Blast colony forming cells in umbilical cord blood: frequency and correlation to other haemopoietic progenitors

Streptomycin-treated adult mice were investigated as a possible model for studying the enteropathogenicity of Aeromonas species. C57BL mice pre-treated with streptomycin (5.0 g/L drinking water, 48 hours) received a single intragastric dose (10(10) bacteria /10.5 mL) of one of six well-characterized, toxin-producing, human diarrhoeal isolates of A. veronii biovar sobria (n = 3) or A. hydrophila (n = 3). Their faeces were examined for Aeromonas for 10 days post-challenge. All strains colonized the antibiotic-treated mice. Colonization did not occur in mice which did not receive streptomycin. Strains of A. hydrophila were recovered in greater numbers than strains of A. veronii biovar sobria, and colonized ( > or = 10(3) cfu/g of faeces) a greater proportion of mice at day 10. Strains of the latter species, however, were more adherent in cell line assays used as models of intestinal adhesion. A. hydrophila strains localized in the large intestine and appeared not to be cell associated. This study, therefore, points to species-related differences in intestinal colonization mechanisms. The streptomycin-treated adult mouse model may prove useful for further investigation of some of these mechanisms. Diarrhoeal symptoms were, however, not produced in this model.     

Elevated levels of lipoprotein(a) in women with pre-eclampsia

Two unusual cases of Aeromonas infection are described, one associated with bacteremia (Aeromonas schubertii) and another in which the organism was recovered from an infected gall bladder (Aeromonas veronii biotype veronii). These strains were initially identified as Vibrio damsela and Vibrio cholerae by the Vitek and API 20E systems, respectively. Use of appropriate screening tests and familiarity with the newer Aeromonas species could prevent initial misidentifications and potential public health consequences.     

Antimicrobial susceptibility patterns of Aeromonas jandaei, A. schubertii, A. trota, and A. veronii biotype veronii

Fifty-six isolates of four Aeromonas species, which have been documented as causative agents of human infections or isolated from human clinical specimens, were subjected to antimicrobial susceptibility testing using a MicroScan WalkAway conventional (overnight incubation) gram-negative panel. The four species tested and the number of isolates of each were as follows: Aeromonas jandaei, 17; A. schubertii, 12; A. trota, 15; and A. veronii biotype veronii, 12. All isolates of A. trota were susceptible to all antimicrobial agents tested, except cefazolin (20% of isolates were resistant) and cefoxitin (13% of isolates were resistant). All isolates of A. schubertii and A. veronii biotype veronii, as well as 88% of A. jandaei isolates, were resistant to ampicillin. Resistance to ampicillin-sulbactam ranged from 25% of A. schubertii strains to 100% of A. veronii biotype veronii strains. Cefazolin resistance ranged from 17% of A. veronii biotype veronii isolates to 59% of A. jandaei isolates. Imipenem resistance was detected in 65% of A. jandaei strains and 67% of A. veronii biotype veronii strains. A. jandaei displayed resistance to piperacillin and ticarcillin in 53 and 71% of the isolates, respectively. A. veronii biotype veronii strains were 100% susceptible to piperacillin and 100% resistant to ticarcillin. These antibiogram data may be useful in establishing the identification of these four species when members of the genus Aeromonas are isolated from human clinical sources.     

