Biomedical subjects

Holger C Scholz

Publications and source records attributed to Holger C Scholz.

12 recordsLinked to original sources

PubMed2006

Real-time PCR using hybridization probes for the rapid and specific identification of Francisella tularensis subspecies tularensis.

Tularemia is a plague-like infection caused by Francisella (F.) tularensis classified as a biological warfare agent. F. tularensis subsp. tularensis is the most virulent subspecies demanding rapid diagnosis. Typing systems for this fastidious bacterium to the subspecies level are laborious and time consuming. Therefore, the aim of this study was to develop a real-time PCR for the rapid and specific identification of F. tularensis subsp. tularensis. The specificity of the assay was determined using a comprehensive panel of Francisella strains, clinically relevant bacteria, and DNA preparations of potential hosts. F. tularensis subsp. tularensis was specifically detected but no other organisms. The range of linearity was determined to be 100 fg to 10 ng, the lower limit of detection was 25 fg of DNA (13 genome equivalents). An internal amplification control PCR system targeting lambda phage DNA was included. Neither the internal amplification control nor host DNA influenced the cycle threshold values obtained for F. tularensis subsp. tularensis. In conclusion, we have developed a highly sensitive and specific assay that can be integrated into real-time PCR-based identification procedures for biological agents. This is a major diagnostic improvement, as all other methods for the specific identification of F. tularensis subsp. tularensis are more time consuming.

Animals↗

PubMed2006

Detection of the reemerging agent Burkholderia mallei in a recent outbreak of glanders in the United Arab Emirates by a newly developed fliP-based polymerase chain reaction assay.

A polymerase chain reaction (PCR) assay targeting the flagellin P (fliP)-I S407A genomic region of Burkholderia mallei was developed for the specific detection of this organism in pure cultures and clinical samples from a recent outbreak of equine glanders. Primers deduced from the known fliP-IS407A sequence of B. mallei American Type Culture Collection (ATCC) 23344(T) allowed the specific amplification of a 989-bp fragment from each of the 20 B. mallei strains investigated, whereas other closely related organisms tested negative. The detection limit of the assay was 10 fg for purified DNA of B. mallei ATCC 23344(T). B. mallei DNA was also amplified from various tissues of horses with a generalized B. mallei infection. The developed PCR assay can be used as a simple and rapid tool for the specific and sensitive detection of B. mallei in clinical samples.

Animals↗

PubMed2006

Brucella endocarditis in prosthetic valves.

Human brucellosis is a multiple organ disease that presents with fever and is most often transmitted via contaminated, unpasteurized goat milk and cheese. In chronic cases, focal complications (eg, spondylitis, neurobrucellosis and endocarditis) are frequently seen. Although the disease may be severely debilitating, the mortality rate is low. Fatal cases are often due to endocarditis. Because Brucella endocarditis is a rare complication (2% to 5%), therapeutic considerations are based on single-case experiences only. Therapy includes long-term antibiotic treatment using combinations of various antimicrobial drugs and surgical valve replacement when required. A case of Brucella endocarditis complicated by the infection of two valvular prostheses implanted after involvement of the mitral and aortic valve due to rheumatic fever is described. The patient was successfully treated by a medical and surgical approach. Therapeutic strategies in Brucella endocarditis are discussed in light of the current literature.

Anti-Bacterial Agents↗

PubMed2006

Description of Pseudochrobactrum gen. nov., with the two species Pseudochrobactrum asaccharolyticum sp. nov. and Pseudochrobactrum saccharolyticum sp. nov.

Two Gram-negative, rod-shaped, oxidase-positive, non-spore-forming, non-motile bacteria (CCUG 46016(T) and CCUG 33852(T)), isolated from a knee aspirate of a 66-year-old man and an industrial glue, respectively, were studied for their taxonomic position. On the basis of chemotaxonomic data [i.e. major ubiquinone (Q-10), major polar lipids (phosphatidylethanolamine, phosphatidylglycerol and phosphatidylcholine) and major fatty acids (C(18 : 1)omega7c and C(19 : 0) cyclo omega8c)] and 16S rRNA gene sequence similarity, both strains belong to the Alphaproteobacteria. The presence of spermidine and putrescine as the predominant polyamines in CCUG 46016(T) were in agreement with its phylogenetic affiliation in the vicinity of the genus Ochrobactrum. 16S rRNA gene sequence similarities between both strains and established species within the genera Bartonella, Ochrobactrum and Brucella were less than 95 %. Although both organisms showed highest 16S rRNA gene sequence similarity to members of Brucella, phenotypic features (including chemotaxonomic features) were more like those of members of the genus Ochrobactrum. Sequence comparison of the recA genes confirmed the separate phylogenetic position of the two strains. On the basis of DNA-DNA pairing results and physiological and biochemical data, the two strains can be clearly differentiated from each other and from all known Ochrobactrum species. It is evident that these organisms represent two novel species in a new genus, Pseudochrobactrum gen. nov., for which the names Pseudochrobactrum asaccharolyticum sp. nov. (the type species, type strain CCUG 46016(T)=CIP 108977(T)) and Pseudochrobactrum saccharolyticum sp. nov. (type strain CCUG 33852(T)=CIP 108976(T)) are proposed.

