Comparative study of Agrobacterium biotypes 1, 2 and 3 by electrophoresis and serological methods

A comparative study by electrophoresis and serology of strains representing the three Agrobacterium biotypes was carried out. Thirteen Spanish isolates and strains from international collections were included. Ten antisera were prepared by using strains from the three biotypes and different types of antigens. The strains were studied by immunodiffusion, indirect immunofluorescence and indirect ELISA. Serological relationship among all the strains was observed, although serological heterogeneity within each of the biotypes occurred. Biotype 3 appears as serologically related to biotypes 1 and 2, having an intermediate position. This observation is in agreement with their biochemical characteristics. Electrophoretic analysis of the three biotypes showed that there was high variability. Three main bands appeared in the six strains studied. One specific band occurred in the biotype 1 strains and another in the biotype 3 strains.     

Taxonomic position of Klebsiella atlantae and Klebsiella edwardsii.

The taxonomic position of two species of the genus Klebsiella (K. atlantae and K. edwardsii) is being introduced. 211 strains of different origin were studied: 80 strains of K. pneumoniae, 46 strains of K. oxytoca, 50 strains of K. atlantae and 35 strains of K. edwardsii. Sixty biochemical characteristics were determined 40 of these pertaining to carbohydrate metabolism. It was discussed whether both are to be considered species or biotypes of another Klebsiella species, however, by determining citrate as carbon source, by MR test and by tests on malonate, gluconate, methyl-xyloside, 1 (--) sorbose, inulin, amylose, methyl-d-mannoside, glycogen, melezitose, VP test, amygdalin, d-tartrate and gas from glucose, we arrived at the conclusion that both could be considered species of the genus. These conclusions were confirmed by the determination of biotypes of both (according to C. Richard). Later, we expect to study their participation in human infective processes and their sensitivity by antibiotic therapy.     

Characterization of Ornithine Decarboxylase-Positive, Nonmotile Strains of the Klebsiella-Enterobacter Group

Thirty-seven strains of ornithine decarboxylase-positive, nonmotile Klebsiella-Enterobacter organisms isolated from 36 patients were studied by biochemical and serological testing. Five strains gave biochemical reactions which conformed closely to those of Escherichia coli; three strains gave positive Quellung reactions to specific Klebsiella antisera. (Two of these were thought to be Enterobacter in spite of this typing reaction.) The remaining 29 strains were classified as Enterobacter. These results demonstrate the necessity of doing both an ornithine decarboxylase test and a motility test to differentiate Klebsiella from Enterobacter. Had only a motility test been done, they all would have been called Klebsiella.     

Biochemical typing as a method of differentiating group B streptococcus]

In epidemiological studies on the group B streptococcus the serological typing is used. The paper present the results of a study on usefulness of biochemical typing for differentiation of the group B streptococcus. For that purpose, 210 strains descended from colonized infants and pregnant women were put to typing with both of mentioned methods. We showed that each of the method distinguishes similar number of biotypes and serotypes. However, ought to be marked that significant number of strains (93.8%) belonged to the three out of eight biochemical types. Similar results were achieved in serological typing, three of the most numerous serotypes contained 81.4% strains. Analysis of the relationship between serological and biochemical types did not reveal statistical association because the strains belonged to various serotypes. Obtained results show that both methods of typing--biochemical and serological, have similar value in differentiation of the strains. The method of biochemical typing is quite simple and can be used in laboratory conditions.     

