Specific Detection of
            <i>Legionella pneumophila</i>
            : Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

Grimm, Dorothee; Merkert, Hilde; Ludwig, Wolfgang; Schleifer, Karl‐Heinz; Hacker, Jörg; Brand, Bettina

doi:10.1128/aem.64.7.2686-2690.1998

Cited by 63 publications

(25 citation statements)

References 28 publications

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“…The foci were localized in the vicinity of blood vessels and resembled bacterial structures in size and shape. To ensure that the observed structures indeed represented bacteria, any unspecific reaction was excluded by probing another section with the S. epidermidis-specific SEP-1 probe and a Legionella pneumophila-specific probe, LEGPNE1 (16), as a negative control. No specific signal of the foci was obtained with the Legionella-specific probe in this control experiment (Fig.…”

Section: Resultsmentioning

confidence: 99%

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

et al. 1999

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Staphylococcus epidermidis and Staphylococcus aureus are the most common causes of medical device-associated infections, including septicemic loosenings of orthopedic implants. Frequently, the microbiological diagnosis of these infections remains ambiguous, since at least some staphylococci have the capacity to reduce their growth rate considerably. These strains exhibit a small-colony phenotype, and often they are not detectable by conventional microbiological techniques. Moreover, clinical isolates ofS. aureus and S. epidermidis adhere to polymer and metal surfaces by the generation of thick, multilayered biofilms consisting of bacteria and extracellular polysaccharides. This study reports improved detection and identification of S. aureusand S. epidermidis by an in situ hybridization method with fluorescence-labeled oligonucleotide probes specific for staphylococcal 16S rRNA. The technique has proven to be suitable for the in situ detection of staphylococci, which is illustrated by the identification of S. epidermidis in a connective tissue sample obtained from a patient with septicemic loosening of a hip arthroplasty. We also show that this technique allows the detection of intracellularly persisting bacteria, including small-colony variants of S. aureus, and the differentiation of S. epidermidisfrom other clinically relevant staphylococci even when they are embedded in biofilms. These results suggest that the 16S rRNA in situ hybridization technique could represent a powerful diagnostic tool for the detection and differentiation of many other fastidious microorganisms.

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Section: Resultsmentioning

confidence: 99%

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

et al. 1999

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“…All samples were heat-fixated to eight-field microscope slides and dehydrated using an increasing ethanol series. After 20 min incubation with 30 mg mL À1 lysozyme in phosphate-buffered saline at 30 1C, hybridization and washing was carried out as described earlier (Grimm et al, 1998;Kaltenpoth et al, 2005). The Coriobacteriumspecific probe COR653 (5 0 -fluorescein isothiocyanate labeled) (Harmsen et al, 2000) and the general eubacterial probe EUB338 (5 0 -Cy3 labeled) (Amann et al, 1990) were used for FISH.…”

Section: Fishmentioning

confidence: 99%

Localization and transmission route of Coriobacterium glomerans, the endosymbiont of pyrrhocorid bugs

Kaltenpoth

Winter

Kleinhammer

2009

FEMS Microbiology Ecology

102

101

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Endosymbiotic gut bacteria play an essential role in the nutrition of many insects. Most of the nutritional interactions investigated so far involve gammaproteobacterial symbionts, whereas other groups have received comparatively little attention. Here, we report on the localization and the transmission route of the specific actinobacterial symbiont Coriobacterium glomerans from the gut of the red firebug, Pyrrhocoris apterus (Hemiptera: Pyrrhocoridae). The symbionts were detected by diagnostic PCRs and FISH in the midgut section M3, in the rectum and in feces of the bugs as well as in the hemolymph of some females. Furthermore, adult female bugs apply the symbionts to the surface of the eggs during oviposition, from where they are later taken up by the hatchlings. Surface sterilization of egg clutches generated aposymbiotic insects and thereby confirmed the vertical transmission route via the egg surface. However, symbionts were readily acquired horizontally when the nymphs were reared in the presence of symbiont-containing eggshells, feces, or adult bugs. Using diagnostic PCRs and partial sequencing of the 16S rRNA gene, closely related bacterial symbionts were detected in the cotton stainer bug Dysdercus fasciatus (Hemiptera: Pyrrhocoridae), suggesting that the symbiosis with Actinobacteria may be widespread among pyrrhocorid bugs.

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“…The fixed and washed cells were hybridized using a previously described protocol by Grimm et al (1998). Hybridization buffer contain 20 nmol l )1 Tris-HCl (pH 7AE6), 0AE9 mol l )1 NaCl and 0AE01% sodium dodecyl sulfate.…”

Section: Hybridization Protocolmentioning

confidence: 99%

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

et al. 2006

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Aims: To confirm the presence of viable Legionella spp. in dental unit waterlines (DUWL) using fluorescent in situ hybridization (FISH) and compare this method with culture approach and also to validate the utility of an enrichment to increase FISH sensitivity. Methods and Results: Water samples from 40 dental units were analysed. Three different techniques for detecting Legionella spp. were compared: (i) culture approach, (ii) direct FISH and (iii) FISH with a previous R2A medium enrichment (R2A/FISH). The FISH detection was confirmed by PCR. The use of the direct FISH does not improve significantly the detection of legionellae when compared with the culture. On the contrary, when R2A/FISH was performed, sensitivity was, respectively, two‐ and threefold higher than that with the direct FISH and culture approach. Using R2A/FISH, 63% of water samples analysed showed a contamination by legionellae. Conclusions: Legionellae detection by direct FISH and R2A/FISH in dental unit water is possible but is more rapid and more sensitive (R2A/FISH) than the culture approach. Significance and Impact of the Study: R2A/FISH showed that several pathogens present in DUWL are viable but may not be culturable. Unlike PCR, R2A/FISH is designed to detect only metabolically active cells and therefore provides more pertinent information on infectious risk.

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Specific Detection of Legionella pneumophila : Construction of a New 16S rRNA-Targeted Oligonucleotide Probe

Cited by 63 publications

References 28 publications

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Detection of Staphylococcus aureus and Staphylococcus epidermidis in Clinical Samples by 16S rRNA-Directed In Situ Hybridization

Localization and transmission route of Coriobacterium glomerans, the endosymbiont of pyrrhocorid bugs

Detection of Legionella spp. by fluorescent in situ hybridization in dental unit waterlines

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