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PubMed2000

16S ribosomal DNA sequence analysis of a large collection of environmental and clinical unidentifiable bacterial isolates.

Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail. However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. In this study, we evaluated the utility of 16S rDNA sequencing as a means to identify a collection of 177 such isolates obtained from environmental, veterinary, and clinical sources. For 159 isolates (89.8%) there was at least one sequence in GenBank that yielded a similarity score of > or =97%, and for 139 isolates (78.5%) there was at least one sequence in GenBank that yielded a similarity score of > or =99%. These similarity score values were used to defined identification at the genus and species levels, respectively. For isolates identified to the species level, conventional identification failed to produce accurate results because of inappropriate biochemical profile determination in 76 isolates (58.7%), Gram staining in 16 isolates (11.6%), oxidase and catalase activity determination in 5 isolates (3.6%) and growth requirement determination in 2 isolates (1.5%). Eighteen isolates (10.2%) remained unidentifiable by 16S rDNA sequence analysis but were probably prototype isolates of new species. These isolates originated mainly from environmental sources (P = 0.07). The 16S rDNA approach failed to identify Enterobacter and Pantoea isolates to the species level (P = 0.04; odds ratio = 0.32 [95% confidence interval, 0.10 to 1.14]). Elsewhere, the usefulness of 16S rDNA sequencing was compromised by the presence of 16S rDNA sequences with >1% undetermined positions in the databases. Unlike phenotypic identification, which can be modified by the variability of expression of characters, 16S rDNA sequencing provides unambiguous data even for rare isolates, which are reproducible in and between laboratories. The increase in accurate new 16S rDNA sequences and the development of alternative genes for molecular identification of certain taxa should further improve the usefulness of molecular identification of bacteria.

Animals↗
PubMed1990

Biochemical identification of Aeromonas genospecies isolated from humans.

One hundred phenotypic characteristics were determined for 138 clinical and environmental Aeromonas strains. Cluster analysis revealed three major phenons equivalent to the A. hydrophila, A. caviae, and A. sobria groups, each of which contained more than one genospecies and more than one named species. An excellent correlation was found between phenotypic identification and classification based on DNA relatedness. DNA hybridization groups within each of the phenotypic groups were also separable by using a few biochemical characteristics. Key tests were production of acid from or growth on D-sorbitol (which separated DNA hybridization group 3 from groups 1 and 2 within the A. hydrophila phenogroup), growth on citrate (which essentially separated DNA hybridization group 4 from groups 5A and 5B within the A. caviae phenogroup), and growth on DL-lactate (which separated DNA hybridization group 1 from groups 2 and 3 within the A. hydrophila phenogroup as well as group 5A from groups 4 and 5B within the A. caviae phenogroup). All except one strain in the A. sobria phenogroup belonged to DNA hybridization group 8. DNA hybridization groups were not equally distributed among clinical and environmental isolates, suggesting that strains of certain DNA hybridization groups might be less virulent than others.

Aeromonas↗
PubMed2003

Prospective study of the performance of vibrational spectroscopies for rapid identification of bacterial and fungal pathogens recovered from blood cultures.

Rapid identification of microbial pathogens reduces infection-related morbidity and mortality of hospitalized patients. Raman spectra and Fourier transform infrared (IR) spectra constitute highly specific spectroscopic fingerprints of microorganisms by which they can be identified. Little biomass is required, so that spectra of microcolonies can be obtained. A prospective clinical study was carried out in which the causative pathogens of bloodstream infections in hospitalized patients were identified. Reference libraries of Raman and IR spectra of bacterial and yeast pathogens highly prevalent in bloodstream infections were created. They were used to develop identification models based on linear discriminant analysis and artificial neural networks. These models were tested by carrying out vibrational spectroscopic identification in parallel with routine diagnostic phenotypic identification. Whereas routine identification has a typical turnaround time of 1 to 2 days, Raman and IR spectra of microcolonies were collected 6 to 8 h after microbial growth was detected by an automated blood culture system. One hundred fifteen samples were analyzed by Raman spectroscopy, of which 109 contained bacteria and 6 contained yeasts. One hundred twenty-one samples were analyzed by IR spectroscopy. Of these, 114 yielded bacteria and 7 were positive for yeasts. High identification accuracy was achieved in both the Raman (92.2%, 106 of 115) and IR (98.3%, 119 of 121) studies. Vibrational spectroscopic techniques enable simple, rapid, and accurate microbial identification. These advantages can be easily transferred to other applications in diagnostic microbiology, e.g., to accelerate identification of fastidious microorganisms.