Deoxyribonucleic acid reassociation in the classification of coagulase-positive staphylococci

DNA-DNA reassociation studies were performed with coagulase-positive staphylococci belonging to the biotypes A, B, C, D, E and F. These studies present genetic evidence for the existence of at least two distinct species within this group of organisms. The common Staphylococcus aureus strains were represented by organisms from biotypes A to D, and their DNA revealed over 80% nucleotide sequence homology under restrictive conditions. Less than 15% DNA homology was detected between strains from biotypes A to D (S. aureus) and those from biotypes E and F. The DNA of organisms from either the biotypes E or F displayed over 70% homology. Together, both biotypes are considered to represent the species S. intermedius. However, DNA homology values dropped to 50–65% between strains from different biotypes. This may justify the separation of S. intermedius biotypes E and F on a subspecies level.Abbreviations O.D. optical density - SSC standard saline citrate buffer (0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) This work was supported by Deutsche Forschungsgemeinschaft     

Identification by 16S rDNA fragment amplification and determination of genetic diversity by random amplified polymorphic DNA analysis of Pasteurella pneumotropica isolated from laboratory rodents

Pasteurella pneumotropica is an opportunistic bacterium frequently isolated from colonies of various laboratory rodents. Identification of this species, including its differentiation into two distinct biotypes (Jawetz and Heyl), is usually based on the use of conventional bacteriologic methods. In this study, a 16S rDNA fragment amplification procedure was developed for use as an alternative method for identification and differentiation of P. pneumotropica. Polymerase chain reaction (PCR) products were two distinctive fragments of 937 and 564 bp specific for biotypes Jawetz and Heyl, respectively. Specificity of PCR products could be achieved by EcoRI cleavage, leading to 596 plus 341-bp and 346 plus 218-bp fragments for each of the amplification products. Use of this procedure confirmed identification of 34 field isolates and allowed definitive identification of some strains that could not have been done by use of bacteriologic examinations. Field isolates subjected to random amplified polymorphic DNA (RAPD) analysis had high genetic diversity among biotype Jawetz strains in contrast to biotype Heyl strains. In conclusion, RAPD could represent an additional means for identification of ambiguous strains of biotype Heyl and a valuable epidemiologic tool for identification of biotype Jawetz strains of P. pneumotropica.     

Use of the 16S-23S ribosomal genes spacer region for the molecular typing of sphingomonads

The ability of sphingomonads in drinking water to cause community- and hospital-acquired opportunistic infections has raised the need to establish reproducible identification assays. In this study, a total of 129 isolates recovered from drinking water with yellow- to orange-pigmented colonies were distributed among 10 biotypes on the basis of colony morphology. Polymorphisms, based on the amplification and restriction digestion of the intergenic transcribed spacer (ITS) region within the 10 assigned biotypes and 18 ATCC reference strains, were used to investigate the ability of this approach to differentiate closely related sphingomonads. ITS size, which ranged between 400 and 1100 bp, did not vary enough among the different genera. However, 16 distinct banding patterns within the ATCC reference strains and 9 within the 10 biotypes were obtained through ITS restriction digestion, and the majority of the tested biotypes produced patterns similar to those generated by the ATCC strains. To our knowledge, this study is not only the first comprehensive record of the size of the ITS region in sphingomonads, it is also the first study that describes the use of ITS restriction digestion to subtype those isolates.     

Identification of species and capsular types of Klebsiella clinical isolates, with special reference to Klebsiella planticola

In the 77 reference strains for Klebsiella K types, there are 17 strains (22.1%) of Klebsiella planticola, 6 strains (7.8%) of Klebsiella oxytoca, 1 strain (1.3%) of Klebsiella terrigena, and 53 strains (68.8%) of Klebsiella pneumoniae. The species K. planticola, which was originally isolated from botanical and aquatic environments and hence thus named, was also identified at high incidence (81 strains, 18.5%) among the 439 recent clinical isolates of Klebsiella species. Among these K. planticola strains of hospital origin, 52 (64%) were isolated from sputum, 17 (21%) from urine, and the remaining 12 (15%) from other sources. The capsular types of these isolates were determined by the gel precipitation reaction. Seventy of 81 K. planticola isolates (86.4%) were typable by antisera to Klebsiella reference strains for K types and the K types of the clinical isolates distributed to 35 kinds of K types. The proportion of typable strains among clinical isolates of K. planticola was very similar to those in K. pneumoniae (87.5%) and K. oxytoca (86.0%).     

