Andreas Essig

Rapid Diagnosis of Bacterial Meningitis by Real-Time PCR and Fluorescence In Situ Hybridization

ABSTRACT Real-time PCR and fluorescence in situ hybridization (FISH) were evaluated as rapid methods for the diagnosis of bacterial meningitis and compared to standard diagnostic procedures. For PCR, a LightCycler approach was chosen, implementing eubacterial and specific PCR assays for the most relevant bacteria. For FISH, a similar probe set containing eubacterial and specific probes was composed of published and newly designed probes. Both methods were evaluated by use of cerebrospinal fluid (CSF) samples from patients with suspected bacterial meningitis. For all microscopy- and culture-positive samples (n = 28), the eubacterial PCR was positive. In addition, all identifiable pathogens were detected with specific PCR assays, according to an algorithm based on the Gram stain. The FISH method detected the pathogen in 13 of 18 positive samples. While the FISH method remained negative for all microscopy- and culture-negative samples (n = 113), the eubacterial PCR was positive for five of these samples. Sequencing of the amplicon revealed the presence of Neisseria meningitidis, Streptococcus agalactiae, and Haemophilus influenzae in three of these five samples. In addition, samples with discordant results by culture and microscopy were successfully investigated by PCR (10 samples) and FISH (5 samples). In conclusion, PCR is a highly sensitive tool for rapid diagnosis of bacterial meningitis. FISH is less sensitive but is useful for the identification of CSF samples showing bacteria in the Gram stain. Based on our results, an approach for laboratory diagnosis of meningitis including PCR and FISH is discussed.

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

ABSTRACT Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.

Survival of Chlamydia pneumoniae-Infected Mono Mac 6 Cells Is Dependent on NF-κB Binding Activity

ABSTRACT The respiratory tract pathogen Chlamydia pneumoniae has been associated with atherosclerosis. Monocytes are supposed to serve as a vehicle for systemic dissemination of intracellular C. pneumoniae from the lung to the artery vessel wall. We were therefore interested in pathogen-induced cellular events associated with NF-κB, a crucial transcription factor for both inflammatory cytokines and antiapoptotic molecules. In this study we demonstrate by electrophoretic mobility shift assay that C. pneumoniaeinfection of the human monocytic cell line Mono Mac 6 induces activation of NF-κB over 48 h, with a maximum level at 1 h postinfection. As shown by supershift assay, the activated NF-κB complex consists of the subunits RelA (p65) and NF-κB1 (p50). Apoptotic host cells were not detected during the early stages of the infection when maximal activation of NF-κB was detected. Pretreatment of Mono Mac 6 with the antioxidant and NF-κB inhibitor PDTC (pyrrolidine dithiocarbamate) induced activation of caspase-3 and led to apoptotic cell death. The C. pneumoniae-induced activation of the NF-κB complex was reduced by PDTC, which in parallel resulted in an increased apoptosis, as quantified by annexin V labeling and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling reaction. In the complete absence of activated NF-κB, when Mono Mac 6 cells were pretreated with the more potent NF-κB inhibitors MG-132 and parthenolide a C. pneumoniae-mediated rescue of cells from induced apoptosis could not be achieved. Our results indicate that activation of NF-κB inC. pneumoniae-infected Mono Mac 6 cells is associated with protection of Mono Mac 6 cells against apoptosis and might thereby contribute to systemic spread of the pathogen.

Evaluation of the Hyplex BloodScreen Multiplex PCR-Enzyme-Linked Immunosorbent Assay System for Direct Identification of Gram-Positive Cocci and Gram-Negative Bacilli from Positive Blood Cultures

ABSTRACT We evaluated the Hyplex BloodScreen PCR-enzyme-linked immunosorbent assay (ELISA) system (BAG, Lich, Germany), a new diagnostic test for the direct identification of gram-negative bacilli and gram-positive cocci from positive blood cultures, with 482 positive BACTEC 9240 blood culture bottles. The test involves amplification of the bacterial DNA by multiplex PCR and subsequent hybridization of the PCR product to specific oligonucleotide probes in an ELISA-based format. The available probes allow the separate detection of Escherichia coli, Pseudomonas aeruginosa, Enterobacter aerogenes, Klebsiella spp., Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis/Enterococcus faecium, Streptococcus pyogenes, and Streptococcus pneumoniae and the staphylococcal mecA gene. The Hyplex BloodScreen test showed an overall sensitivity of 100% for the identification of gram-negative bacilli and 96.6 to 100% for the identification of gram-positive cocci (S. aureus, 100%; S. epidermidis, 97.2%; Enterococcus faecalis/Enterococcus faecium, 96.6%; and Streptococcus pneumoniae, 100%). The specificities of the test modules ranged from 92.5 to 100% for gram-negative bacilli and 97.7 to 100% for gram-positive cocci (Escherichia coli, 92.5%; Pseudomonas aeruginosa, 98.5%; Klebsiella spp., 100%; Enterobacter aerogenes, 100%; S. aureus, 100%, S. epidermidis, 97.7%; Enterococcus faecalis/Enterococcus faecium, 99.6%; Streptococcus pyogenes, 100%; and Streptococcus pneumoniae, 99.3%). The result of the mecA gene detection module correlated with the result of the phenotypic oxacillin resistance testing in all 38 isolates of Staphylococcus aureus investigated. In conclusion, the Hyplex BloodScreen PCR-ELISA system is well suited for the direct and specific identification of the most common pathogenic bacteria and the direct detection of the mecA gene of Staphylococcus aureus in positive blood cultures.