Medicine, population and war

The colonization mechanisms of enteropathogenic Aeromonas strains are poorly characterized, but recent studies indicate that some filamentous structures are intestinal adhesins. This study describes the purification and characterization of a long, flexible pilus from a gastroenteritis-associated strain of Aeromonas veronii biovar sobria. SDS-PAGE analysis (various conditions) of pili preparations yielded a pilin protein band of approximately 21 kDa. Its N-terminal amino acid sequence was unambiguous and homologous with those of type IV pilins. Immunogold electron microscopy with rabbit antisera produced against this pilin protein (SFP) decorated single pili and rope-like bundles of pili on the bacterial surface. These were seen more frequently on strains grown at 22 degrees C compared with 37 degrees C and in liquid rather than on solid medium. SFP was not detected on any of 104 strains of Aeromonas (different species and sources) from our culture collection, although morphologically similar structures were seen on a number of these strains. This finding and differences among other published amino acid sequences show that Aeromonas type IV pili are antigenically diverse. Bundle-forming type IV 'class B' pili are important in the virulence of other enteropathogenic bacteria. The N-terminal amino acid sequence of the Aeromonas SFP, however, showed closer homology to the type IV 'class A' pilins. Studies are in progress to investigate the role of SFP in Aeromonas virulence.     

Molecular cloning of an Australian isolate of hepatitis C virus

A total of 208 strains of Aeromonas were isolated by monthly sampling from two estuaries (one provided with, and the other devoid of a waste-water treatment system) on the Italian coast of the Adriatic sea between September 1994 and August 1995. Biotyping at the species level allowed the identification of 96 strains (46%) as Aer. caviae, 46 (22%) as Aer. sobria, 33 (16%) as Aer. hydrophila and 25 (12%) as Aer. veronii. Eight strains (4%) were regarded as unnamed aeromonads. Aeromonas caviae was the most prevalent species in water with a high degree of pollution, while Aer. hydrophila strains were more commonly isolated from cleaner water. Aeromonas sobria and Aer. veronii were equally distributed in both estuaries. There was no correlation between temperature and numbers of aeromonads in either estuary. Using a biochemical fingerprinting method, strains were divided into similarity groups (PhP-types) based on their biochemical phenotypes. Several different PhP-types were found in each estuary, yielding a high diversity for these strains. However, some identical PhP-types were also found in both estuaries and at different times of the year, indicating that certain Aeromonas strains can survive more widely varying physico-chemical conditions. The production of toxins capable of causing cytoskeletal-dependent changes in the morphology of Chinese hamster ovary (CHO) cells was detected in 14 strains and appeared to be dependent on the season.     

Identification and molecular characterization of two tandemly located flagellin genes from Aeromonas salmonicida A449

Two tandemly located flagellin genes, flaA and flaB, with 79% nucleotide sequence identity were identified in Aeromonas salmonicida A449. The fla genes are conserved in typical and atypical strains of A. salmonicida, and they display significant divergence at the nucleotide level from the fla genes of the motile species Aeromonas hydrophila and Aeromonas veronii biotype sobria. flaA and flaB encode unprocessed flagellins with predicted Mrs of 32,351 and 32,056, respectively. When cloned under the control of the Ptac promoter, flaB was highly expressed when induced in Escherichia coli DH5alpha, and the FlaB protein was detectable even in the uninduced state. In flaA clones containing intact upstream sequence, FlaA was barely detectable when uninduced and poorly expressed on induction. The A. salmonicida flagellins are antigenically cross-reactive with the A. hydrophila TF7 flagellin(s) and evolutionarily closely related to the flagellins of Pseudomonas aeruginosa and Vibrio anguillarum. Electron microscopy showed that A. salmonicida A449 expresses unsheathed polar flagella at an extremely low frequency under normal laboratory growth conditions, suggesting the presence of a full complement of genes whose products are required to make flagella; e.g., immediately downstream of flaA and flaB are open reading frames encoding FlaG and FlaH homologs.     

Evolving concepts regarding the genus Aeromonas: an expanding Panorama of species, disease presentations, and unanswered questions

It has been almost 10 years since a major review on the association of Aeromonas with human disease has been published. During that period the number of valid species in the genus has grown to 14, with a new family (Aeromonadaceae) established to house this genus. Despite this explosion in the number of new genomospecies, only five (Aeromonas hydrophila, A. caviae, A. veronii, A. jandaei, and A. schubertii) are currently recognized as human pathogens. New syndromes attributed to this genus include hemolytic uremic syndrome, burn-associated sepsis, and a variety of respiratory tract infections, including epiglottitis. Convincing evidence suggests that some aeromonads do cause gastroenteritis, but it is presently unclear whether many of the strains isolated from feces are involved in diarrheal disease. Many questions regarding this genus remain unanswered.     

