Conventional Identification Tests Research Articles

Case Report: Soft tissue infection with Burkholderia thailandensis capsular variant: case report from the Lao PDR

Background Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandenisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

Case Report: Soft tissue infection with Burkholderia thailandensis capsular variant: case report from the Lao PDR

Background Burkholderia thailandensis is an environmental bacteria closely related to Burkholderia pseudomallei that rarely causes infection in humans. Some environmental isolates have shown to express a capsular polysaccharide known as B. thailandensis capsular variant (BTCV), but human infection has not previously been reported. Although B. thailandednisis has been identified in environmental samples in Laos before, there have not been any human cases reported. Case A 44-year-old man presented to a district hospital in Laos with a short history of fever and pain in his left foot. Physical examination identified a deep soft-tissue abscess in his left foot and an elevated white blood count. A deep pus sample was taken and melioidosis was suspected from preliminary laboratory tests. The patient was initially started on cloxacillin, ceftriaxone and metronidazole, and was then changed to ceftazidime treatment following local melioidosis treatment guidelines. Laboratory methods A deep pus sample was sent to Mahosot Hospital microbiology laboratory where a mixed infection was identified including Burkholderia sp. Conventional identification tests and API 20NE were inconclusive, and the B. pseudomallei-specific latex agglutination was positive. The isolate then underwent a Burkholderia species specific PCR which identified the isolate as B. thailandensis. The isolate was sent for sequencing on the Illumina NovaSeq 6000 system and multi-locus sequence typing analysis identified the isolate had the same sequence type (ST696) as B. thailandensis E555, a strain which expresses a B. pseudomallei-like capsular polysaccharide. Conclusion This is the first report of human infection with B. thailandensis in Laos, and the first report of any human infection with the B. thailandensis capsular variant. Due to the potential for laboratory tests to incorrectly identify this bacteria, staff in endemic areas for B. thailandensis and B. pseudomallei should be aware and ensure that appropriate confirmatory methods are used to differentiate between the species.

Polymerase Chain Reaction Detection of Salmonella spp. in Fecal Samples of Pet Birds

The aims of this study were 1) to determine the prevalence of Salmonella in clinically ill birds in aviaries in Ankara, Turkey, and 2) to compare conventional culture and polymerase chain reaction (PCR) for detection of Salmonella in feces from clinically ill pet birds. In the study, 185 fecal samples (feces and/or swabs) collected from the pet birds kept in the seven different aviaries in the city of Ankara were investigated for the existence of Salmonella spp. by bacterial isolation and PCR. The conventional isolation and identification methods were performed for Salmonella isolation from fecal cultures. Suspected colonies were confirmed with the Salmonella polyvalent O antiserum and serogrouped with Salmonella group-specific antiserum. PCR was performed after the fecal swabs were incubated for 18 hr in 10 ml of tetrathionate broth. Three (1.63%) out of 185 fecal samples were found to harbor Salmonella spp. by conventional identification tests and were found to belong to serogroup B. Five (2.7%) swab samples were found to harbor Salmonella DNA by PCR tests. As a conclusion, PCR following incubation of clinical samples in pre-enrichment broth seemed to be a fast and practicable method for Salmonella spp. diagnosis when compared to protracted labor-intensive conventional culture techniques.

Repetitive-sequence-PCR-based DNA fingerprinting using the Diversilab system for identification of commonly encountered dermatophytes.