Bacteria↗
PubMed2004

Detection of amino acid and peptide transmitters in physiologically identified brainstem cardiorespiratory neurons.

Most of the CNS neurons that regulate circulation and respiration reside in regions of the brain characterized by extreme cellular heterogeneity (nucleus of the solitary tract, reticular formation, parabrachial nuclei, periaqueductal gray matter, hypothalamus, etc.). The chemical neuroanatomy of these regions is correspondingly complex and teasing out specific circuits in their midst remains a problem that is usually very difficult if not impossible to solve by conventional tract-tracing methods, Fos methodology or electrophysiology in slices. In addition, identifying the type of amino acid or peptide transmitter used by electrophysiologically recorded neurons has been until recently an especially difficult task either for lack of a specific marker or because such markers (many peptides for example) are exported to synaptic terminals and thus undetectable in neuronal cell bodies. In this review, we describe a general purpose method that solves many of these problems. The approach combines juxtacellular labeling in vivo with the histological identification of mRNAs that provide definitive neurochemical phenotypic identification (e.g. vesicular glutamate transporter 1 or 2, glutamic acid decarboxylase). The results obtained with this method are discussed in the general context of amino acid transmission in brainstem cardiorespiratory pathways. The presence of markers of amino acid transmission in specific aminergic pre-sympathetic neurons is especially emphasized as is the extensive co-localization of markers of GABAergic and glycinergic transmission in the brainstem reticular formation.

Amino Acid Transport Systems, Neutral↗
PubMed2001

The broad autism phenotype: a complementary strategy for molecular genetic studies of autism.

The genetic liability for autism appears to be expressed not only as the full syndrome of autism, but in milder, qualitatively similar characteristics that collectively have been referred to as constituting the broad autism phenotype. Identification of components of the broad autism phenotype that segregate independently in relatives of autistic individuals may provide an index of genes that, when present together, may interact to produce autism. Inclusion of information on the broad autism phenotype in relatives, in linkage studies of autism, may provide a potentially important, complementary approach for detecting the genes causing this condition.

Autistic Disorder↗
PubMed2004

Phenotypic and genotypic identification of Candida spp. isolated from hospitalized patients.

As candidosis incidence continue to rise, quick laboratory identification of Candida species is becoming increasingly important for a growing population of patients at-risk. RAPD techniques were used on samples of Candida obtained from patients hospitalized at Santa Casa de Misericordia in Belo Horizonte (SCMBH) Brazil, from March 1998 to December 2000 and then compared with the results of phenotypic identification techniques. Two hundred and forty two yeasts were isolated and phenotypically identified as follows: Candida albicans (105), Candida tropicalis (62), Candida parapsilosis (28), Candida glabrata (19), Candida krusei (8), Candida guilliermondii (5) and Candida spp. (15). Samples from the three most frequent species isolated were selected randomly in order to compare the phenotypic and genotypic analyses. Genotypic analysis using RAPD primer M13 (F/R) displayed the best results of all test samples. There was both agreement and consistency between phenotypic and genotypic analysis using RAPD, demonstrating that is possible to apply this method for the identification of Candida species.

Candida↗
PubMedDate not supplied

Propagation and characterization of lymphocytes from rejecting human cardiac allografts.