Nucleotide and amino acid sequences of the metallo-beta-lactamase, ImiS, from Aeromonas veronii bv. sobria

The Aeromonas veronii bv. sobria metallo-beta-lactamase gene, imiS, was cloned. The imiS open reading frame extends for 762 bp and encodes a protein of 254 amino acids with a secreted modified protein of 227 amino acids and a predicted pI of 8.1. To confirm the predicted sequence, purified ImiS was digested and the resulting peptides were identified, yielding an identical sequence for ImiS, with 98% identity to CphA. Both possessed the putative active-site sequence Asn-Tyr-His-Thr-Asp at positions 88 to 92, which is unique to the Aeromonas metallo-beta-lactamases.     

Aeromonas popoffii sp. nov., a mesophilic bacterium isolated from drinking water production plants and reservoirs

We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.     

The cellular fatty acid composition of bacteria in the family Vibrionaceae

It is shown that strains of Vibrio cholerae of serovar O1, biovar eltor, subtype Ogawa, museum strains V. cholerae of serovar O1 and NAG-vibrios (isolated from various sources: sea, river and sewage water, canal water and people) possess identical composition of cell fatty acids with prevailing hexadecenoic, hexadecanoic and octadecenoic acids. Being identical, fatty acid profiles of V. parahaemolyticus and V. alginolyticus, are close to that of V. cholerae differing from the latter mainly by the higher content of dodecanoic acid. Similarity of Aeromonas sp. and Vibrio strains in the fatty acid composition proves phylogenetic relation-ship of these bacteria. Fatty acid composition of Plesiomonas shigelloides cells characterized by the presence of methylenhexadecanoic acid as well as by similarity with Vibrio and Aeromonas by the content of most fatty acids confirms a supposition of R. R. Colwell on the intermediate status of genus Plesiomonas between the families Enterobacteriaceae and Vibrionaceae. Independent of the growth medium, the strains Vibrio. Aeromonas and Plesiomonas preserved a fatty-acid profile, inherent in them, with variations mainly in the content of fatty acids with the odd number of carbon atoms. Allowing for relative stability of fatty acid composition and its peculiarity in certain taxonomic groups of the studied bacteria, the above test may be used as additional objective criterion to identify the representatives of Vibrionaceae family.     

Differentiation of Brucella melitensis, B. ovis and B. suis biovar 2 strains by use of membrane protein- or cytoplasmic protein-specific gene probes

The possibility of differentiating Brucella species and biovars by Southern blot hybridization of agarose gel-electrophoresed HindIII-digested genomic DNA with membrane protein- or cytoplasmic protein-specific gene probes was investigated on 92 reference and field strains representative of all known species and biovars. Based on the RFLP pattern observed, three gene probes, i.e. br25, 39ugpa and omp16 coding for membrane or cytoplasmic proteins differentiated B. melitensis, B. ovis and B. suis biovar 2 strains from each other and from the other Brucella species and biovars. Thus, the use of these specific gene probes could contribute, in addition to previously identified species- or biovar-specific markers, to the molecular identification and typing of Brucella isolates.     