The performance of repetitive-sequence-based PCR (rep-PCR) using the DiversiLab system for identification of dermatophytes commonly isolated in a clinical laboratory was assessed by comparing results to those of conventional tests (colony morphology, microscopic examination of slide cultures, and, for suspected Trichophyton species, use of additional media). Sixty-one cultures were tested in phase 1, the feasibility portion of the study; 64 additional cultures were tested in phase 2, the validation portion conducted to assess reproducibility and confirm accuracy. Discrepancies were resolved by repeating rep-PCR and conventional tests and, in phase 2, sequencing the internal transcribed spacers. After initial testing of the cultures in phase 1 (excluding one contaminated culture), agreement between conventional tests and rep-PCR was 90% (54 of 60). Agreement was 98.3% after resolution of discrepancies, and in all but one case the initial rep-PCR result was correct. After initial testing of cultures in phase 2 (excluding one discarded and one contaminated culture), agreement between rep-PCR and conventional testing was 88.7% (55 of 62). After discrepancies were resolved, agreement was 100%. Initial rep-PCR results were correct, except for one Microsporum canis culture containing two colony variants, which could not be initially identified by rep-PCR. The performance of the DiversiLab system for identification of the dermatophytes commonly encountered in a clinical mycology laboratory-Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, and M. canis-was excellent. Moreover, the DiversiLab system is technically simple and provides results in < 24 h once a pure culture is available for testing, which is considerably more rapid than conventional identification tests.

Impact of genotypic studies on mycobacterial taxonomy: the new mycobacteria of the 1990s.

The advancement of genetic techniques has greatly boosted taxonomic studies in recent years. Within the genus Mycobacterium, 42 new species have been detected since 1990, most of which were grown from clinical samples. Along with species for which relatively large numbers of strains have been reported, some of the new species of mycobacteria have been detected rarely or even only once. From the phenotypic point of view, among the new taxa, chromogens exceed nonchromogens while the numbers of slowly and rapidly growing species are equivalent. Whereas conventional identification tests were usually inconclusive, an important role was played by lipid analyses and in particular by high-performance liquid chromatography. Genotypic investigations based on sequencing of 16S rRNA gene have certainly made the most important contribution. The investigation of genetic relatedness led to the redistribution of the species previously included in the classically known categories of slow and rapid growers into new groupings. Within slow growers, the intermediate branch related to Mycobacterium simiae and the cluster of organisms related to Mycobacterium terrae have been differentiated; among rapid growers, the group of thermotolerant mycobacteria has emerged. The majority of species are resistant to isoniazid and, to a lesser extent, to rifampin. Many of the new species of mycobacteria are potentially pathogenic, and there are numerous reports of their involvement in diseases. Apart from disseminated and localized diseases in immunocompromised patients, the most frequent infections in immunocompetent people involve the lungs, skin, and, in children, cervical lymph nodes. The awareness of such new mycobacteria, far from being a merely speculative exercise, is therefore important for clinicians and microbiologists.

Burden of unidentifiable mycobacteria in a reference laboratory.

Modern identification techniques at the genomic level have greatly improved the taxonomic knowledge of mycobacteria. In adjunct to nucleic acid sequences, mycobacterial identification has been endorsed by investigation of the lipidic patterns of unique mycolic acids in such organisms. In the present investigation, the routine use of high-performance liquid chromatography (HPLC) of mycolic acids, followed by the sequencing of the 16S rRNA, allowed us to select 72 mycobacterial strains, out of 1,035 screened, that do not belong to any of the officially recognized mycobacterial species. Most strains (i.e., 47) were isolated from humans, 13 were from the environment, 3 were from animals, and 9 were from unknown sources. The majority of human isolates were grown from the respiratory tract and were therefore most likely not clinically significant. Some, however, were isolated from sterile sites (blood, pleural biopsy, central venous catheter, or pus). Many isolates, including several clusters of two or more strains, mostly slow growers and scotochromogenic, presented unique genetic and lipidic features. We hope the data reported here, including the results of major conventional identification tests, the HPLC profiles of strains isolated several times, and the whole sequences of the 16S rRNA hypervariable regions of all 72 mycobacteria, may encourage reporting of new cases. The taxonomy of the genus Mycobacterium is, in our opinion, still far from being fully elucidated, and the reporting of unusual strains provides the best background for the recognition of new species. Our report also shows the usefulness of the integration of novel technology to routine diagnosis, especially in cases involving slow-growing microorganisms such as mycobacteria.

Automated Detection of Hydrogen Sulfide Release from Thiosulfate by Salmonella spp.