In recent years heart transplantation has become an increasingly common therapeutic measure for cases of heart failure not amenable to other forms of management. Despite major advances, graft rejection continues to be the main cause of transplant failure. To expand our knowledge of the rejection process, we have grown lymphocytes from biopsy specimens obtained from potentially rejecting allografts. The growth of the lymphocytes requires the presence of interleukin-2 and was shown to correlate well with histologic criteria in identifying rejection (p less than 0.003, chi-square analysis). Of 125 biopsy specimens, 61 (49%) became positive for lymphocyte growth within 30 days. Of the biopsy specimens that were positive on culture, 54 (88%) were histologically diagnosed as rejecting, and seven (11%) were histologically nonrejecting. Of the 64 specimens that were negative on culture, 28 (44%) were histologically negative for rejection, and 36 (56%) were positive for some grades of rejection. Of these culture negative, microscopically positive specimens, however, 31 (86%) were associated with mild (grade 3) or equivocal (grade 2) rejection. Discrepancies between culture and histology appeared to be related to degree of rejection, quantity of sample, and sampling variation. Phenotypic identification of cultured cells showed that the suppressor/cytotoxic phenotype was the predominant cell type in all cultures (mean +/- standard error = 83% +/- 3% of cells). Antihuman mature inducer/helper T cell (OKT4+) T-lymphocytes in culture were associated with biopsy specimens obtained immediately before onset or at the beginning of rejection. Preliminary studies testing in vitro sensitivities of lymphocytes to antithymocyte globulin (ATG) and other control immunosuppressants not believed to function by direct cytolysis (methylprednisolone, cyclosporine, azathioprine) were undertaken. These yielded reproducible dose-response curves, and the drug concentration producing 50% cytotoxicity (LD50) was calculated for each drug-culture combination. The mean LD50 for ATG was in the range of in vivo therapeutic concentrations, whereas LD50 values for control drugs were threefold to twelvefold higher than therapeutic concentrations. Resistance to the effects of ATG showed a tendency to correlate with prolongation of rejection; however, a similar tendency was noted for control drugs, suggesting the possibility of a nonspecific mechanism of resistance to the effects of ATG. The technique of interleukin-2 lymphocyte culture from rejecting allografts promises to further our understanding of mechanisms of graft rejection.

Adult↗
PubMedDate not supplied

[Molecular identification of species within the Mycobacterium tuberculosis complex using regions of difference].

The differentiation within the Mycobacterium tuberculosis complex (MTBC) based on phenotypic methods is long and does not give an unambiguous result in every case whereas the advance in genetic knowledge leads to new views. Thus, regions of difference (RD), that seem to characterize the species of the MTBC, have been identified. Amplification methods, targeted on these zones, have been developed then. The study of four regions (RD1, RD5, RD9, RD10) has been done on 64 isolates formerly identified thanks to phenotypic methods. Genotypic results confirm phenotypic identifications except in one case. This strain initially identified as M. tuberculosis and isolated from a Gabonese patient, corresponds, according to genotypic identification, to M. africanum. Since phenotypic characterization of M. africanum is difficult, this method would allow to accurately determine the true prevalence of this specie. Moreover, the study of RD10 doesn't seem to be informative. The amplification of only two RD, RD1 and RD9 carries out the identification of all M. tuberculosis and M. bovis BCG isolates. M. bovis and M. africanum will be then identified thanks to RD5. Thus, this easy and rapid method of identification of the major species of MTBC seems to be appropriated for a routine use.

Antitubercular Agents↗
PubMed2005

Homofermentative lactic acid bacteria of a traditional cheese, Comlek peyniri from Cappadocia region.