Typing of rhizobia by PCR DNA fingerprinting and PCR-restriction fragment length polymorphism analysis of chromosomal and symbiotic gene regions: application to Rhizobium leguminosarum and its different biovars

Characterization of 43 strains of Rhizobium leguminosarum biovars viciae, trifolii, and phaseoli was performed by two methodologies based on PCR amplification, i.e., PCR DNA fingerprinting of interrepeat sequences and restriction fragment length polymorphism (RFLP) analysis of PCR -amplified chromosomal and symbiotic gene regions. Groupings generated by PCR DNA fingerprinting with either extragenic palindromic repetitive primers or two different single random primers were correlated with similar levels of resolution. Although less discriminating, PCR-RFLP analysis of intergenic spacer between genes coding for 16S and 23S rRNA (16S and 23S rDNA) yielded intraspecific polymorphisms. The classification of strains was independent of the biovar status and was in agreement with those obtained by PCR DNA fingerprinting. Intrabiovar variation within symbiotic gene regions was detected by PCR-RFLP analysis of nifDK and nodD gene regions, but the strains were grouped according to the biovar. The rDNA intergenic spacer and nif primers were verified to be universal for rhizobial species by testing of various reference strains, whereas the nod primers designed in this study were biovar or species specific for R. leguminosarum and Rhizobium etli. Classifications of R. leguminosarum strains by the PCR-based methods were correlated with those previously obtained by conventional total DNA restriction profile comparisons and RFLP analysis using chromosomal and symbiotic gene probes. Ranges of discriminating powers were also equivalent between the two approaches. However, the PCR-based methods are much less time-consuming and are therefore more convenient.     

Identification of species of Brucella using Fourier transform infrared spectroscopy

Fourier transform infrared spectroscopy (FTIR) is a technique that has been used over the years in chemical analysis for the identification of substances and is one that may be applied to the characterisation of microorganisms. The marked tendency of Brucella towards variation in the smooth rough phase, together with the laboriousness and risk involved in the methods used in their identification, make their classification difficult. We studied the type strains of the different species and biovars of Brucella and 11 isolates of human origin of Brucella melitensis, six corresponding to biovar 1, one to biovar 2 and five to biovar 3. The results of linear discriminant analysis performed using the data provide an above 95% likelihood of correct classification, over half of which are in fact above 99% for the vast majority of Brucella strains. Only one case of B. melitensis biovar 1 has been incorrectly classified. The rest of the microorganisms studied (Staphylococcus aureus, Strteptococcus pyogenes, Enterococcus faecalis, Corynebacterium pseudodiphtheriticum, Clostridium perfringens, Escherichia coli, Acinetobacter calcoaceticus and Pseudomonas aeruginosa) have been classified correctly in all cases to a likelihood of over 80%. In the graphic representation of the analysis, a grouping of these can be seen in clusters, which include the different species. One of these comprises B. melitensis, another Brucella abortus, and another wider one is made up of Brucella suis. The Brucella canis, Brucella ovis and Brucella neotomae strains appear separate from the previously described groups.     

MUMPS--an economical and efficient time-sharing system for information management

A total of 203 characters has been determined for 68 strains of Aeromonas belonging to the Aeromonas hydrophila-A. punctata group. The results have been subjected to computer analysis using the coefficient of Jaccard-Sneath and the strains clustered by the method of aggregation according to the variance. The 68 strains can be divided into two well-segregated classes on the basis of 59 variable characters, of which seven are of diagnostic value. The two classes are considered as two separate species. The first one (42 strains) is assigned to the type species of the genus, A. hydrophila, and it appears that the species name, A. punctata, is an illegitimate synonym for A. hydrophila. The second (26 strains) constitutes a new species for which the name A. sobria sp. nov. is proposed. The type strain of this new species has been deposited under the reference CIP7433 (our strain 208).     

Plaque formation by and plaque cloning of Chlamydia trachomatis biovar trachoma

A new technique for the induction of plaque formation by Chlamydia trachomatis biovar trachoma applicable to the titration of infectivity and cloning of biovar trachoma was established. Three novel strains were cloned and confirmed to be free of glycogen inclusions. The lack of glycogen accumulation correlated with the absence of a 7.5-kb plasmid, which is highly conserved in other strains of C. trachomatis. Although the growth efficiency of these plasmid-free strains was slightly lower than that of plasmid-positive strains, possession of the plasmid and glycogen accumulation were not essential for the survival of C. trachomatis.     