Hydrogen sulfide production is used in conventional tests for identification and differentiation of Salmonella spp. from other species of Enterobacteriaceae, and a black precipitate on agar media is the indicator of the reaction. Selective liquid media were formulated for automated optical detection of H2S in salmonellae using the BioSys instrument. The media contained thiosulfate and ferric ammonium citrate, and production of H2S caused copious black pigmentation of the broth. Combination of the H2S indicators with dulcitol or xylose as fermentable carbohydrate, lysine, omithine or arginine to induce decarboxylase activity, and Tergitol 4 as inhibitor selectively identified six Salmonella spp. by a sharp drop in transmittance at 585 nm. The time for detection of transmittance changes was inversely proportional to initial numbers of CFU in the media: 10 h for 105 CFU/ml and 17 h for 101 CFU/ml. No detection was observed in six non-Salmonella species of Enterobacteriaceae tested.

Predictive values of species' identifications from the Vitek Gram-Positive Identification Card using clinical isolates of coagulase-negative staphylococci.

The Gram-Positive Identification Card (GPI, bioMerieux Vitek) was compared to conventional tests for identification of 616 clinical isolates of coagulase-negative staphylococci. All tests were inoculated with subcultures from single isolated colonies. The predictive values of GPI species' identifications (the number of the system's correct calls divided by the total number of calls for each species) were 100% (12 of 12) for Staphylococcus capitis, 100% (79 of 79) for S saprophyticus, 98.4% (246 of 250) for S epidermidis and 96.0% (120 of 125) for S haemolyticus, but only 64.5% (69 of 107) for the other species analyzed. When an infrequently encountered Staphylococcus species is named using the GPI, the identification should be confirmed using additional tests.

Testing for Pseudomonas syringae pv. atrofaciens and Xanthomonas campestris pathovars on cereals in Italy

During recent years, recurrent attacks of basal glume rot/leaf blight attributed to Pseudomonas syringae pv. atrofaciens have been observed on cultivars of durum wheat grown in the Po valley (IT). The main aim of this study was to test for this pathogen and Xanthomonas campestris pathovars in commercial seed lots of cereals produced in different regions of Italy, as well as in symptomatic plants collected in the field. None of the analyses led to the detection of xanthomonads. In testing for P.s. atrofaciens, representative bacterial isolates were selected and characterized; this was done by combining conventional identification tests and computerized densitometric analysis of electrophoretic patterns of cell proteins obtained using the SDS‐PAGE technique. P.s. atrofaciens was detected in seed samples (barley, wheat and durum wheat) and symptomatic plants (durum wheat) grown in northern and central Italy. The possible pathogenic role of other unrelated fluorescent pseudomonads in this interaction was also examined. Difficulties in discriminating pvs syringae and atrofaciens of P. syringae are discussed and the uncertain taxonomic relationship between these two pathovars is emphasized.

Simplified techniques for identifying foodborne yeasts

Four problematic areas associated with the identification of foodborne yeasts are discussed. These consist of (1) the inability of conventional identification tests to recognize some common and important foodborne yeasts characterized by genomic differences (e.g., Saccharomyces cerevisiae, S. bayanus and S. pastorianus); (2) the delay in application of non-traditional identification methods such as DNA fingerprinting, chromosome karyotyping, protein electrophoretic patterns and fatty acid profiles for routine identification purposes; (3) the lack of commercially available manual or automated identification systems dedicated to the diagnosis of foodborne yeasts; and (4) the disregard for considering ecological frequency of yeasts in computerized probabilistic identification systems.

Identification of atypical strains ofStreptococcus pneumoniae by a specific DNA probe

A specific DNA probe containing a 0.65 Kb fragment coding for the aminoterminal region of the major pneumococcal autolysin (amidase) was constructed, deleting the region involved in the specific recognition of cell wall choline residues. The high specificity of this probe was demonstrated in tests with Streptococcus pneumoniae related species including Streptococcus oralis, which contains choline in its cell wall. The probe was used to characterize pneumococcal isolates showing atypical responses in conventional identification tests. The hybridization obtained with 27 atypical pneumococci and 11 of 17 isolates presumptively identified as viridans streptococci confirmed that the probe is suitable for diagnostic use.