Three immunity types of klebicins which use the cloacin DF13 receptor of Klebsiella pneumoniae

We have investigated a group of bacteriocinogenic strains used in the epidemiological investigation of Klebsiella infections. Transfer of plasmids from these strains to laboratory strains allowed the identification of three klebicins which use the cloacin DF13 receptor in Klebsiella, but are of three distinct immunity types. These klebicins use the ferric-aerobactin receptor determined by ColV-K30 in Escherichia coli, which is also used by cloacin DF13. We propose to call them group A klebicins, of immunity types A1, A2 and A3. On the basis of immunity, cloacin DF13 also belongs to the klebicin A1 group.     

Biotyping and resistotyping for epidemiological tracing of the fecal origin of Klebsiella pneumoniae in urinary infections

Twenty-four (82.7%) out of 29 patients suffering from hospital acquired urinary infections by Klebsiella pneumoniae had the same species in their faeces. Biotyping of 24 urinary and 219 fecal strains of K. pneumoniae resulted in 50 different biotypes - an average of four biotypes per fecal sample. Ten patients (34.4%) had the same biotype in urine and faeces without any correlation with previous vesical catheterization (p greater than 0.05). Using resistotyping to four chemical compounds selected among 34 tested substances (brilliant green, malachite green, potassium tellurite and mercuric chloride) 16 different resistotypes were found. Fourteen patients (58.3%) presented the same resistotype in urine and faeces but only in five patients was there correlation with simultaneous biotyping identity. Simultaneous occurrence of identical biotypes or resistotypes in faeces and urine occurred in only 54.2% of cases. However, there was a significant association between resistance ot mercuric and tellurite ions in fecal and urinary strains isolated from the same patient (p less than 0.001).     

An electron microscopic study of surface polysaccharides in Bacteroides

The electron microscopic appearance of the cell surface of Bacteroides strains and Klebsiella pneumoniae stained with ruthenium red or colloidal iron is described. The effect of polymyxin B (PMB) was also registered. It was found that all Bacteroides strains have a polysaccharide lined 'micro-capsule' external to the outer membrane which could aggregate and form blebs. The blebs so formed were distinct from other types of bleb formed in Klebsiella involving the outer membrane and induced by PMB. Such types of PMB alterations were not induced in Bacteroides.     

Characterization by numerical taxonomy and ribotyping of Vibrio splendidus biovar I and Vibrio scophthalmi strains associated with turbot cultures

Twelve Vibrio strains were examined phenotypically in 91 biochemical characters and genotypically by ribotyping. Ten were isolated from sea water and two from diseased turbot (Scophthalmus maximus). All isolates originated from one experimental system located in Ría de Vigo (Galicia, north-west Spain). Different type strains were used for comparative purposes. The taxonomic position was analysed with the NTSYST-pc and similarities among strains were calculated by the Simple Matching coefficient (SSM). rRNA gene restriction patterns were performed with the HindIII enzyme. The SSM coefficient separated the 12 Vibrio strains into two groups which included strains that showed a SSM coefficient quite similar to V. splendidus biovar 1 (ATCC 33125) and V. scophthalmi (CECT 4638). None of 91 phenotypical characters were specific in distinguishing both species. The ribotyping confirmed the taxonomic classification of strains. The pathogenicity of each strain was evaluated; 10 environmental strains were avirulent and two, isolated from diseased turbot, were virulent. Different biotypes and ribotypes were found among the avirulent isolates. This work showed ribotyping to be a valuable tool for identification and confirmed the necessity of extending the ribotype database within closely related Vibrio species in order to clarify the taxonomic position.     