Studies on the bacterial flora of fish which are potential pathogens for human. Virulence factors of potential human pathogen isolated

Tests for various virulence factors, such as production of haemolysin on sheep blood agar plate, cytotoxin on HeLa cell line and enterotoxin in GM-1 ELISA and suckling mouse assay model, were done among the various strains of Aeromonas spp., Vibrio spp., Plesiomonas shigelloides and Esch. coli isolated from fresh water fish samples. Invasive properties of the isolates were also seen by using Sereny test. Haemolysin production was observed in 85.7% of Aeromonas, all (100%) of Vibrios, 13.3% of Esch. coli and none (0%) of P. shigelloides strains. Cytotoxin production was demonstrated in 60.8% of Aeromonas, 38.4% of Vibrios and none (0%) of P. shigelloides and Esch. coli strains. About 8% of Vibrio spp., were found positive for LT in GM-1 ELISA method whereas, none of the Aeromonas spp., Plesiomonas and Esch. coli. strains were found positive for LT and ST in GM-1 ELISA. By suckling mouse assay model 43.4% strains of Aeromonas were found positive for enterotoxin production whereas, strains of Vibrio spp., Plesiomonas and Esch. coli yielded negative results. Sereny test for invasive property was found negative in all the strains tested. The isolates from fish possess various virulence factors which contributes for pathogenicity in order to cause various diseases to susceptible individual.     

Assessment of apparent vena caval penetration by the Greenfield filter

Bacteria belonging to the family Vibrionaceae were suspended using saline and a solution prepared from a marine-cations supplement. The effect of this on the profile of oxidized substrates obtained when using Biolog GN MicroPlates was investigated. Thirty-nine species belonging to the genera Aeromonas, Listonella, Photobacterium, and Vibrio were studied. Of the strains studied, species of Listonella, Photobacterium, and Vibrio could be expected to benefit from a marine-cations supplement that contained Na+, K+, and Mg2+. Bacteria that are not of marine origin are usually suspended in normal saline. Of the 39 species examined, 9 were not included in the Biolog data base and were not identified. Of the 30 remaining species, 50% were identified correctly using either of the suspending solutions. A further 20% were correctly identified only when suspended in saline. Three species, or 10%, were correctly identified only after suspension in the marine-cations supplemented solution. The remaining 20% of species were not correctly identified by either method. Generally, more substrates were oxidized when the bacteria had been suspended in the more complex salts solution. Usually, when identifications were incorrect, the use of the marine-cations supplemented suspending solution had resulted in many more substrates being oxidized. Based on these results, it would be preferable to use saline to suspend the cells when using Biolog for identification of species of Vibrionaceae. A salts solution containing a marine-cations supplement would be preferable for environmental studies where the objective is to determine profiles of substrates that the bacteria have the potential to oxidize. If identifications are done using marine-cations supplemented suspending solution, it would be advisable to include reference cultures to determine the effect of the supplement. Of the Vibrio and Listonella species associated with human clinical specimens, 8 out of the 11 studied were identified correctly when either of the suspending solutions was used.     

A simple and inexpensive method for the identification of Staphylococcus epidermidis and Staphylococcus hominis

Eight hundred and ninety-two strains of Staphylococcus species were identified by means of desferrioxamine susceptibility and fermentation results of three carbohydrates, with the API Staph system (bioMérieux, France) as reference method. No identification could be obtained for 34 strains with API Staph. Of the remaining 858 strains, identical identification was obtained with 842 (98.1%). All 707 strains identified as Staphylococcus epidermidis or Staphylococcus hominis by the API Staph system were found to be desferrioxamine susceptible, and all but 5 (3.3%) of 151 strains identified as other staphylococcal species were found to be resistant, yielding an identification correlation of 99.4% for desferrioxamine. The five additional strains which were susceptible to desferrioxamine were identified as Staphylococcus capitis (2 strains), Staphylococcus lugdunensis (2 strains), and Staphylococcus warneri (1 strain) by API Staph, and as Staphylococcus epidermidis (1 strain), Staphylococcus hominis (3 strains), and one other staphylococcal species by the experimental system.     