Adhesives↗

PubMed2006

Genotyping of Ochrobactrum anthropi by recA-based comparative sequence, PCR-RFLP, and 16S rRNA gene analysis.

A recA-PCR restriction fragment length polymorphism assay was developed to study intraspecies variation among Ochrobactrum anthropi. Primers deduced from the known recA gene sequence of the genetically closely related genus Brucella allowed the specific amplification of a 1065 bp recA fragment from each of the 38 O. anthropi and the eight Brucella strains investigated. RecA was also amplified from the type strains of O. intermedium, O. tritici, and O. lupini but could not be generated from O. grignonense and O. gallinifaecis. Subsequent comparative recA sequence- and HaeIII-recA restriction fragment length polymorphism analysis identified nine different genospecies among the tested 38 O. anthropi isolates, whereas the recA sequences of the Brucella spp. were indistinguishable. Furthermore, Brucella spp., O. anthropi, O. intermedium, and O. tritici were clearly separated from each other by means of their recA sequences and HaeIII restriction patterns. Five strains of uncertain species status listed in the Culture Collection University of Göteborg bacterial culture collection as O. anthropi were characterized by recA analysis, and their phylogenetic position within the Brucella-Ochrobactrum group was determined. In summary, recA-sequence analysis provides a new reliable molecular subtyping tool to study the phylogeny of the Ochrobactrum taxon at both the inter- and intraspecies level.

Animals↗

PubMed2006

Development of a 5'-nuclease real-time PCR assay targeting fliP for the rapid identification of Burkholderia mallei in clinical samples.

BACKGROUND: Burkholderia mallei is a potential biological agent that causes glanders or farcy in solipeds, a disease notifiable to the Office International des Epizooties (OIE). The number of reported outbreaks has increased steadily during the last decade, but diagnosis is hampered by the low bacterial load in infected tissues and excretions. METHODS: We developed a B. mallei-specific 5'-nuclease real-time PCR assay that targets the fliP gene of B. mallei and includes an internal amplification control. Specificity was assessed with 19 B. mallei strains, 27 Burkholderia pseudomallei strains, other Burkholderia strains of 29 species, and clinically relevant non-Burkholderia organisms. RESULTS: Amplification products were observed in all B. mallei strains but in no other bacteria. The linear range of the B. mallei real-time PCR covered concentrations from 240 pg to 70 fg of bacterial DNA/reaction. The detection limit was 60 fg of B. mallei DNA. The clinical applicability of the assay was demonstrated by use of organ samples from diseased horses of a recent outbreak that was reported to the OIE by the United Arab Emirates in 2004. CONCLUSIONS: Compared with conventional PCR, our rapid 5'-nuclease real-time PCR assay for the specific identification of B. mallei has a lower risk of carryover contamination and eliminates the need for post-PCR manipulations. This real-time PCR assay also shortens the turnaround time for results and has the potential for automation.

Animals↗

PubMed2005

Immunoproteomic characterization of Brucella abortus 1119-3 preparations used for the serodiagnosis of Brucella infections.

The diagnosis of brucellosis is mainly based on the detection of anti-LPS antibodies. Due to substantial similarity of the O-polysaccharide of Brucella LPS to that of various other Gram-negative bacteria, serological tests of samples containing high amounts of LPS lack specificity. Hence, the development of assays based on more specific protein antigens is an essential subject in brucellosis research. The aim of this study was proteomic characterization of various antigen preparations of the diagnostic reference strain Brucella abortus 1119-3 and the identification of immunogenic proteins suitable for serological assays. Seventeen out of 383 protein spots of B. abortus 1119-3 were identified to be immunogenic by 2-D immunoblotting. These immunogenic spots were assigned to 6 proteins by MALDI-MS and nLC-ESI-MS/MS: Cu-Zn SOD, BCSP31, L7/L12, GroEL, GroES, and DnaK. All immunogenic proteins were present in three different antigen preparations investigated, i.e. native antigen, standard agglutination and commercially available agglutination antigen. 2-D immunoblotting of bacteria cross-reacting with Brucellae in agglutination tests proved that cross-reactivity of proteins is negligible. Surface enhanced laser desorption/ionization mass spectrometry (SELDI-MS) spectra also differentiated B. abortus clearly from cross-reacting bacteria. The combination of SELDI-MS analysis with the specificity of antibody binding will improve the identification of Brucella specific immunogenic proteins.

Animals↗

PubMed2005

Failure of a short-term antibiotic therapy for human brucellosis using ciprofloxacin. A study on in vitro susceptibility of Brucella strains.

