Bacterial isolates
K. pneumoniae resistance to carbapenems is defined as resistance to imipenem, meropenem or ertapenem. In total, 102 CRKP strains were identified from 674 (15.13%) KP strains collected from December 2020 to January 2022. The clinical characteristics of the 102 patients with CRKP infections are presented in Table 1. The mean±SD age of patients with CRKP infection was 75.89±16.17 (4-104 years), with an absolute male predominance of 76.47% (n=78). The CRKP strains were mainly derived from sputum specimens (68 strains, 66.67%), urine specimens (17 strains, 16.67%) and blood specimens (5 strains, 4.90%). Patients with CRKP infection were mainly located in intensive care units (ICUs, 55.88%), respiratory medicine units (PCCMs, 14.71%), and general surgery units (11.76%). The clinical diagnoses were mostly pulmonary infection, including community-acquired pneumonia (n=28, 27.45%), severe pneumonia (n=19, 18.63%), chronic obstructive pulmonary disease (n=15, 14.71%) and pneumonia (n=12, 11.76%).
Table 1. Clinical characteristics of patients with CRKP infections
|
Variable
|
n(%)
|
Demographics
|
|
|
age (years), mean±SD
|
75.89±16.17
|
|
male
|
78 (76.47)
|
Location at time of culture
|
|
|
Intensive Care Medicine (ICU)
|
57 (55.88)
|
|
Respiratory Medicine (PCCM)
|
15 (14.71)
|
|
General Surgery
|
12 (11.76)
|
|
Emergency Medicine Ward
|
7 (6.86)
|
|
Gastroenterology
|
4 (3.92)
|
|
Hematology Ward
|
3 (2.94)
|
|
Orthopedic
|
3 (2.94)
|
|
Pediatrics
|
1 (0.98)
|
Infection source
|
|
|
Sputum
|
68 (66.67)
|
|
Urine
|
17 (16.67)
|
|
Blood
|
5 (4.90)
|
|
Bronchoalveolar lavage solution
|
4 (3.92)
|
|
Pharyngeal swab
|
2 (1.96)
|
|
Decubitus ulcers
|
2 (1.96)
|
|
Secretions
|
2 (1.96)
|
|
Catheter
|
1 (0.98)
|
|
Peritoneal puncture fluid
|
1 (0.98)
|
Comorbidity
|
|
|
Community-acquired pneumonia
|
28 (27.45)
|
|
Severe pneumonia
|
19 (18.63)
|
|
Chronic obstructive pulmonary disease
|
15 (14.71)
|
|
Pneumonia
|
12 (11.76)
|
|
Septicemia
|
10 (9.80)
|
|
Cerebral hemorrhage recovery
|
7 (6.86)
|
|
Cerebral infarction sequelae
|
4 (3.92)
|
|
Type 2 diabetes
|
3 (2.94)
|
|
Gallbladder stones
|
2 (1.96)
|
|
Dementia
|
1 (0.98)
|
|
Epilepsy
|
1 (0.98)
|
Antimicrobial susceptibility
The antimicrobial susceptibility of CRKP to carbapenems, cephalosporins, quinolones, aminoglycosides, sulfonamides and tetracycline is shown in Table 2. The resistance rates of CRKP to imipenem, meropenem and ertapenem were 82.98%, 79.52% and 73.58%, respectively. The carbapenem-resistant isolates had MICs ranging from 0.25 to >16 mg/L, MIC50 >16 mg/L, and MIC90 >16 mg/L for imipenem; MICs ranging from 0.0625 to >32 mg/L, MIC50 >32 mg/L, and MIC90 >32 mg/L for meropenem; and MICs ranging from 0.12 to >8 mg/L, MIC50 >8 mg/L, and MIC90 >8 mg/L for ertapenem. The majority of the CRKP strains were extremely resistant to commonly used noncarbapenem antibiotics. Even the three compounds with the greatest inhibitory activity, MNO, CSL and AMK, inhibited CRKP by only 30.95%, 24.0% and 20.73%, respectively. In contrast, 85.57%-100% of CRKP strains were resistant to the other β-lactam antibiotics SAM (100%), CZO3 (100%), CTT (94.87%), CRO (95.6%), TZP (86.59%), CAZ (91.84%), FEP (85.57%), AMP (100%), AMC (91.23%), TIC (100%), PIP (100%), CFT (100%), CZO (100%), CXM (93.10%), ATM (95.74%), FOX (86.21%), CFM (100%), CTX (100%) and CXA (91.84%). The CRKP strains also exhibited high resistance rates to quinolones NAL (85.71%), OFX (87.50%), CIP (93.62%), LVX (91.84%), NIT (95%), TCY (100%), and SXT (88.89) and aminoglycosides GEN (95.74%) and TOB (95.74%).