Comlek peyniri is a typical artisanal cheese in Central Anatolia. This type of cheese was made by using the indigenous lactic acid bacteria (LAB) flora of cow or ewes' milk. Majority of the samples were taken from fresh cheese because the aim was to isolate homofermentative LAB. Initially 661 microbial isolates were obtained from 17 cheese samples. Only 107 were found to be homofermentative LAB. These isolates were selected and identified by using both phenotypic and molecular methods. Phenotypic identification included curd formation from skim milk, catalase test, Gram staining and light microscopy, growth at different temperatures and salt concentrations, arginine hydrolysis, gas production from glucose, and carbohydrate fermentation. Molecular identification was based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of the 16S rRNA gene-ITS (internally transcribed spacer) region. By combining the phenotypic and molecular identification results, isolates belonging to each of the following genera were determined at species or subspecies level: 54 Lactococcus lactis subsp. lactis, 21 Enterococcus faecium, 3 Ec. faecalis, 2 Ec. durans, 10 Ec. sp., 15 Lactobacillus paracasei subsp. paracasei, and 2 Lb. casei strains. Technological characterisation was also performed by culturing each of the strains in UHT skim milk, and by monitoring pH change and lactic acid production at certain time intervals through the 24 h incubation. Results of the technological characterisation indicated that 33% of the isolates (35 strains) were capable of lowering the pH of UHT milk below 5.3 after 6 h incubation at 30 degrees C. Thirty four of these strains were Lc. lactis subsp. lactis, and only one was an Ec. faecium strain.

Animals↗
PubMed2003

Taxonomy, ecology and antibiotic resistance of enterococci from food and the gastro-intestinal tract.

Apart from genotypic identification methods, there is a need for reliable conventional phenotypic identification schemes for simple and rapid determination of enterococcal species in food or in the gastro-intestinal tract (GIT). Only a limited number of enterococcal species is of importance for the ecology of the GIT or the food microflora, including E. faecalis, E. faecium, E. durans/hirae, E. gallinarum and E. casseliflavus. After genus identification the differentiation within these species can include, e.g. mannitol and arabinose fermentation and growth at 50 degrees C. Widely used commercial identification systems may fail to precisely identify rare species. Ecological aspects should also be taken into account. In the human GIT E. faecium is the most common species whereas in most animal species E. faecalis is at least present in the same amount. Especially in foods of animal origin (cheese, pork meat, beef, poultry meat) also E. faecalis is very frequent. This is of special interest as glycopeptide resistance is most often found in human clinical E. faecium strains as well as in E. faecium from the environment or animal samples and less frequent in E. faecalis strains. EU experts propose as safety criteria for probiotics in feed additives the exclusion of resistances or the lack of transferability. This proposal can also be applied to enterococci in foods. Specific resistances must be excluded, but transferability or acquisition of resistance (e.g. vancomycin) cannot be excluded per se. However, technologically used strains should differ from clinical strains concerning their resistance patterns and transfer rates.

Anti-Bacterial Agents↗
PubMed2003

Natural antimicrobial susceptibilities and biochemical profiles of Yersinia enterocolitica-like strains: Y. frederiksenii, Y. intermedia, Y. kristensenii and Y. rohdei.

The natural susceptibility of 131 Yersinia strains of Y. frederiksenii (n=38), Y. intermedia (n=48), Y. kristensenii (n=26) and Y. rohdei (n=19) to 70 antibiotics was tested. Minimum inhibitory concentration (MIC) values were determined with a microdilution procedure in IsoSensitest broth (all strains) and cation-adjusted Mueller Hinton broth (some strains). All species were naturally sensitive or sensitive and of intermediate susceptibility to tetracyclines, aminoglycosides, acylureidopenicillins, numerous cephalosporins, carbapenems, aztreonam, quinolones, chloramphenicol, folate-pathway inhibitors, nitrofurantoin, and fosfomycin. Uniform natural resistance was found with penicillin G, oxacillin, several macrolides, lincosamides, streptogramins, glycopeptides, rifampicin and fusidic acid. Species-specific differences in susceptibility affecting clinical assessment criteria were seen with aminopenicillins (in the presence and absence of beta-lactamase inhibitors), ticarcillin and some cephalosporins. Major medium-dependent susceptibilities were found with fosfomycin. beta-Lactam MIC susceptibility patterns suggested that most strains of the species tested produce both class A and class C (AmpC) beta-lactamases that are characteristic for the species. The present study describes a database concerning the natural susceptibility of some Y. enterocolitica-like species to a wide range of antibiotics, which can be applied to validate forthcoming antibiotic susceptibility tests of these strains and might contribute to their identification. An evaluation of 30 biochemical tests that secured phenotypic identification to the Yersinia species level is presented.

