Table 1.
Organisms and growth conditions used in this study.
Table 2.
FISH-probes used in this study.
Fig 1.
FISH with urea-based buffers is specific and similar in signal intensity to hybridizations with formamide.
(A) Specific detection of E. coli by the probe Gam42a (red), N. meningitidis by the probe Bet42a (green), and both organisms by the EUB probe (blue) in hybridizations with urea. In the lower right corner, an overlay of the three images is shown. Absence of yellow color indicates that there was no unspecific double hybridization of Cy3 (red)—and Fluos (green)—labelled probes. Bar 10 μm. (B) Ratios of signal intensities after hybridizations with urea and formamide for six different probes. Ratios above 1 indicate higher signal intensities in hybridizations with urea-based reagents; ratios below 1 indicate higher signal intensities in hybridizations with formamide. Ratios are based on at least three replicates and the standard deviation is shown for each ratio. (C) Ratios for the signal intensities after hybridization with urea and formamide-based reagents with different fluorophores. Ratios are based on at least three replicates (min. 670 cells/replicate) and the standard deviation is shown for each ratio.
Fig 2.
Influence of lyophilization and prolonged storage on the probe signal intensity in urea-based hybridizations.
The Cy3-labeled Gam42a, Cy5-labeled Bet42a and FLUOS-labeled EUB probes were lyophilized and used in hybridizations either directly after lyophilization or after storage at room temperature for 28 days. Signal intensities were compared to signal intensities of freshly prepared reagents without lyophilization. A ratio of 1 means no change, while ratios below 1 indicate negative influence of lyophilization or storage. E. coli was used as target organism for Gam42a, N. meningitidis for Bet42a and both organisms as target for the EUB probe. The means and the standard deviation based on at least three replicates are shown.
Fig 3.
Detection of Bacillus anthracis by NotiFy-FISH under field conditions.
Signals for Bacillus anthracis detected by the probes Bac1157 (left) and EUB (middle) after hybridization with urea-based lyophilized reagents and DAPI staining (right) were recorded with a conventional smartphone through the lens of a portable, battery-operated, fluorescence LED microscope.
Fig 4.
Two-step workflow for the identification of thirteen different organisms by DOPE-FISH.
Probes that were designed and evaluated in this study are underlined in black. Note that B. mallei is detected by the probes Bmal and Bboth, while B. pseudomallei is detected by Bboth only. F. tularensis belongs to the Gammaproteobacteria, but is detected by the probe Bet42a. Bacterial FISH images are drawn to scale and represent 10 μm squares each.
Fig 5.
Identification of biological agents on skin surface after sampling by tape, in powder samples and in a spore suspension using the NotiFy-algorithm.
E. coli was detected on porcine skin in the first hybridization by the Gam42a probe (labeled in Cy5 and FLUOS, turquoise color in the overlay) and by the probe Esco864 (labeled in Cy5) in the second hybridization. Additional bacteria were detected in the first hybridization by the Bet42a probe (labeled in Cy3 and FLUOS, yellow color in the overlay) and DAPI or by DAPI alone, but could not be identified by the algorithm. DAPI-signals for bacteria other than E. coli were also observed in the second hybridization. In powder samples, Y. pestis was identified by the Gam42a probe (labeled in Cy5 and FLUOS, turquoise color in the overlay) in the first hybridization and by the probe Ypest1531LNA (labeled in Cy3, red color in the overlay) in the second hybridization. Autofluorescent powder material can be observed in all three channels. B. anthracis spores were detected after pretreatment to break the spore coat with the probe LGC354B (labeled in Cy5 and Cy3, violet color in the overlay) in the first and with the Bac1157 probe (labeled in Cy3) in the second hybridization. Not all spores were accessible to FISH-probes after the pretreatment, resulting in only DAPI signals but no FISH signals for some of the spores. Bars 10 μm.
Fig 6.
Application of the diagnostic algorithm for the identification of bacteria in clinical samples.
E. coli was identified in an infected skin lesion, Brucella spp.in a blood culture and Burkholderia pseudomallei in a swab of an infected patient. In the infected skin lesion, bacteria that were only detect by DAPI, but not by any of the other probes used were present (arrows). Efficient probe binding was controlled with a positive control containing a mixture of known bacteria (control). Shown are images acquired after hybridization with formamide-based (wound infection and control) or urea-based reagents (blood culture and swab). E. coli was detected by the probe Gam42a (labeled in Cy5 and FLUOS, turquoise color in the overlay) in the first hybridization and identified by the probe Esco864 (labeled in Cy5) in the second hybridization. Brucella was identified by Bru996 (labeled in FLUOS) in the first hybridization. B. pseudomallei was detected by the probe Bet42a (labeled in Cy3 and FLUOS, yellow color in the overlay) in the first hybridization and identified with the probe Bboth (labeled in Cy3) in the second hybridization. In the control overlay, specific binding of the probes used in the first hybridization is shown by detection of B. anthracis in pink, Y. pestis in turquoise, B. suis in green, L. borgpetersenii in red, B. pseudomallei in yellow and R. slovaca in blue. The probes and fluorophores resulting in signals in the respective sample are indicated on the far right under a representation of the additive mixing of colors. Bars 10 μm.