2.1 Clinical information of CRKP strains
A total of 162 non-repetitive strains of CRKP were isolated from clinical specimens in SYS Memorial Hospital from 2015 to 2019. The average age of patients was 56.3 ± 20.7 years old, among which 115 were male patients.
2.1.1 The annual detection amount of CRKP strains
From 2015 to 2019, the detection amount of CRKP showed an overall increasing trend year by year, and there was a rapid rising peak in 2017, as shown in Figure 1A.
2.1.2 Distribution of CRKP strains in different specimen types
The top four specimen types of the 162 strains of CRKP were sputum, urine, ascites/abdominal drainage fluid and blood, accounting for 45.7% (74/162), 13.0% (21/162), 11.1% (18/162) and 9.9% (16/162), respectively, as shown in Figure 1B. The number of the first sputum was more than three times higher than the number of the second urine.
2.1.3 Distribution of CRKP strains in different departments
In terms of inpatient ward distribution of CRKP strains, the top three departments were, the ICU, neurology/neurosurgery and rehabilitation departments, accounting for 41.4% (67/162), 10.5% (17/162) and 8.0% (13/162), respectively, as shown in Figure 1C. The number of CRKP strains in ICU was much higher than in other departments, and was about four times higher than that in neurology/neurosurgery, the secondary department.
2.1.4 Composition of departments that detected CRKP annually
From 2015 to 2019, with the increase in the detection amount of CRKP strains, the detection departments also increased year by year, among which, the ICU, hepatopancreatobiliary surgery and hematology department detected CRKP every year, and the detection amount of CRKP in the ICU department was the largest every year. The sudden increase in the detection amount in 2017 mainly occurred in the ICU department, as shown in Table S2.
2.1.5 Distribution of specimen types in the first six departments that detected CRKP
By analyzing the distribution of specimen types in the first six departments which detected CRKP, it was found that CRKP could be detected from all parts of the patient in ICU, even vaginal secretions, of which sputum, ascites/abdominal drainage fluid, blood and catheter were the most commonly detected specimen types. Neurology/neurosurgery patients most commonly detected CRKP from sputum specimens. Rehabilitation patients most commonly detected CRKP in sputum and urine specimens. Urology/nephrology patients most commonly detected CRKP in urine specimens. Hematologic patients often detected CRKP in blood, catheters, and feces. Hepatopancreatobiliary surgery patients often detected CRKP from ascites/abdominal drainage fluid and bile, as shown in Table S3.
2.2 Antimicrobial sensitivity profile
In 162 strains of CRKP, no strains were detected to be resistant to colistin or polymyxin B, while the resistance rate of tigecycline was 10% and the resistance rate of other commonly used antibiotics were all higher than 50%, as shown in Figure 1D.
2.3 The MIC and MBC results of CRKP strains
2.3.1 Analysis of resistance of CRKP strains to seven chemical disinfectants commonly used in clinic
The MIC and MBC results of seven clinically used chemical disinfectants against CRKP strains were expressed by diluted multiples. The higher the diluted multiple is, the lower the MIC and MBC values are. MIC50 and MIC90 of the CRKP strains were compared with the MIC values of the standard strains ATCC 29522 and ATCC 700603. If the diluted multiple was reduced, it indicated that the CRKP strains showed resistance to the disinfectant. Analyze the MBC data in the same way. The results are shown in Table 1.
Table 1. The MIC and MBC results of CRKP strains against seven kinds of disinfectants (dilution multiples)
Disinfectants
|
MICs
|
MIC50
|
MIC90
|
QC strains
|
MBCs
|
MBC50
|
MBC90
|
QC strains
|
ATCC 700603
MIC
|
ATCC 25922
MIC
|
ATCC 700603
MBC
|
ATCC 25922
MBC
|
0.1% Benzalkonium bromide
|
16-256
|
64
|
32
|
32
|
128
|
1-128
|
16
|
2
|
4
|
32
|
4% Aqueous chlorhexidine
|
256-32768
|
2048
|
512
|
1024
|
16384
|
16-32768
|
512
|
32
|
256
|
512
|
75% Alcohol
|
4-64
|
8
|
8
|
8
|
8
|
2-64
|
4
|
4
|
4
|
4
|
Entoiodine II
|
64-8192
|
512
|
256
|
256
|
16384
|
16-4096
|
128
|
32
|
128
|
8192
|
2% Glutaraldehyde
|
8-128
|
32
|
16
|
64
|
64
|
4-64
|
16
|
8
|
32
|
64
|
2000 mg/L Chlorine-containing disinfectants
|
2-16
|
8
|
4
|
8
|
8
|
2-8
|
8
|
4
|
8
|
8
|
3% Hydrogen peroxide
|
256-2048
|
1024
|
512
|
1024
|
2048
|
64-2048
|
512
|
256
|
512
|
1024
|
Note: MIC50 represents the MIC value required to inhibit the growth of 50% of the tested bacteria, and MIC90 represents the MIC value required to inhibit the growth of 90% of the tested bacteria. MBC50 represents the MBC value required to kill 50% of the tested bacteria, and MBC90 represents the MBC value required to kill 90% of the tested bacteria. MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration; QC, quality control; ATCC, American type culture collection.
