Qualitative and quantitative analysis of tectoridin in I. japonica extract
The standard tectoridin as a control, I. japonica aqueous extracts and ethanol extract under UV light color reaction were: gray under visible light, brown and fluorescent spots under UV light. Ammonia reaction were: ammonia smoked yellow or brownish yellow fluorescent spots. Aluminium trichloride colorimetric determination phenomena were: Yellowish green fluorescent spots under UV light. Hydrochloric acid - magnesium reaction was: foam was purple. The reaction results were positive, indicating that I. japonica aqueous extracts and ethanol extract contains flavonoids.
MIC of penicillin G and cefotaxime sodium to E. coli
E-test results showed that the MIC of penicillin G and cefotaxime sodium to 183 clinical isolates of E. coli resistant to β-lactam antibiotics respectively were ≥ 32 mg/mL and ≥ 16 mg/mL. These results concord with the mensuration of hospitals which used by microtube dilution methods. Therefore, the isolates were resistant to penicillin G and cefotaxime sodium.
Detection of ESBLs
PCR results showed that TEM, CTX-M,KPC and/or SHV genes all can be detected in 183 E. coli resistant to β-lactam antibiotics (Table 2). TEM (83.1%, 152/183) gene had the highest detection rate, then CTX-M (77.1%, 141/183), SHV (8.2%, 15/183), KPC had the lowest detection rate (2.2%, 4/183). As a result, TEM and CTX-M gene detection rate were significantly higher than SHV and KPC gene. 31.7% (58/183) strains carried KPC, TEM, CTX-M or SHV single gene, 68.3% (125/183) strains carried two or more ESBLs genes among the total. Therefore, the detection rate of strains who carried two or more ESBLs genes was significantly higher than the detection rate of strains who carried only one gene. 92.0% (115/125) strains carried TEM and CTX-M genes (TEM+CTX-M) among the total, and these strains were significantly higher than the number of strains who carried TEM+SHV (4.8%, 6/125) or TEM+CTX-M+SHV (3.2%, 4/125).
Owing to the gene expression mechanism of single gene strains relative to polygenic strains is relatively simple and clear, we selected E001 (TEM+), E002 (CTX-M+), E003 (KPC+), E004 (SHV+), E005 (TEM+), E006 (CTX-M+), E007 (CTX-M+), E008 (TEM+), E009 (SHV+), E010 (TEM+) to investigate the variation of β-lactam antibiotic sensitivity of these 10 strains, after the pre-incubated at 37℃ for 30 min of I. japonica aqueous extracts (250 mg/mL, 125 mg/mL and 62.5 mg/mL) and ethanol extracts (100 μg/mL, 50 μg /mL and 25 μg /mL). At the same time, we selected E001 (TEM+), E002 (CTX-M+), E003 (KPC+) and E004 (SHV+), four strains to detect the effect of I. japonica extracts to expression of ESBLs single resistance gene.
MIC and MBC of I. japonica extracts
The bactericidal results of I. japonica aqueous extracts, ethanol extracts to 183 Escherichia coli, respectively in Microtube dilution methods were MIC ≥ 500 mg/mL and ≥ 200 µg/mL, MBC ≥ 1000 mg/mL and ≥ 400 µg/mL. To provide an experimental basis for determining the working concentration of I. japonica aqueous extracts (250 mg/mL, 125 mg/mL and 62.5 mg/mL) and ethanol extracts (100 mg/mL, 50 mg/mL and 25 mg/mL), as well as exploring the variation of β-lactam antibiotic sensitivity for I. japonica extracts to E. coli, and the drug resistance gene expression (Table 3).
TABLE 2 ESBLs gene types of 183 b-lactam antibiotics-resistant isolates
Gene type
|
strains (n)
|
(%)
|
TEM
|
27
|
14.8
|
CTX-M
|
22
|
12.0
|
SHV
|
5
|
2.7
|
KPC
|
4
|
2.2
|
TEM+CTX-M
|
115
|
62.8
|
TEM+SHV
|
6
|
3.3
|
TEM+CTX-M+SHV
|
4
|
2.2
|
TABLE 3 MIC and MBC of I. japonica to 183 E. coli
|
aqueous extracts (mg/mL)
|
ethanol extracts (mg/mL)
|
MIC
|
≥ 500
|
≥ 200
|
MBC
|
≥ 1000
|
≥ 400
|
Effect of I. japonica extracts to penicillin G and cefotaxime sodium
After the incubation of 250 mg/mL, 125 mg/mL, and 62.5 mg/mL I. japonica aqueous extracts for 0.5 h, we knew that only E003 (KPC+) invariant resistant to penicillin G at 250 mg/mL I. japonica aqueous extracts, and the remaining 9 isolates were resistant to sensitive. Such as the MIC of penicillin G to E001, E004, E006 and E009 from the original 32 µg/mL to 8 µg/mL, to E002 and E005 from the original 32 µg/mL to 4 µg/mL, to E007 and E010 from the original 64 µg/mL to 16 µg/mL, while to E008, the MIC from the original 128 µg/mL changed to 16 µg/mL. While, I. japonica aqueous extracts (125 mg/mL) could only enhance the susceptibility of E002 from the original 32 µg/mL to 8 µg/mL, I. japonica aqueous extracts (62.5 mg/mL) had hardly any effect on resistant E. coli to penicillin G (Table 4).