Discrimination of Staphylococcus aureus biotypes by pulsed-field gel electrophoresis of DNA macro-restriction fragments

AIMS: To examine whether pulsed-field gel electrophoresis (PFGE) of DNA macro-restriction fragments could provide better discrimination among the different biotypes previously described within the species Staphylococcus aureus than the traditional biochemical approach. METHODS AND RESULTS: Seventy three Staph. aureus strains from various sources (human, animal or food origin) and belonging to eight biotypes, including the poultry-like biotype, tentatively designated as an 'abattoir' biotype, were genotyped by PFGE after SmaI digestion of DNA. The PFGE patterns were compared using the average linkage matching method (UPGMA) with the Dice coefficient. A total of 61 PFGE patterns were observed, showing between 31 and 100% similarity. In most cases, strains with the same biotype were grouped specifically into one, two or three separate sub-clusters. Strains from the 'abattoir' biotype were clustered in one separate sub-cluster. CONCLUSIONS: The PFGE typing is useful to distinguish the traditional biotypes of Staph. aureus and has a more discriminatory power than the biochemical typing. SIGNIFICANCE AND IMPACT OF THE STUDY: The PFGE typing confirms the 'abattoir' biotype as a separate group on a genetic level and is well suited to investigate modes of staphylococcal contamination of food.     

Differentiation of Salmonella enterica serotype gallinarum biotype pullorum from biotype gallinarum by analysis of phase 1 flagellin C gene (fliC)

Salmonella enterica serotype gallinarum biotype gallinarum and biotype pullorum are non-motile and pathogenic avian strains. Biotype gallinarum causes fowl typhoid and biotype pullorum is the cause of pullorum disease in chickens. The two biotypes could be differentiated based on biochemical characteristics. However, conventional culture and biochemical assays are time-consuming, laborious and need sterile laboratory practices. Although the two biotypes, gallinarum and pullorum are non-motile, they possess the phase 1 flagellin C gene. The variable regions of the flagellin C gene from 41 biotype pullorum and 52 biotype gallinarum were amplified by colony-PCR and analyzed by single strand conformational polymorphism (SSCP) method. Differences in SSCP electrophoretic patterns were confirmed by nucleotide sequencing. In addition, PCR-RFLP with Hinp1I was also successfully applied to differentiate the two biotypes. These results suggested that the variable regions of fliC could be used as a genetic marker to differentiate biotype gallinarum from biotype pullorum.     

Phenotypic Characterization and Deoxyribonucleic Acid Homologies of Pseudomonas solanacearum

Twenty-six strains and colony variants of Pseudomonas solanacearum belonging to four described biotypes were characterized, by using 169 phenotypic characters previously found useful in distinguishing among strains of other Pseudomonas species. Deoxyribonucleic acid (DNA) hybridization (intra- and interspecific DNA-DNA hybridizations) was performed by using the in vitro "DNA competition" technique. P. solanacearum appears to be a moderately homogeneous species, which is, at most, only remotely related to all other species of the genus studied to date. The four biotypes are not clearly distinct from one another with respect to nutritional characters or DNA homologies. Discrepancies between acid production and growth with some carbohydrates were noted. Difficulties were encountered in certain DNA competition experiments and some problems of the methodology are discussed.     

