D), which differ from one another in the reactivity of somatic antigens (heat-stable antigens).