TABLE 4 Effect of penicillin G on MIC of E. coli after I. japonica aqueous extracts
Strain
|
ESBLs gene
|
Effect of I. japonica aqueous extracts on the MIC of E. coli isolates resistant to penicillin G (mg/mL)
|
0
|
62.5
|
125
|
250
|
E001
|
TEM
|
32
|
16
|
16
|
8*
|
E002
|
CTX-M
|
32
|
32
|
8*
|
4*
|
E003
|
KPC
|
64
|
64
|
64
|
32
|
E004
|
SHV
|
32
|
32
|
32
|
8*
|
E005
|
TEM
|
32
|
32
|
16
|
4*
|
E006
|
CTX-M
|
32
|
32
|
16
|
8*
|
E007
|
CTX-M
|
64
|
32
|
32
|
16*
|
E008
|
TEM
|
128
|
128
|
64
|
16*
|
E009
|
SHV
|
32
|
16
|
16
|
8*
|
E010
|
TEM
|
64
|
64
|
32
|
16*
|
penicillin G≤ 8 μg/mL was sensitive, > 8 μg/mL was resistant; *compared with before the inhibitor, P< 0.05
After the incubation of 100 mg/mL, 50 mg/mL, and 25 mg/mL of the I. japonica ethanol extracts for 0.5 h, we knew that only E003 (KPC+) invariant resistant to penicillin G at 100 mg/mL I. japonica ethanol extracts, the remaining 9 isolates were resistant to sensitive. Such as the MIC of penicillin G to E001 and E002 from the original 32 µg/mL to 4 µg/mL, to E004, E005, E006 and E009 from the original 32 µg/mL to 8 µg/mL; to E007 and E010 from the original 64 µg/mL to 8 µg/mL or 16 mg/mL , to E008, the MIC from the original 128 µg/mL changed to 16 µg/mL, to E003, the MIC still is 64 µg/mL. While, I. japonica ethanol extracts (50 mg/mL) could only enhance the susceptibility of E001 from the original 32 µg/mL to 8 µg/mL, I. japonica ethanol extracts (25 mg/mL) had hardly any effect on resistant E. coli to penicillin G (Table 5).
TABLE 5 Effect of Penicillin G on MIC of E. coli after I. japonica ethanol extracts
strain
|
ESBLs gene
|
Effect of I. japonica ethanol extracts on the MIC of E. coli isolates resistant to penicillin G (mg/mL)
|
0
|
25
|
50
|
100
|
E001
|
TEM
|
32
|
16
|
8*
|
4*
|
E002
|
CTX-M
|
32
|
32
|
32
|
4*
|
E003
|
KPC
|
64
|
64
|
64
|
64
|
E004
|
SHV
|
32
|
32
|
16
|
8*
|
E005
|
TEM
|
32
|
32
|
32
|
8*
|
E006
|
CTX-M
|
32
|
16
|
16
|
8*
|
E007
|
CTX-M
|
64
|
64
|
32
|
8*
|
E008
|
TEM
|
128
|
128
|
128
|
16*
|
E009
|
SHV
|
32
|
32
|
32
|
8*
|
E010
|
TEM
|
64
|
64
|
16
|
16*
|
cefotaxime sodium≤ 8 μg/mL are sensitive, > 8 μg/mL are resistant; *compared with before the inhibitor, P< 0.05
After the incubation of I. japonica aqueous extracts for 0.5 h, we knew that only E003 (KPC+) invariant resistant to cefotaxime sodium at 250 mg/mL I. japonica aqueous extracts, the remaining 9 isolates were resistant to sensitive. Such as the MIC of cefotaxime sodium to E001 and E002 from the original 16 µg/mL to 4 µg/mL, to E004, E006, E007 and E009 from the original 32 µg/mL to 8 µg/mL. I. japonica aqueous extracts (125 mg/mL) could enhance the susceptibility of E001, E002, E005, E007 and E010 to cefotaxime sodium. While I. japonica aqueous extracts (62.5 mg/mL) had hardly any effect on resistant E. coli to cefotaxime sodium (Table 6).
After the incubation of I. japonica ethanol extracts for 0.5 h, we knew thar only E003 (KPC+) invariant resistant to cefotaxime sodium at 100 mg/mL I. japonica ethanol extracts, the remaining 9 isolates are resistant to sensitive. I. japonica ethanol extracts (50 mg/mL) can enhance the susceptibility of E002, E004, E007 and E008 to cefotaxime sodium. I. japonica ethanol extracts (25 mg/mL) had hardly any effect on resistant E. coli to cefotaxime sodium (Table 7).
