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.Brucellae are gram-negative bacteria which cause serious infections in animals and humans. Four species, namely, Brucella abortus, Brucella canis, Brucella suis, and especially Brucella melitensis, are able to infect humans and cause a systemic disease called brucellosis that is endemic in Mediterranean countries, the Middle East, Africa, and Latin America. DNA hybridization and multilocus enzyme electrophoresis studies show a high level of homology among the Brucella spp., indicating that the genus Brucella comprises only one species with several biovars (6,15,17). A reliable and early diagnosis of brucellosis is of major importance in order to initiate an adequate therapy. Culture of blood and other specimens, such as bone marrow aspirates and biopsy specimens, is the gold standard for diagnosis of human brucellosis (16,17). However, culture and phenotypic identification are hampered by the slow and fastidious growth of the organisms and by possible misleading results of commercial biochemical identification systems (4). In addition, brucellae are associated with a high risk of laboratory-acquired infections since they are easily transmitted by aerosols and the infectious dosage is very low. Intensive handling of the organism in the laboratory often precedes identification as Brucella spp., and thus, protective measures are taken too late (8,12). Serological methods for the detection of Brucella-specific antibodies are available, but their usefulness is limited by a high prevalence of Brucellaspecific antibodies in areas where brucellosis is endemic, low sensitivity, especially in acute infections, and cross-reactions with other gram-negative bacteria (6, 17).The development of new diagnostic techniques that facilitate rapid detection and identification of brucellae and minimize the risk of laboratory infection is of great practical importance. Fluorescence in situ hybridization (FISH) using fluorescently labeled oligonucleotide probes complementary to unique target sites on the rRNA has already been successfully implemented in the field of clinical microbiology, including the detection and identification of pathogens in blood cultures and cerebrospinal fluid (9, 13). FISH is a rapid and easy-to-perform method that directly visualizes bacteria without prior amplification or cultivation steps.Here, we describe a new FISH assay that facilitates rapid and specific detection of all human-pathogenic species of Brucella spp. that can be applied directly to positive blood cultures. The FISH assay was evaluated by means of 17 reference strains of brucellae, 19 other relevant bacterial species (43 isolates), and 33 positive blood cultures, including 2 from patients with brucellosis.The FISH probe Bru-996 (5Ј-CCA CTA ACC GCG ACC GGG ATG), targeting ...

Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent <i>In situ </i>hybridization

“…11 Several reports show that FISH has already been successfully applied for the detection of E. coli, H. pylori, Staphylococcus aureus and Brucella spp. [5][6][7][8] However, this is the Þ rst report on detection of acinetobacters and Pseudomonas aeruginosa using FISH in Malaysia.…”

“…In addition, culture and sensitivity assays to determine the antibiotic proÞ le of the infecting agent could take up to an additional 24 h. Fluorescent in situ hybridization (FISH) has been used to detect various bacteria in clinical samples. [4][5][6][7][8] The general principle of this method is the use of a ß uorescently labelled oligonucleotide probe that speciÞ cally hybridizes to the target sequence of 16S rRNA, thereby enabling visualization of the whole bacterium with a ß uorescence microscope. The aim of this study was to evaluate the speciÞ city and sensitivity of FISH for the identiÞ cation of Acinetobacter spp.…”

The relationships between Brucella melitensis predilection sites, bacterial loads in vivo, and the agglutinating antibody response in experimentally infected sheep

“…Subsequently, the bound probe was detected with 1:500 alkaline phosphatase-conjugated anti-DIG antibody and developed into color in BCIP/NBT solution by applying the DIG Detection Kit (Roche). Finally, the sections were counterstained with nuclear fast red and mounted under coverslips (12). Tissues from the noninfected sheep were used as a negative control.…”

“…After that, the sample sections were permeabilized in 0.1 N HCl for 30 min, rinsed in distilled water, acetylated in triethanolamine (TEA)-hydrochloride (HCl) buffer (0.5 M TEA-HCl, 0.75 M NaCl, pH 8.0) with 0.25% acetic anhydride for 10 min, and rinsed in distilled water. Finally, the sample sections were dehydrated in 70% ethanol for 5 min and allowed to air-dry (12).To decrease the background, the slides were allowed to prehybridize at 55 °C for 1 h with hybridization buffer containing 50% deionized formamide, 10% dextran sulfate, 0.1% sodium pyrophosphate, 0.05% sodium dodecyl sulfonate, 2X SSC (0.3 M sodium chloride, 0.03 M sodium citrate, pH 7.0), 5X Denhardt's solution, and 1 mg/mL denatured salmon sperm DNA (Sigma). Each of 10 sections was hybridized in 0.5 mL of hybridization mixture consisting of 50% deionized formamide, 10% blocking reagent, 10% dextran sulfate, 5X Denhardt's solution, 4X SSC, 10 mg/ mL denatured salmon sperm, and 5 pg/µL DIG-labeled DNA probe at 60 °C for 18-20 h in a humid box to prevent drying out.…”

“…All the samples were cut into two pieces; one piece was fixed in 10% neutral buffered formalin for in situ hybridization (ISH) (12) and the other was used for bacteriological examination.…”