3.1. dormancy induction increased the formation of persisters in the mixed biofilm
Compared with the negative control group, the dormancy induced by tetracycline, gentamicin, rifampicin, 5-FC, fluconazole and CCCP significantly increased the level of S.epidermidis persisters (P < 0.01), and the level of persisters of the tetracycline group was the highest (P < 0.01). The dormancy induced by 5-Fc, and caspofungin significantly increased the level of C.albicans persisters (P < 0.01), and the level of persisters of the 5-Fc group was the highest (P < 0.01)(Table 1).The bacterial solution of mixed biofilm in blank control group (not induced dormancy, not sterilized), negative control group (after induced dormancy, but not sterilized), and the mixed biofilm bacterial solution obtained by reculture in the experimental group(after dormancy induction, and after sterilization), the results of strain identification were S. epidermidis and C.albicans. There was no contamination of bacteria in the operation process. According to the drug sensitivity test, the S.epidermidis strain before inducing dormancy was resistant to erythromycin, clindamycin, oxacillin, and penicillin; and was sensitive to ciprofloxacin, tetracycline, gentamicin, rifampin, linezolid, moxifloxacin, nitrofurantoin, levofloxacin, tigecycline, and vancomycin.
Table 1
The colony count of S.epidermidis and C.albicans in each group after dormancy induction(± s)
group
|
Dormancy inducing drugs
|
Drugs to kill non persister bacteria (ciprofloxacin + amphotericin B)
|
S.epidermidis
(×104 CFU)
|
C.albicans
(×104 CFU)
|
1
|
tetracycline
|
yes
|
46.17 ± 0.85*
|
0.39 ± 0.052
|
2
|
gentamicin
|
yes
|
39.27 ± 1.10
|
0.67 ± 0.073
|
3
|
rifampicin
|
yes
|
1.87 ± 0.20
|
0.51 ± 0.062
|
4
|
5-Fc
|
yes
|
1.51 ± 0.22
|
21.96 ± 0.86**
|
5
|
caspofungin
|
yes
|
1.14 ± 0.23
|
14.75 ± 0.65
|
6
|
fluconazole
|
yes
|
2.41 ± 0.15
|
8.41 ± 0.59
|
7
|
CCCP
|
yes
|
2.23 ± 0.43
|
0.53 ± 0.078
|
8(negative control)
|
none
|
yes
|
1.03 ± 0.21
|
0.73 ± 0.077
|
9(Blank control)
|
none
|
none
|
73.26 ± 1.02
|
42.16 ± 0.89
|
*Compared with the negative control group, the dormancy induced by tetracycline, gentamicin, rifampicin, 5-FC, fluconazole and CCCP significantly increased the level of S. epidermidis persisters(P < 0.01), and the level of persisters of the tetracycline group was the highest(P < 0.001). |
**Compared with the negative control group, the dormancy induced by 5-Fc, caspofungin, andcaspofunginsignificantly increased the level of C.albicans persisters(P < 0.01), and the level of persisters of the 5-Fc group was the highest(P < 0.001). |
The C.Albicans strain was sensitive to amphotericin B, fluconazole, 5-FC, caspofungin, and itraconazole. Induction of dormancy did not result in resistance of S.epidermidis and C.albicans to the antimicrobials tested. After treatment with ciprofloxacin and amphotericin B, the survival of some S.epidermidis and C.albicans was not caused by antibiotic resistance due to drug-resistant mutation (Table 2).
Table 2
The agents susceptibility to S.epidermidis and C.albicans before and after dormancy induction
Antimicrobial drug
|
MICbefore induction(μg/mL)
|
MICafter induction(μg/mL)
|
S. epidermidis
|
|
|
Ciprofloxacin
|
≤ 0.5
|
≤ 1
|
Tetracycline
|
≤ 1
|
≤ 2
|
Gentamicin
|
≤ 4
|
≤ 4
|
Rifampin
|
≤ 0.5
|
≤ 1
|
Linezolid
|
≤ 2
|
≤ 2
|
Moxifloxacin
|
≤ 0.25
|
≤ 0.25
|
Nitrofurantoin
|
≤ 16
|
≤ 16
|
Levofloxacin
|
≤ 0.125
|
≤ 0.125
|
Tigecycline
|
≤ 0.125
|
≤ 0.125
|
Vancomycin
|
≤ 2
|
≤ 2
|
Erythromycin
|
> 8
|
> 8
|
Clindamycin
|
> 8
|
> 8
|
Oxacillin
|
> 4
|
> 4
|
Penicillin
|
> 0.5
|
> 0.5
|
C.albicans
|
|
|
Amphotericin B
|
≤ 0.5
|
≤ 1
|
Fluconazole
|
≤ 1
|
≤ 2
|
5-FC
|
≤ 4
|
≤ 4
|
Caspofungin
|
≤ 0.125
|
≤ 0.125
|
Itraconazole
|
≤ 0.125
|
≤ 0.125
|
The sensitivity and specificity of PCR-SSCp assay were 71.4% and 100%, respectively.
|
3.2. Morphological and structural changes of persisters in the mixed biofilm under SEM
Under SEM, the blank control group, the negative control group, and the experimental group all showed that mixed growth of C.albicans and S.epidermidis in the mixed biofilms. The hyphae and spores of C.albicans formed the reticular structure skeleton of the mixed biofilm. In the skeleton, S.epidermidis adheres to the surface of C.albicans, gathers together to form a mass, dense and complex hybrid biofilm. The morphological characteristics are consistent with the previous research results of the research group[18]. In the experimental group, the number of mixed biofilm reduced, the density of structure decreased, the clumpy colonies decreased, and the number of bacteria and fungi is reduced(Fig. 1A, B).
The mixed biofilm treated with 5-FC and caspofungin to induce dormancy, although the ratio of yeast phase to mycelium phase of C.albicans did not change, but the surface of C.albicans has a convex morphological change, with a diameter of about 70%-80% of the host bacteria(Fig. 1C, D), the change was noted to occur in both the yeast and the mycelial phases. In the negative control group, the C.albicans in the mixed biofilm had almost no such morphological changes(Fig. 1E). Afterinduced by tetracycline and gentamicinin the experimental group, the S.epidermidis showed reduced cell division and smaller volume than normal cells(Fig. 1F, G). In the mixed biofilm in the blank control group, there were a large number of mitotic cells with uniform size (Fig. 1H).
3.3. Changes of persisters and mixed biofilms under CLSM
In the blank control group, the CSLM images showed that mixed biofilm adhered to the surface of biomaterials, the bacteria on the surface of the biofilm are mainly living, which emit green fluorescence, and are densely distributed in clumps. The average thickness of biofilm is about 37.1µm(Fig. 2A). In the negative control group, the mixed biofilm was dissolved and destroyed, and a large number of dead bacteria were seen, which emit red fluorescence. The live bacteria were distributed on the surface of the mixed biofilm in a dot shape, and hardly gather in clumps, the average thickness of biofilm is about 6.3µm(Fig. 2B). In the experimental group, the structure of the mixed biofilm was partially destroyed, the average thickness is about15.7µm. The mixed biofilm with high content of persistent bacteria did not dissolve under the killing of antibiotics. The structure of the mixed biofilm was complete and dense. On the surface of the biomaterials, there were still living bacteria gathered into clumps. The mixed biofilm with low content of persistent bacteria dissolved in large area (Fig. 2C).