TABLE 6 Effect of cefotaxime sodium on MIC of E. coli after I. japonica aqueous extracts
strain
|
ESBLs gene
|
Effect of I. japonica aqueous extracts on the MIC of E. coli isolates resistant to cefotaxime sodium (mg/mL)
|
0
|
62.5
|
125
|
250
|
E001
|
TEM
|
16
|
16
|
4*
|
4*
|
E002
|
CTX-M
|
16
|
16
|
4*
|
4*
|
E003
|
KPC
|
32
|
32
|
16
|
32
|
E004
|
SHV
|
32
|
32
|
16
|
8*
|
E005
|
TEM
|
32
|
16
|
8*
|
4*
|
E006
|
CTX-M
|
32
|
16
|
16
|
8*
|
E007
|
CTX-M
|
32
|
32
|
8*
|
8*
|
E008
|
TEM
|
64
|
64
|
64
|
8*
|
E009
|
SHV
|
32
|
32
|
32
|
8*
|
E010
|
TEM
|
64
|
64
|
16*
|
16*
|
penicillin G≤ 8 μg/mL are sensitive, > 8 μg/mL are resistant; *compared with before the inhibitor, P< 0.05
Effect of I. japonica extracts to the gene expression of ESBLs:
with 16S RNA as a reference, compared with the blank control group, 1/4 MIC penicillin G or cefotaxime sodium could induce more than 10 times elevation of TEM, SHV and CTX-M, while the gene expression of KPC was almost unchanged. We could find that 1/4 MIC antibiotics could induce elevation of the gene expression (Figure 1).
TABLE 7 Effect of cefotaxime sodium on MIC of E. coli after I. japonica ethanol extracts
strain
|
ESBLs gene
|
Effect of I. japonica ethanol extracts on the MIC of E. coli isolates resistant to cefotaxime sodium (mg/mL)
|
0
|
25
|
50
|
100
|
E001
|
TEM
|
16
|
16
|
8
|
4*
|
E002
|
CTX-M
|
16
|
16
|
4*
|
4*
|
E003
|
KPC
|
32
|
32
|
32
|
32
|
E004
|
SHV
|
32
|
16
|
8
|
4*
|
E005
|
TEM
|
32
|
16
|
16
|
4*
|
E006
|
CTX-M
|
32
|
32
|
8
|
4*
|
E007
|
CTX-M
|
32
|
32
|
8
|
8*
|
E008
|
TEM
|
64
|
64
|
16
|
8*
|
E009
|
SHV
|
32
|
32
|
16
|
8*
|
E010
|
TEM
|
64
|
64
|
32
|
16*
|
cefotaxime sodium≤ 8 μg/mL are sensitive, > 8 μg/mL are resistant; *compared with before the inhibitor, P< 0.05
When I. japonica aqueous extracts (250 mg/mL, 125 mg/mL or 62.5 mg/mL) and ethanol extracts (100 mg/mL, 50 mg/mL or 25 mg/mL) worked separately on E001(TEM+), 250 mg/mL I. japonica aqueous extracts and 100 mg/mL, 50 mg/mL ethanol extracts could decreased more than 50% of gene expression, demonstrating that I. japonica extracts in these concentrations could interrupt the antibiotics-induced elevation of ESBLs-mRNAs (Figure 2).
When I. japonica aqueous extracts (250 mg/mL, 125 mg/mL or 62.5 mg/mL) and ethanol extracts (100 mg/mL, 50 mg/mL or 25 mg/mL) worked separately on E002 (CTX-M+), 250 mg/mL I. japonica aqueous extracts and 100 mg/mL, 50 mg/mL ethanol extracts could decreased more than 40% of gene expression, demonstrating that I. japonica extracts in these concentrations could interrupt the antibiotics-induced elevation of ESBLs-mRNAs (Figure 3).
When I. japonica aqueous extracts (250 mg/mL, 125 mg/mL or 62.5 mg/mL) and ethanol extracts (100 mg/mL, 50 mg/mL or 25 mg/mL) worked separately on E004 (SHV+), 250 mg/mL I. japonica aqueous extracts and 100 mg/mL, 50 mg/mL ethanol extracts could decreased more than 50% of gene expression, demonstrating that I. japonica extracts in these concentrations could interrupt the antibiotics-induced elevation of ESBLs-mRNAs (Figure 4).
Effect of I. japonica extracts to the HK in TCSS:
The protein phosphorylation of E. coli E001 (TEM+), E002 (CTX-M+) and E004 (SHV+) significantly promoted, because of the induction of 1/4 MIC penicillin G and cefotaxime sodium, while the E003 (KPC+) was almost unchanged. (Figure 5) (P< 0.05)
250 mg/mL I. japonica aqueous extracts and 100 mg/mL ethanol extracts pretreated E001 (TEM+), E002 (CTX-M+) and E004 (SHV+), respectively. The phenomenon of protein phosphorylation promoted induced by 1/4 MIC penicillin G and cefotaxime sodium was inhibited. (P< 0.05). While 150 mg/mL I. japonica aqueous extracts or 50 μg/mL ethanol extracts pretreated E. coli, only the phenomenon of protein phosphorylation promoted induced by 1/4 MIC cefotaxime sodium was inhibited. (P< 0.05), yet little influence on the protein phosphorylation induced by 1/4 MIC penicillin G (Figure 6). HK inhibitor Closantel (132.5 μg/mL) was used as the positive control.