1 INTRODUCTION
2 RESULTS
2.1 Characteristics of the recombinant bioluminescent PAO1-lux strain
Fig.1 Characteristics of the bioluminescent PAO1-lux strain. A Growth curves of the PAO1-lux and PAO1 strains. Bacteria were cultured in LB medium on 96-well plate for 24 h at 37 °C. The OD at 600 nm was measured at every 0.5 h time points. The data were representative of three independent experiments with similar results. B Light detection of the PAO1-lux and PAO1 strains using a Bio-Rad imaging system under white light and luminescent modes. Bacteria were cultured on LB plates for 24 h. C Light detection of the PAO1-lux strain cultured in vitro using a multi-mode microplate reader. Data are reported as relative light units (RLU) and wells with sterile saline solution were used to determine the background value, which was subtracted from the bioluminescence values from the wells with bacteria. The corresponding bacterial counts were determined by CFU counting. The data are mean values of three independent experiments |
2.2 Bioluminescence stability of the PAO1-lux strain
2.3 Effect of culture conditions on growth and bioluminescence of the PAO1-lux strain
Fig.2 Characteristics of the bioluminescence and growth curves of the PAO1-lux strain under different nutritional conditions and different pH conditions. A The PAO1-lux strain was cultured at 37 °C for 24 h in LB medium with the pH conditions of 6.2 and 7.2. The OD and RLU values of the 96-well microplate were detected by a Multi-Mode Reader every 0.5 h. B The bioluminescence pictures of the PAO1-lux strain at a single time point for 6 h were collected using a Bio-Rad imaging system. C The PAO1-lux strain was cultured at 37 °C for 24 h in M9 medium with the pH conditions of 6.2 and 7.2. The OD and RLU values of the 96-well microplate were detected by a Multi-Mode Reader every 0.5 h. D The PAO1-lux strain was cultured in M9 medium with different pH conditions (pH = 6.2, 7.2 and 8.2) and the picture was observed by the imaging system at 6 h |
2.4 Evaluation of bacterial adhesion on 96-well microplates by bioluminescence and crystal violet staining method
2.5 Evaluation of bacterial adhesion on different materials by bioluminescence and CFU counting method
Fig.3 Bacterial adhesion to the six kinds of materials as determined by the bioluminescence method (A) and the CFU counting method (B, C). A The RLU values of the PAO1-lux strain that adhered to the six kinds of materials. B The CFU counts of the PAO1-lux strain that adhered to the six kinds of materials. C Bacterial adhesion of the PAO1-lux strain to the six kinds of materials were determined using the agar dilution assay. Five 10-fold serial dilutions of each sample grown overnight were spotted (10 μL per spot) onto LB agar. The dilution ratio of Zn, Al, glass groups from 10−1 to 10−5, and medical gauzes, PE gloves, latex gloves groups from 10−2 to 10-6. The plates were incubated for 16 h to 18 h at 37 °C |
2.6 Effect of Triton X-100 on the growth and bioluminescence of the PAO1-lux strain
Fig.4 Determination of bacterial adhesion on the materials surface after Triton X-100 treated. A Effect of Triton X-100 in different concentrations on the growth and bioluminescence of the PAO1-lux strain. The PAO1-lux strain was cultured in LB liquid medium containing 0.1%, 0.05%, 0.025% or 0.0125% Triton X-100. The OD at 600 nm and RLU values were measured after 6 h and 24 h. B The changes of bacterial adhesion on six kinds of medical device materials’ surfaces were determined by bioluminescence method after Triton X-100 treatment. C Imaging of bacterial adhesion on the irregular materials’ surfaces by a Bio-Rad imaging system under white light and luminescent modes. D The changes of bacterial adhesion on six kinds of medical device materials' surfaces were determined by CFU counting method after Triton X-100 treatment |
2.7 Evaluation of bacterial adhesion on materials’ surfaces after Triton X-100 treatment
3 DISCUSSION
Tab.1 Comparison of bacterial adhesion testing methods |
CFU counting | Crystal violet staining | ATP | Bioluminescence | |
---|---|---|---|---|
Accuracy | RSD ≤ 30% | RSD ≤ 30% | RSD ≤ 30% | RSD ≤ 15% |
Range | 1 to 10 8 cells | 10 4 to 10 9 cells | 10 2 to 10 8 cells | 10 3 to 10 8 cells |
Sensitivity | 1 to 10 2 cells | 10 3 to 10 4 cells | 10 2 cells | 10 3 cells |
Specificity | Strong | Weak(Dye can be adsorbed to the abiotic materials) | Weak(Non-microbial ATP can interfere with detection) | Strong |
Testing time | 2 d | 3–4 h | 1–2 h | 1–2 h |
Testing steps | 4 steps | 4 steps | 3 steps | 2 steps |
Invasive detection | No | Yes | Yes | No |
Real time detection | No | No | No | Yes |
Applicability for materials of irregular shape | No | No | Yes | Yes |
Applicability for batch testing | No | Yes | Yes | Yes |
Testing equipment requirements | Low | Medium | High | High |
Reference | Meireles et al. 2015; Pantanella et al. 2011 | Honraet et al. 2005; Martin and An 2000; Stepanovic et al. 2000 | Dostalek and Branyik 2018; Harber et al. 1983; Robrish et al. 1978 |
4 CONCLUSION
5 MATERIALS AND METHODS
5.1 Materials and reagents
5.2 Bacterial strains and bacterial cultures
Tab.2 Reference strains and plasmids used in this study |
Bacterial strain or plasmid | Genotype or phenotype | Source or reference |
---|---|---|
E. coli strains | ||
DH5α | F - mcrA Δ( mrr-hsdRMS-mcrBC)φ80 lacZ ΔM15 ΔlacX74 deoR recA1 endA1 araD139 Δ( ara leu) 7697 galU galK λ - rpsL nupG | Invitrogen |
P. aeruginosa strains | ||
PAO1 | Wild type, standard strain | This study |
PAO1- lux | Recombinant strain | This study |
Plasmids | ||
pMS402 | Expression reporter plasmid carrying the promoterless luxCDABE; Kn r, Tmp r | Liang et al. 2008 |
pKD- algU | pMS402 containing algU promoter region, Kn r, Tmp r | This study |
Mini-CTX | Integration plasmid, Tet R | Hoang et al. 2000 |
pRK2013 | Broad-host-range helper vector; Tra +, Kn r | Schweizer 1992 |