Detection of Acinetobacter spp. in Blood Cultures by an Improved Fluorescent in Situ Hybridization Assay

Abstract

Fluorescent in situ hybridization (FISH) allows rapid detection of microorganisms. We aimed (i) to evaluate the sensitivity and specificity of FISH for the detection of Acinetobacter spp. in blood culture specimens and (ii) to test the simultaneous application of two genus-specific probes labeled with the same fluorochrome to increase the fluorescent signal intensity and improve the detection of Acinetobacter spp. Three hundred and twenty blood culture specimens were tested via both the conventional laboratory methods and FISH to detect Acinetobacter spp. The specimens were examined separately with each genus-specific probe Aci and ACA, and also using a mixture of the both probes Aci and ACA. In all examinations, probe EUB338 was used accompanied by Aci and ACA. The specificity of FISH was 100% (97.5% confidence interval [CI] = 98.7% - 100%). The sensitivity of FISH by the use of probe Aci was 96.4% (95% CI = 81.7% - 99.9%), whereas, the sensitivity of this technique by the use of probe ACA as well as by the combination of both probes Aci and ACA was 100% (97.5% CI = 87.7% - 100%). Moreover, simultaneous hybridization by probes Aci and ACA increased the fluorescent signal of Acinetobacter spp. cells to 3+ in 13 specimens. In conclusion, FISH, particularly using a combination of Aci and ACA, is a highly accurate method for the detection of Acinetobacter spp. in blood cultures. Furthermore, simultaneous hybridization by the both probes Aci and ACA can increase the fluorescent signal intensity of Acinetobacter spp. cells in some blood culture specimens and facilitate the detection of these microorganisms.

Identification of Stenotrophomonas maltophilia in Blood Cultures by Fluorescence in Situ Hybridization

“…High background fluorescence might be the cause of false-negative result of FISH in this specimen. Sometimes the presence of a large amount of materials such as protein and blood cells emits a strong background fluorescence that may mask the specific fluorescent signal of bacteria and impede the interpretation of slides 7,9 . Background fluorescence is a limitation of FISH, which has also been reported in other samples such as sputum samples obtained from patients with cystic fibrosis 10…”

“…For FISH, an aliquot of each blood culture specimen was fixed with 4% paraformaldehyde (Sigma-Aldrich), as described previously 9,10 . Two oligonucleotide probes, Stemal and EUB338, which were synthesized and 5′-labeled (Metabion, Planegg/Steinkirchen, Germany), were used in the present study.…”

“…Thereafter, the specimens or control bacterial strains were hybridized at 46°C for 90 minutes by application of 10 μL of hybridization buffer (0.9 M NaCl, 20 mM Tris-HCl [pH 8], 0.01% SDS, 30% formamide) containing a mixture of the probes EUB338-Fluo and Stemal-Cy3. Stringent washing was carried out using a washing buffer (20 mM Tris-HCl [pH 8], 0.01% SDS, 112 mM NaCl) at 48°C for 15 minutes 9,12 . Afterward, DNA was stained with 1 μg/mL 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Roche, Mannheim, Germany) for 5 minutes.…”

“…9,12 Afterward, DNA was stained with 1 μg/mL 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI; Roche, Mannheim, Germany) for 5 minutes. Finally, the slides were washed with phosphate-buffered saline, air-dried, mounted with a fluorescent mounting medium (DAKO, Glostrup, Denmark), 5,6,9 and observed with an epifluorescence microscope (Nikon 80i, Tokyo, Japan) equipped with a DS-5Mc-L1 digital camera system. Microscopy was performed by 2 expert researchers who were blinded to the results of the conventional methods.…”

Association of Antibiotic Alterations in Gut Microbiota With Decreased Osseointegration of an Intramedullary Nail in Mice With and Without Osteomyelitis

“…In this study, we used the EUB338 probe (5′-GCTG CCTCCCGTAGGAGT-3′) (Biomers, Ulm, Germany) to identify the location and quantify the number of microbes in bone tissue sections (18)(19)(20). Briefly, after conventional dewaxing and rehydration of the sections with bone specimen, 0.2 mol/L hydrochloric acid was applied to the tissue for 15 min, followed by 0.5% Triton for 8 min, and washed twice by PBS for 5 min.…”