流感嗜血杆菌的分型研究

目的分析同济医院分离的流感嗜血杆菌的生物学分型及荚膜基因分型,了解本地区分离的流感嗜血杆菌的主要流行株。方法2012年1月1日至2012年12月31日从华中科技大学同济医学院附属同济医院分离流感嗜血杆菌100株。根据脲酶、吲哚和鸟氨酸脱羧酶试验对流感嗜血杆菌进行传统的生物学分型,分为Ⅰ～Ⅷ八个生物型。回顾患者病史资料,分析生物学分型和流感嗜血杆菌所引起的疾病之间的关系。用流感嗜血杆菌荚膜编码基因（bexA）和a—f型特异性荚膜基因设计引物,采用PCR技术对流感嗜血杆菌进行荚膜基因检测。通过生物学分型和荚膜基因分型结果的比对,探讨两者之间的关联。结果分离的100株流感嗜血杆菌生物学分型结果如下：Ⅲ型6株,Ⅳ型28株,Ⅴ型1株,Ⅵ型54株,Ⅶ型11株。未分离到Ⅰ型、Ⅱ型和Ⅷ型。分析患者的临床诊断,发现主要流行株Ⅵ型流感嗜血杆菌主要引起患者肺炎（包括支气管肺炎和新生儿肺炎）和支气管炎（包括毛细支气管炎和喘息性支气管炎）。荚膜基因分型结果显示,未分离到b型和b-型流感嗜血杆菌。共分离到1株f型,其余99株均为无荚膜抗原的不可分型流感嗜血杆菌。生物学分型和荚膜分型之间无明显的相关性。结论该院分离的流感嗜血杆菌主要为生物型Ⅵ型。回顾患者病史,发现Ⅵ型主要引起肺炎和支气管炎。荚膜基因分型显示,本地区分离的流感嗜血杆菌主要为不可分型流感嗜血杆菌。生物学分型和荚膜基因分型之间无明显相关性。     

A taxonomic study of clinical isolates of Pseudomonas pickettii, 'P. thomasii' and 'group IVd' bacteria

On the basis of cluster analysis, average similarities within and between groups, and DNA base composition of selected strains, Pseudomonas pickettii appeared to be a distinct species comprising several biotypes. Although some or all of these biotypes, represented by subclusters in our computer study, may ultimately warrant recognition as separate species, our results were not conclusive enough to warrant such a proposal at present. Most strains tentatively named 'P. thomasii' could be included in P. pickettii so that the name P. pickettii, which was validly published whilst 'P. thomasii' was not, takes priority over 'P. thomasii'. Strains of Group Va (Tatum et al., 1974) examined were also included in P. pickettii. Group IVd (King, 1964) did not appear to be a natural group but some of the strains could also be included in P. pickettii.     

Phenotypic characterization ofBeneckea anguillara biotypes I and II

Twenty-five strains of marine bacteria pathogenic for fish, which had been shown by means of in vitro DNA/DNA hybridization to comprise two biotypes of the speciesBeneckea anguillara, were subjected to a phenotypic characterization. A numerical analysis of the data segregated the strains into two phenotypically distinct clusters coincident with the biotypes established by DNA homology studies. The two biotypes were distinguishable fromPhotobacterium and other species ofBeneckea by a number of unrelated phenotypic properties.     

Utilization of D-xylose by wild-type strains of Salmonella typhimurium.

Enzyme studies of strains of Salmonella typhimurium representing biotypes that utilized D-xylose rapidly (xylose strong) or slowly (xylose weak) showed that they were different in the utilization of D-xylose because the xylose-weak strains were deficient in the transport of D-xylose. This observation is consistent with the idea that strains of the different xylose-weak biotypes, e.g. biotypes 17 to 32, were descended from strains of xylose-strong types, particularly from biotype 1.     

Species and biotype identification ofSerratia strains associated with insects

Forty-eightSerratia strains associated with insects were, identified to species level and biotyped according to recent taxonomic schemes. Each strain was submitted to 36 biochemical tests, including 23 carbon source utilization tests. Twenty-eight strains were assigned to eight biotypes ofSerratia marcescens: A1a, A2a, and A6a (pigmented biotypes: 18 strains); and A3a, A3b, A4a, A5, and TCT (nonpigmented biotypes: 10 strains). However, biotypes A8a, A8b, and A8c, which are frequently involved in nosocomial infections, were not found in insects. Ninetten strains were identified asS. liquefaciens (S. proteamaculans) biotypes C1a (12 strains), C1c (4 strains), C1d (2 strains), and one atypicalS. liquefaciens strain. Only one strain was identified asS. marinorubra (a nonchitinolytic species). The recent emergence ofSerratia in human pathology calls for a reevaluation of the idea of usingSerratia to biologically control insects.