BACKGROUND: Human brucellosis is characterized byfocal complications, chronic courses, and therapeutic failures. METHODS: In a relapsed case of brucellosis after short-term antibiotic therapy using doxycycline and ciprofloxacin two Brucella strains were isolated, before and after treatment. In vitro susceptibilities of both isolates were determined by E tests including a great variety of antibiotics. In a killing rate experiment the bactericidal activities of doxycycline, streptomycin, rifampin and ciprofloxacin as single agents and in combinations were determined. RESULTS: Lowest MIC values were measured for doxycycline and ciprofloxacin. MICs did not change under therapy. Streptomycin alone exhibited the most effective killing within 6 h, whereas the other single agents did not show bactericidal activity. Doxycycline plus ciprofloxacin was the most active combination in vitro. CONCLUSION: Routine susceptibility testing of Brucellae is not obligatory as most of the 'traditional' anti-Brucella antibiotics are active in vitro and bactericidal efficacy may differ in vivo.

Anti-Infective Agents↗

PubMed2005

Growth characteristics of Bacillus anthracis compared to other Bacillus spp. on the selective nutrient media Anthrax Blood Agar and Cereus Ident Agar.

Anthrax Blood Agar (ABA) and Cereus Ident Agar (CEI) were evaluated as selective growth media for the isolation of Bacillus anthracis using 92 B. anthracis and 132 other Bacillus strains from 30 species. The positive predictive values for the identification of B. anthracis on ABA, CEI, and the combination of both were 72%, 71%, and 90%, respectively. Thus, less than 10% of all species were misidentified using both nutrient media. Species which might be misidentified as B. anthracis were B. cereus, B. mycoides, and B. thuringiensis. Particularly, 30% of B. weihenstephanensis strains were misidentified as B. anthracis.

Agar↗

PubMed2005

Rapid presumptive identification of Burkholderia pseudomallei with real-time PCR assays using fluorescent hybridization probes.

Burkholderia pseudomallei (the etiologic agent of melioidosis) can cause pyogenic or granulomatous lesions in almost any organ. Septicemia has a case fatality rate of >40%. Early diagnosis and appropriate antibiotic therapy are crucial for survival, but cultivation, biochemical identification, and conventional PCR of B. pseudomallei are time consuming. We established real-time PCR assays using fluorescent hybridization probes targeting the 16S rDNA, the flagellin C (fliC) and the ribosomal protein subunit S21 (rpsU) genes. The test sensitivity and specificity were assessed with a representative panel of 39 B. pseudomallei, 9 B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. The detection limit for the 16S rDNA, fliC, and rpsU assay was 40, 40, and 400 genome equivalents per reaction, however, in spiked blood samples it was 300, 300, and 3000, respectively. Specificity, positive and negative predictive value of the assays was 100%. In conclusion, we recommend the use of the 16S rDNA and/or fliC real-time PCR assays for the rapid identification of B. mallei and B. pseudomallei in positive blood cultures or from suspicious bacterial colonies.

Burkholderia pseudomallei↗

PubMed2005

Identification of brucella species and biotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).

Brucellosis is a worldwide zoonosis causing reproductive failures in livestock and a severe multi-organ disease in humans. The genus Brucella is divided into seven species and various biotypes differing in pathogenicity and host specificity. Although Brucella spp. represent a highly homogenous group of bacteria, RFLPs of selected genes display sufficient polymorphism to distinguish Brucella species and biovars. PCR-RFLP analysis shows excellent typeability, reproducibility, stability, and epidemiological concordance. Consequently, PCR-RFLP assays of specific gene loci can serve as tools for diagnostic, epidemiological, taxonomic, and evolutionary studies. Various PCR-RFLPs used for the identification of Brucella species and biotypes are reviewed.

Bacterial Outer Membrane Proteins↗

PubMed2004

Development of 5' nuclease real-time PCR assays for the rapid identification of the burkholderia mallei//burkholderia pseudomallei complex.

Burkholderia pseudomallei is the causative agent of melioidosis and was classified as a biologic agent by the Centers for Disease Control and Prevention (Atlanta, GA). Acute melioidosis has a case fatality rate of >40%, and septicemia is fatal in up to 90%. The aim of the study was to design 5'-nuclease real-time PCR assays for the rapid and reliable identification of the B. mallei/B. pseudomallei complex. Real-time PCR assays using TaqMan probes targeting the 16S rDNA and fliC were developed on an ABI Prism 7000 sequence detection system (Applied Biosystems, Foster City, CA). Specificity was assessed with 64 B. pseudomallei, nine B. mallei, 126 other Burkholderia strains of 29 species, and 45 clinically relevant non-Burkholderia organisms. Sensitivity, specificity, and positive and negative predictive value of the assays were 100%. Discrimination between B. pseudomallei and B. mallei, an organism which can be regarded as a clone of B. pseudomallei, could not be achieved. A probit analysis revealed that 7.5 and 52 genome equivalents (GE) of B. pseudomallei could be detected using the fliC and the 16S rDNA assays (P = .05), respectively. In spiked blood samples, the detection limit was approximately 300 and 3.000 GE for fliC and the 16S rDNA, respectively. In conclusion, we recommend the simultaneous use of the 16S rDNA and fliC real-time PCR assays for the rapid and specific identification of the B. mallei/B. pseudomallei complex in positive blood cultures or from suspicious bacterial colonies allowing the early onset of appropriate antibiotic therapy.

Animals↗