Identification and Plant Interaction of a Phyllobacterium sp., a Predominant Rhizobacterium of Young Sugar Beet Plants

The second most abundant bacterium on the root surface of young sugar beet plants was identified as a Phyllobacterium sp. (Rhizobiaceae) based on a comparison of the results of 39 conventional identification tests, 167 API tests, 30 antibiotic susceptibility tests, and sodium dodecyl sulfate-polyacrylamide gel electrophoretic fingerprints of total cellular proteins with type strains of Phyllobacterium myrsinacearum and Phyllobacterium rubiacearum. It was found on 198 of 1,100 investigated plants between the 2nd and 10th leaf stage on three different fields in Belgium and one field in Spain. Densities ranged from 2 x 10 to 2 x 10 CFU/g of root. Five isolates exerted a broad-spectrum in vitro antifungal activity. DNA-DNA hybridizations showed that Phyllobacterium sp. does not contain DNA sequences that are homologous with the attachment genes chvA, chvB, the transferred-DNA (T-DNA) hormone genes iaaH and ipt from Agrobacterium tumefaciens, iaaM from A. tumefaciens and Pseudomonas savastanoi, or the nitrogenase genes nifHDK from Klebsiella pneumoniae. Phyllobacterium sp. produces indolylacetic acid in in vitro cultures and induces auxinlike effects when cocultivated with callus tissue of tobacco. When Phyllobacterium sp. was transformed with a Ti plasmid derivative, it gained the capacity to induce tumors on Kalanchoe daigremontiana. The potential role of Phyllobacterium sp. in this newly recognized niche is discussed.

Staphylococcus aureus Identification Characteristics and Enterotoxigenicity

ABSTRACTEnterotoxigenicity and conventional tests for identification of Staphylococcus aureus were correlated for strains of staphylococci isolated from foods. These strains were examined for enterotoxin production, colonial morphology on Baird‐Parker agar, coagulase activity with rabbit and pig plasma, thermostable nuclease (TNase) production, lysostaphin sensitivity and anaerobic utilization of glucose and mannitol. Enterotoxins A,B,C,D, and E were produced singly or in combination by 100 of the S. aureus strains; 51 strains produced no enterotoxin. False‐negative rates in identifying the enterotoxigenic group as typical S. aureus were as follows: 11% for colonial morphology on Baird‐Parker agar, 8% for coagulase activity with Difco rabbit plasma, 7% for TNase production, 4% for lysostaphin sensitivity and 2% and 4%, respectively, for the anaerobic utilization of glucose and mannitol. Consequently, none of these tests was reliable for differentiating toxigenic from nontoxigenic S. aureus..

Hydrolytic activity in the genusSchizosaccharomyces lindner

A total of 22 strains of known species of the genusSchizosaccharomyces Lindner were evaluated by numerical taxonomy based on conventional identification tests. The results of numerical taxonomy were supplemented by a determination of activity of extracellular hydrolytic enzymes, especially 1,3-β-D-glucanase, RNAase and DNAase. All the strains tested were capable of utilizing the 1,4-capable of utilizing the 1,4-α-D-glucan tamarind. Study of life cycles of these organisms showed that extracellular hydrolytic enzymes were present mainly at the time of maturation of asci and release of their walls. Strains forming four-spore asci could be comprised in the single speciesSchizosaccharomyces pombe Lindner as two varieties:S. pombe var.pombe andS. pombe var.malidevorans. The two varieties differ in maltose fermentation. Urease is produced by all strains irrespective of the life cycle phase. A number of hydrolytic enzymes are not produced by the genusSchizosaccharomyces (e.g. amylolytic enzymes) despite the fact that oligomers of the maltose type are utilized. Species of the genusSchizosaccharomyces lack also xylanase, cellulases, mannase, and are incapable of degrading carrageenan and acid polysaccharides.