Sequence homology between 4qter and 10qter loci facilitates the instability of subtelomeric KpnI repeat units implicated in facioscapulohumeral muscular dystrophy

An analysis of 13 immunoglobulin A1 (IgA1) protease genes (iga) of strains of Streptococcus pneumoniae, Streptococcus oralis, Streptococcus mitis, and Streptococcus sanguis was carried out to obtain information on the structure, polymorphism, and phylogeny of this specific protease, which enables bacteria to evade functions of the predominant Ig isotype on mucosal surfaces. The analysis included cloning and sequencing of iga genes from S. oralis and S. mitis biovar 1, sequencing of an additional seven iga genes from S. sanguis biovars 1 through 4, and restriction fragment length polymorphism (RFLP) analyses of iga genes of another 10 strains of S. mitis biovar 1 and 6 strains of S. oralis. All 13 genes sequenced had the potential of encoding proteins with molecular masses of approximately 200 kDa containing the sequence motif HEMTH and an E residue 20 amino acids downstream, which are characteristic of Zn metalloproteinases. In addition, all had a typical gram-positive cell wall anchor motif, LPNTG, which, in contrast to such motifs in other known streptococcal and staphylococcal proteins, was located in their N-terminal parts. Repeat structures showing variation in number and sequence were present in all strains and may be of relevance to the immunogenicities of the enzymes. Protease activities in cultures of the streptococcal strains were associated with species of different molecular masses ranging from 130 to 200 kDa, suggesting posttranslational processing possibly as a result of autoproteolysis at post-proline peptide bonds in the N-terminal parts of the molecules. Comparison of deduced amino acid sequences revealed a 94% similarity between S. oralis and S. mitis IgA1 proteases and a 75 to 79% similarity between IgA1 proteases of these species and those of S. pneumoniae and S. sanguis, respectively. Combined with the results of RFLP analyses using different iga gene fragments as probes, the results of nucleotide sequence comparisons provide evidence of horizontal transfer of iga gene sequences among individual strains of S. sanguis as well as among S. mitis and the two species S. pneumoniae and S. oralis. While iga genes of S. sanguis and S. oralis were highly homogeneous, the genes of S. pneumoniae and S. mitis showed extensive polymorphism reflected in different degrees of antigenic diversity.     

Multipoint identification of Enterobacteriaceae: report of the British Society for Microbial Technology collaborative study

AIMS: To evaluate the accuracy and reproducibility of multipoint identification schemes in a multicentre trial. METHODS: Forty two strains of Enterobacteriaceae were distributed to 22 laboratories for identification by routine multipoint methods. Analysis of results enabled inter- and intralaboratory reproducibility of a variety of tests, and the ability of laboratories to identify individual organisms to be determined. RESULTS: Interlaboratory reproducibility of most of the biochemical tests was acceptable. The least reproducible tests, both within and between laboratories, were citrate utilisation, production of urease and beta galactosidase, detection of motility, and decarboxylation of lysine and ornithine. Inconsistent results for these tests were often associated with misidentified strains. Most laboratories performed identifications satisfactorily. Most isolates (72.1%) were identified correctly to species level; 9.6% were incorrectly identified, and 6.4% could not be identified at all. The most difficult organisms to identify were Citrobacter freundii, Enterobacter cloacae, Hafnia alvei and Aeromonas hydrophila. Strains of Enterobacter, Serratia sp, and Providencia sp were difficult to speciate. Several laboratories could not identify organisms exhibiting at least one atypical biochemical reaction. CONCLUSION: This study emphasises the need for quality control of media and reagents for multipoint identification of Gram negative enteric bacilli.