Animals↗
PubMed2000

Simplified phenotypic tests for identification of Acinetobacter spp. and their antimicrobial susceptibility status.

Acinetobacter spp. have been found to be responsible for an increasing number of nosocomial infections. During a 16-month period, 22 patients hospitalised mainly in the respiratory intensive care unit (RICU), paediatric and other medical wards were investigated either for infection or colonisation by Acinetobacter spp. Of the 45 isolates of Acinetobacter detected among the total of 425 non-fermenters encountered, 24 representative isolates were selected for extended phenotypic identification. Four environmental isolates were also included in the study. These 28 isolates were typed by biotyping and antibiotyping, which helped in delineating the Acinetobacter spp. into 12 phenotypes and two distinct antibiotypes respectively. A sudden increase of cases of acinetobacter infection suggested that three outbreaks during the study period were due to phenotypes 1 and 2 of A. calcoaceticus-A. baumannii complex (Acb). Strains of Acb-complex showed multiple drug resistance and were sensitive only to netilmicin. A comparatively high proportion of resistance to amikacin (48%) was also detected among these strains by the agar dilution method. The RICU environment was recognised as an important reservoir for the resistant outbreak strain (Acb-1) which was probably leading to persistent colonisation and recurrent infections.

Acinetobacter↗
PubMed2001

First isolation of Candida dubliniensis in Rio Grande do Sul, Brazil.

Candida dubliniensis is a newly recognized species closely phylogenetically related to Candida albicans and is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. In this paper we report the isolation of three strains of C. dubliniensis, from AIDS patients, in the state of Rio Grande do Sul (Brazil). The phenotypic identification was based on germ tube emission, abundant production of chlamydospores, assimilation of sucrose but not of xylose and the inability to grow at 42 degrees C. Randomly amplified polymorphic DNA (RAPD) analysis and genomic DNA sequencing confirmed the distinct genetic nature C. dubliniensis. Topics related to the epidemiology, isolation, phenotypical and genotypical identification of C. dubliniensis are also discussed.

AIDS-Related Opportunistic Infections↗
PubMed2003

Simplified panel of assimilation tests for identification of Acinetobacter species.

A total of 66 Acinetobacter isolates obtained from JIPMER hospital wards were subjected to phenotypic identification schemes involving 25-test and a simplified 13-test panel of carbon utilization or assimilation tests. Reference strains belonging to different DNA groups (n=24) were also tested. Identification was done using numerical approach based on a matrix constructed of phenotypic data published elsewhere and the strains were assigned to different DNA groups according to classification of Tjernberg & Ursing. Sixty-six strains tested represented 10 DNA groups in matrix of large test panel; at a probability level of 0.95. Much simplified scheme of 13 assimilation test panel failed to differentiate some isolates with in A. calcoaceticus-A. baumannii complex (Acb-complex) unlike extended panel. In all, from the large panel 95% of isolates were identified correctly among all the isolates and it did not identify 5% of isolates. From the small panel, a total of 89% of isolates were identified correctly and it could not identify 11% of isolates. Reduced number of assimilation tests to 13 from the large panel bought reduction in identification percentage rate by only 6%. It is impossible for many bacterial diagnostic labs worldwide to perform large panel of carbon utilization tests in routine practice. Simplified panel of assimilation tests suggested here seems to be the best alternative method for identification of Acinetobacter species.

Acinetobacter↗
PubMed1998

Identification of classical Propionibacterium species using 16S rDNA-restriction fragment length polymorphisms.

