Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis

Schabereiter-Gurtner, Claudia; Maca, Saskia; Kaminsky, Stephan; Rölleke, Sabine; Lubitz, Werner; Barisani-Asenbauer, Talin

doi:10.1016/s0732-8893(02)00401-7

Cited by 22 publications

(12 citation statements)

References 29 publications

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“…It has also been suggested that corneal biopsies are more sensitive than the corneal sampling technique used in this study for the isolation of obligate anaerobic bacteria 38 . Additionally, some obligate anaerobic bacteria identified as pathogens of the human cornea have proven impossible to isolate with current microbiologic techniques and can only been identified with molecular diagnostics 31,39 . If similar bacteria were present within the animals of this study, the experimental methods would not have permitted their identification.…”

Section: Discussionmentioning

confidence: 95%

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals

Ledbetter¹,

Scarlett²

2008

Veterinary Ophthalmology

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Section: Discussionmentioning

confidence: 95%

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals

Ledbetter¹,

Scarlett²

2008

Veterinary Ophthalmology

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“…PCR-DGGE has previously been used for analysis of the human microbiota of the gastrointestinal tract (10,16,20), vagina (4), oral cavity (12), and eye (13). However, this is the first report of the application of this technique to samples from the sinuses.…”

Section: Case Reportmentioning

confidence: 99%

Non-Culture-Based Analysis of Bacterial Populations from Patients with Chronic Rhinosinusitis

et al. 2005

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Middle meatus aspirates from patients with chronic rhinosinusitis were analyzed by bacterial culture, denaturing gradient gel electrophoresis (DGGE), and antibiotic sensitivity techniques. DGGE detected a greater bacterial diversity than culture methods. Although resistance to antibiotics was low, there was evidence of changes in the composition of the bacterial microbiota over time, and the presence of noncultured bacteria was demonstrated. CASE REPORTUsing a glass canula, aspirates were taken from the middle meatus of six patients who had previously undergone surgery for chronic rhinosinusitis. These patients had continued infection after surgery despite ongoing nasal toilet and antibiotic treatment. The antibiotics prescribed to the patients within the 2 months prior to the aspirates being taken are listed in Table 1. Four of the patients had a second aspirate taken 2 to 3 months after the first sample. The aspirates were first suspended in 2 ml phosphate-buffered saline and then thoroughly homogenized by being vortexed before being divided into two equal volumes for use in culture-based and DNA-based analyses. For the isolation of staphylococci, mannitol salt agar (Difco, Sparks, MD) was incubated at 37°C aerobically. Total bacterial populations were isolated on chocolate agar (Columbia agar base [Difco] with 5% heat-lysed defibrinated sheep blood [New Zealand Venous Supplies, Tuakau, New Zealand]) by incubation at 37°C either aerobically, in air supplemented with 5% CO 2 , or in an anaerobic atmosphere (85% N 2 , 10% H 2 , 5% CO 2 ). For the isolation of Streptococcus pneumoniae, sheep blood agar (Columbia agar base containing 5% defibrinated sheep blood) with 5 g/ml gentamicin (Sigma, St. Louis, MO) was used with anaerobic incubation at 37°C. Representative bacterial isolates were then identified by

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“…Due to the universal character, broad‐range real‐time PCR is more vulnerable to contamination of reagents than species‐specific real‐time PCR, which affects sensitivity. When only little amounts of the pathogen DNA compete with the contaminating DNA, the amplification of the pathogen can either be suppressed or mixed sequences are generated which cannot be sequenced without prior separation of the PCR products by cloning or DGGE‐analysis (Schabereiter‐Gurtner et al. 2002).…”

Section: Discussionmentioning

confidence: 99%

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis

et al. 2008

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Aim: Introduction of a protocol for broad‐range diagnosis of bacterial infections, which remain negative in culture. Methods and Results: The new TaqMan real‐time PCR assay amplifies part of the 16S rRNA gene. Species are identified by subsequent sequencing and phylogenetic blast analysis. The analytical sensitivity showed to be 50 fg DNA per PCR. The lowest detectable bacterial cell concentration in blood was 1000 CFU per 200 μl EDTA‐blood. The utility in clinical routine diagnosis was evaluated by testing 136 clinical specimens. Bacterial pathogens were detected in 33 samples (24·3%) either by culture or molecular diagnosis. In 10 culture negative cases, pathogens such as Mycoplasma timone/orale, Ureaplasma parvum/urealyticum, Treponema pallidum, different streptococci and staphylococci were identified by molecular diagnosis only. Conclusions: The introduced broad‐range real‐time PCR protocol showed to be useful in the clinical routine in cases where bacterial infection was highly anticipated but culture remained negative. However, the obtained data have to be always interpreted with caution and in conjunction with the clinical data, crossing‐point values and with the Blast result of both the sample and the controls. Significance and Impact of the Study: This work introduces a new and well‐evaluated broad‐range real‐time PCR protocol for diagnosis of bacterial infections.

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Investigation of an anaerobic microbial community associated with a corneal ulcer by denaturing gradient gel electrophoresis and 16S rDNA sequence analysis

Cited by 22 publications

References 29 publications

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals

Isolation of obligate anaerobic bacteria from ulcerative keratitis in domestic animals

Non-Culture-Based Analysis of Bacterial Populations from Patients with Chronic Rhinosinusitis

Evaluation of a protocol for molecular broad-range diagnosis of culture-negative bacterial infections in clinical routine diagnosis

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