Table 2. Antimicrobial susceptibility of CRKP to Carbapenems, cephalosporins, quinolones, aminoglycosides, sulfonamides, tetracycline
|
Antibiotic
|
R(%)
|
I(%)
|
S(%)
|
MIC50 (mg/L)
|
MIC90 (mg/L)
|
MIC range (mg/L)
|
IPM
|
82.98(78/94)
|
0(0/94)
|
17.02(16/94)
|
>16
|
>16
|
0.25 to >16
|
MEM
|
79.52(66/83)
|
6.02(5/83)
|
14.46(12/83)
|
>32
|
>32
|
0.0625 to >32
|
ETP
|
73.58(39/53)
|
1.89(1/53)
|
24.53(13/53)
|
>8
|
>8
|
0.12 to >8
|
ESBL
|
15.56(14/90)
|
|
|
|
|
|
SAM
|
100(39/39)
|
0(0/39)
|
0(0/39)
|
>32
|
>32
|
>32
|
CZO3
|
100(8/8)
|
0(0/8)
|
0(0/8)
|
>64
|
>64
|
>64
|
CTT
|
94.87(37/39)
|
0(0/39)
|
5.13(2/39)
|
>64
|
>64
|
4 to >64
|
CRO
|
95.6(87/91)
|
0(0/91)
|
4.4(4/91)
|
>64
|
>64
|
0.25 to >64
|
TZP
|
86.59(71/82)
|
3.66(3/82)
|
9.76(8/82)
|
>128
|
>128
|
4 to >128
|
CAZ
|
91.84(90/98)
|
0(0/98)
|
8.16(8/98)
|
>64
|
>64
|
0.12 to >64
|
FEP
|
85.57(83/97)
|
7.22(7/97)
|
7.22(7/97)
|
>32
|
>64
|
0.12 to >64
|
AMP
|
100(29/29)
|
0(0/29)
|
0(0/29)
|
>32
|
>32
|
16 to >32
|
AMC
|
91.23(52/57)
|
0(0/57)
|
8.77(5/57)
|
>32
|
>32
|
2 to >32
|
TIC
|
100(7/7)
|
0(0/7)
|
0(0/7)
|
>32
|
>32
|
>32
|
PIP
|
100(8/8)
|
0(0/8)
|
0(0/8)
|
>32
|
>32
|
>32
|
CFT
|
100(7/7)
|
0(0/7)
|
0(0/7)
|
>16
|
>16
|
>16
|
CZO
|
100(33/33)
|
0(0/33)
|
0(0/33)
|
>64
|
>64
|
4 to >64
|
CXM
|
93.10(54/58)
|
0(0/58)
|
6.90(4/58)
|
>64
|
>64
|
1 to >64
|
ATM
|
95.74(45/47)
|
0(0/47)
|
4.26(2/47)
|
>64
|
>64
|
2 to >64
|
FOX
|
86.21(50/58)
|
0(0/58)
|
13.79(8/58)
|
>64
|
>64
|
4 to >64
|
CFM
|
100(8/8)
|
0(0/8)
|
0(0/8)
|
2
|
2
|
2
|
CTX
|
100(7/7)
|
0(0/7)
|
0(0/7)
|
2
|
2
|
2
|
CSL
|
76.00(57/75)
|
0(0/75)
|
24.00(18/75)
|
>64
|
>64
|
8 to >64
|
NAL
|
85.71(6/7)
|
0(0/7)
|
14.29(1/7)
|
32
|
32
|
16 to 32
|
OFX
|
87.50(7/8)
|
0(0/8)
|
12.50(1/8)
|
4
|
4
|
2 to 4
|
CIP
|
93.62(44/47)
|
0(0/47)
|
6.38(3/47)
|
4
|
4
|
0.25 to 4
|
LVX
|
91.84(90/98)
|
2.04(2/98)
|
6.12(6/98)
|
8
|
8
|
0.12 to 8
|
NIT
|
95.00(19/20)
|
5.00(1/20)
|
0(0/20)
|
>512
|
>512
|
64 to >512
|
TCY
|
100(8/8)
|
0(0/8)
|
0(0/8)
|
8
|
8
|
8
|
MNO ND30
|
17.86(15/84)
|
51.19(43/84)
|
30.95(26/84)
|
/
|
/
|
/
|
SXT
|
88.89(80/90)
|
0(0/90)
|
11.11(10/90)
|
>320
|
>320
|
20 to >320
|
GEN
|
95.74(45/47)
|
0(0/47)
|
4.26(2/47)
|
>16
|
>16
|
1 to >16
|
TOB
|
95.74(45/47)
|
0(0/47)
|
4.26(2/47)
|
>16
|
>16
|
1 to >16
|
AMK
|
79.27(65/82)
|