The phenotypic identification of the classical propionibacteria is essentially still problematic and alternative techniques for the identification of the various species are required. A rapid and sensitive technique for the routine identification of the classical propionibacteria, based on the amplification of 16S rRNA genes using the polymerase chain reaction and the subsequent restriction endonuclease digestion of the PCR products, was previously described. Although this technique enabled differentiation between the various classical species examined it was only evaluated on a limited number of type and reference strains. During this study, the taxonomic relationship between 135 Propionibacterium strains from diverse ecological niches, representing four classical species was investigated using this PCR/RFLP technique. Visual differentiation between the classical Propionibacterium was possible after restriction endonuclease digestion of the PCR products obtained using primers 16sP1-16sP4 and 16sP3-16sP4 with the restriction endonucleases HaeIII, AluI and HpaIII, respectively. With the exception of strains independently identified as "P. rubrum" and "P. sanguineum", the results of this study confirm the consolidation of the "old" species into the various classical species as they currently exist. It was therefore concluded that the PCR/RFLP protocol is suitable for the rapid and routine identification of the classical propionibacteria.

Bacterial Typing Techniques↗
PubMed2001

Phenotypic and molecular identification and clustering of lactic acid bacteria and yeasts from wheat (species Triticum durum and Triticum aestivum) sourdoughs of Southern Italy.

The microflora of 25 wheat sourdoughs from the Apulia region, Southern Italy, was characterized. The sourdoughs were mainly produced from Triticum durum wheat. The number of lactic acid bacteria and yeasts ranged from ca. log 7.5 to log 9.3 colony forming units (cfu)/g and from log 5.5 to log 8.4 cfu/g, respectively. About 38% of the 317 isolates of lactic acid bacteria were identified by conventional physiological and biochemical tests. Phenotypic identification was confirmed by 16S rDNA and 16S/23S rRNA spacer region PCR. Overall, 30% of the isolates were identified as Lactobacillus sanfranciscensis, 20% as Lb. alimentarius, 14% as Lb. brevis, 12% as Leuconostoc citreum, 7% as Lb. plantarum, 6% as Lactococcus lactis subsp. lactis, 4% as Lb. fermentum and Lb. acidophilus, 2% as Weissella confusa and 1% as Lb. delbrueckii subsp. delbrueckii. Some of these species have not been previously isolated from sourdoughs. Since bakers yeast is widely used in sourdough production, Saccharomyces cerevisiae was largely found. The phenotypical relationships within the main lactic acid bacteria identified were established by using cluster analysis. A microbial map of the 25 sourdoughs was plotted showing characteristic associations among lactic acid bacteria and differences in the lactic acid bacteria species which were mainly due to the species of wheat flour, use of bakers yeast and type of bread.

Bread↗
PubMed1998

Phenotypic, genotypic and pathogenic variation among streptomycetes implicated in common scab disease.

This study examines the diversity of streptomycetes causing potato scab using phenotypic identification, 16S rRNA sequence, and pathogenicity measurements. Pathogenicity did not correlate well with taxonomic identification, nor with phylogenetic position. This result suggests that neither phenotypic nor 16S characteristics are diagnostic for scab-causing strains. Additionally, phenotypic characterizations were not fully consistent with phylogenetic relationships. This incongruency highlights the difficulties of taxonomy within the streptomycetes and affirms the need for better integration of genetic and phenotypic characters in the systematics of this group.

DNA, Bacterial↗
PubMedDate not supplied

[Identification of the Pseudomonas genus bacteria by computer analysis].

A computer program for the simplified phenotypic identification of Pseudomonas has been developed. The information concerning 66 species included in up-to-date Pseudomonas genus characterized by 113 tests was accumulated in a database. The identification key is represented in interactive mode on a website http://www.imv.kiev.ua/PsmIK/default.htm. The program was used for the identification of 46 Pseudomonas strains isolated from rhizosphere. For 23 more strains unidentified by conventional technique, the level of similarity was 67-74%. This fact allows suggesting that they might be representatives of new Pseudomonas species.

Databases, Factual↗