2.3.2 Recommended concentration for CRKP disinfection
We referred to China's nosocomial infection control industry standards and combined with the maximum MBC values of seven disinfectants in our study, in order to giving recommended concentrations for CRKP disinfection. Detailed results are shown in Table 2.
Table 2. Recommended concentration for CRKP disinfection according to health industry standards of the People's Republic of China
Note: a. Presents maximum MBC values of seven disinfectants in our study. b. Referring to regulation of disinfection technique in healthcare settings. c. Referring to regulation for cleaning and disinfection management of environmental surface in healthcare. d. Referring to regulation for cleaning and disinfection technique of flexible endoscope. e. If MBCMAX is less than or equal to healthcare industry standard requirements, it is marked green, otherwise, it is marked red and listed in accordance with recommended concentrations provided by MBCMAX. MBC, minimum bactericidal concentration.
2.3.3 Comparisons of the MICs and MBCs of CRKP strains isolated from different wards and different specimens for each disinfectant
The results of MIC and MBC of CRKP strains against seven commonly used clinical disinfectants were divided into two groups according to ICU ward and non-ICU wards, and statistically analyzed by Mann-Whitney U test of two independent samples (Figure 2). Only the MIC results of aqueous chlorhexidine were found to be statistically different between the ICU ward and non-ICU wards (p <0.05) (Figure 2B), the MIC and MBC results of other disinfectants showed no statistical difference between ICU ward and non-ICU wards. On the other hand, the results of MIC and MBC were divided into four groups as invasive specimen (bile, catheter, ascites/abdominal drainage fluid, blood), skin and soft tissue, urine and sputum, and statistically analyzed by Kruskal-Wallis test. But no statistical difference between different specimen groups was found (Figure 3).
2.4 Analysis of efflux pump genes
2.4.1 Detection of efflux pump genes
The detection rates of the three efflux pump genes (oqxA , oqxB and qacE△1-sul1 ) were 68.8% (44/64), 15.6% (10/64) and 89.1% (57/64), respectively. Partial results as shown in Figure S1-S3. There were 5 positive detection patterns among the 3 efflux pump genes of the 64 strains of CRKP, among which the qacE△1-sul1+oqxA positive pattern was the dominant one, accounting for 48.4%, followed by the single gene qacE△1-sul1 positive one, accounting for 28.1%, as shown in Table 3.
Table 3. Positive detection patterns of efflux pump genes
Positive detection patterns
|
Positive numbers (n)
|
Percentage (%)
|
qacE△1-sul1+oqxA
|
31
|
48.4
|
qacE△1-sul1
|
18
|
28.1
|
qacE△1-sul1+oqxA+oqxB
|
8
|
12.5
|
oqxA
|
3
|
4.7
|
oqxA+oqxB
|
2
|
3.1
|
2.4.2 Correlation between efflux pump genes and disinfectant resistance
The results of MIC and MBC of the disinfectants were divided into two groups according to the negative and positive genes of efflux pump, and the t test of two independent samples was used for statistical analysis. There was a statistical difference in the MIC values of 0.1% benzalkonium bromide between the negative and positive oqxA gene groups (p < 0.05), while the MIC values of entoiodine II was significantly different between negative and positive qacE△1-sul1 gene groups (p < 0.05), and there was no statistical difference between the negative and positive efflux pump gene groups among the other disinfectants (detailed in Table 4).
Table 4. Comparison of MIC and MBC values of disinfectants in negative and positive groups of efflux pump genes (p value)
Genes
|
Numbers
|
0.1% Benzalkonium bromide
|
4% Aqueous chlorhexidine
|
75% Alcohol
|
Entoiodine II
|
2% Glutaraldehyde
|
2000 mg/L Chlorine-containing disinfectants
|
3% Hydrogen peroxide
|
|
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
MIC
|
MBC
|
|
oqxA
|
+:44
|
0.046*
|
0.22
|
0.89
|
0.78
|
0.24
|
0.43
|
0.93
|
0.95
|
0.15
|
0.61
|
0.16
|
0.37
|
0.63
|
0.82
|
|
-:20
|
|
oqxB
|
+:10
|
0.72
|
0.88
|
0.55
|
0.65
|
0.98
|
0.96
|
0.31
|
0.30
|
0.74
|
0.75
|
0.23
|
0.35
|
0.16
|
0.13
|
|
-:54
|
|
qacE△1-sul1
|
+:57
|
0.59
|
0.62
|
0.13
|
0.61
|
0.76
|
0.92
|
0.037*
|
0.22
|
0.76
|
0.69
|
0.87
|
0.71
|
0.60
|
0.58
|
|
-:7
|
|
Note: * p<0.05. MIC, minimum inhibitory concentration; MBC, minimum bactericidal concentration.