Inquilinus limosus in Patients with Cystic Fibrosis, Germany

We identified Inquilinus limosus, a recently described α-proteobacterium, in sputum of 2 patients with cystic fibrosis whose respiratory tracts were persistently colonized for >9 months. We present data on the epidemiology, antimicrobial susceptibility, and molecular characteristics of I. limosus.

Chlamydia pneumoniae induces the expression of inhibitor of apoptosis 2 (c-IAP2) in a human monocytic cell line by an NF-kappaB-dependent pathway.

Members of the inhibitor of apoptosis protein family are important factors that regulate apoptotic cell death. As demonstrated both by RT-PCR and Western Blot analysis C. pneumoniae infection of the human monocytic cell line Mono Mac 6 induces the expression of mRNA and protein of the cellular inhibitor of apoptosis 2 (c-IAP2). Blocking NF-kappaB DNA-binding activity by the proteasome inhibitor MG-132 results in decrease of C. pneumoniae-induced c-IAP2 expression. Therefore, C. pneumoniae may exploit the NF-kappaB pathway to induce expression of an antiapoptotic host cell protein that may contribute to intracellular survival of the pathogen.

Rapid Discrimination of Haemophilus influenzae, H. parainfluenzae, and H. haemolyticus by Fluorescence In Situ Hybridization (FISH) and Two Matrix-Assisted Laser-Desorption-Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF-MS) Platforms

Background Due to considerable differences in pathogenicity, Haemophilus influenzae, H. parainfluenzae and H. haemolyticus have to be reliably discriminated in routine diagnostics. Retrospective analyses suggest frequent misidentifications of commensal H. haemolyticus as H. influenzae. In a multi-center approach, we assessed the suitability of fluorescence in situ hybridization (FISH) and matrix-assisted laser-desorption-ionization time-of-flight mass-spectrometry (MALDI-TOF-MS) for the identification of H. influenzae, H. parainfluenzae and H. haemolyticus to species level. Methodology A strain collection of 84 Haemophilus spp. comprising 50 H. influenzae, 25 H. parainfluenzae, 7 H. haemolyticus, and 2 H. parahaemolyticus including 77 clinical isolates was analyzed by FISH with newly designed DNA probes, and two different MALDI-TOF-MS systems (Bruker, Shimadzu) with and without prior formic acid extraction. Principal Findings Among the 84 Haemophilus strains analyzed, FISH led to 71 correct results (85%), 13 uninterpretable results (15%), and no misidentifications. Shimadzu MALDI-TOF-MS resulted in 59 correct identifications (70%), 19 uninterpretable results (23%), and 6 misidentifications (7%), using colony material applied directly. Bruker MALDI-TOF-MS with prior formic acid extraction led to 74 correct results (88%), 4 uninterpretable results (5%) and 6 misidentifications (7%). The Bruker MALDI-TOF-MS misidentifications could be resolved by the addition of a suitable H. haemolyticus reference spectrum to the systems database. In conclusion, no analyzed diagnostic procedure was free of errors. Diagnostic results have to be interpreted carefully and alternative tests should be applied in case of ambiguous test results on isolates from seriously ill patients.

Rapid Detection of Brucella spp. in Blood Cultures by Fluorescence In Situ Hybridization

ABSTRACT Brucellosis is a severe systemic disease in humans. We describe a new 16S rRNA-based fluorescence in situ hybridization assay that facilitates rapid and specific detection of all human pathogenic species of Brucella and that can be applied directly to positive blood cultures.

Rapid Identification of Clinically Relevant Enterococcus Species by Fluorescence In Situ Hybridization

ABSTRACT A fluorescence in situ hybridization assay for the rapid identification of clinically relevant enterococci (Enterococcus faecalis, E. faecium, E. gallinarum, the VanC-type resistance group) was developed and evaluated with 33 reference strains, 68 clinical isolates, and 58 positive blood cultures. All probes showed excellent sensitivities and specificities.

Expanded spectrum of Nocardia species causing clinical nocardiosis detected by molecular methods

Nocardia species isolated from seven patients with clinical infection were investigated by conventional biochemical methods and 16S rRNA gene sequencing. Three isolates were identified as recently described species (i.e., N. paucivorans, N. abscessus and N. veterana). We provide data on the epidemiology, clinical significance and antimicrobial susceptibility of these newly described Nocardia species.