0(0/82)
|
20.73(17/82)
|
>64
|
>64
|
2 to >64
|
CXA
|
91.84(45/49)
|
0(0/49)
|
8.16(4/49)
|
>64
|
>64
|
1 to >64
|
R, resistant; I, intermediate; S, susceptible; IPM, Imipenem; MEM, Meropenem; ETP, Ertapenem; ESBL, Extended spectrum β-lactamase; SAM, Ampicillin/Sulbactam; CZO3, Cefazolin (other); CTT, Cefotetan; CRO, Ceftriaxone; TZP, Piperacillin/tazobactam; CAZ, ceftazidime ; FEP, cefepime; AMP, Ampicillin; AMC, Amoxicillin/Clavulanic Acid; TIC, Ticarcillin; PIP, Piperacillin; CFT, Cefalotin; CZO, Cefazolin; CXM, Cefuroxime; ATM, Aztreonam; FOX, Cefoxitin; CFM, Cefixime; CTX, Cefotaxime; CSL, Cefoperazone/Sulbactam; NAL, Nalidixic acid; OFX, Ofloxacin; CIP, Ciprofloxacin; LVX, Levofloxacin; NIT, Nitrofurantoin; TCY, Tetracycline; MNO, Minocycline; SXT, Compound trimethoprim; GEN, Gentamicin; TOB, Tobramycin; AMK, Amikacin; CXA, Cefuroxime Axetil.
Virulence assay
Resistance profile of 23 CR-HVKP is present in Table 3. Most of CR-HVKP strains are XDR, three of the CR-HVKP strains also produced ESBL. As shown in Table 4, twenty-three (22.55%) of the 102 CRKP isolates were positive for both the string test and gene markers. The occurrence of HVKP gene markers was as follows: ybtS (100%, n =23), mrkD (100%, n =23), fimH (100%, n =23), iucA (95.65%, n =22), entB (95.65%, n =22), iutA (91.30%, n =21), fepA (91.30%, n =21), iroN (91.30%, n =21), and prmpA (8.90%, n =2). The gene markers iroB and prmpA2 were missing in all the isolates. In 86.96% (n=20) of the CR-HVKP strains, virulence genes (iucA, iutA, fepA, entB, iroN, ybtS, mrkD, and fimH) coexisted. In 8.90% (n=2) of the CR-HVKP strains, virulence genes (iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH, and prmpA) coexisted. However, the virulence genes iucA, ybtS, mrkD, and fimH, as well as entB, ybtS, mrkD, and fimH, were present in only 4.35% (n=1) of the bacteria. Capsule K1 was detected in 43.48% (n=10) of the CR-HVKP strains. The mean±SD age of patients with CR-HVKP infection was 75.89±16.17 years (64-104 years). Patients with CR-HVKP infection are mainly located in the ICU. CR-HVKP strains are mainly derived from sputum and urine.
Table 3. Resistance profile of 23 highly virulent k.pneumoniae.
|
|
No.
|
Isolates
|
MIC of antimicrobials
|
ESBL
|
Resistance profile
|
Degree of resistance
|
Mer (mg/L)
|
IMP (mg/L)
|
1
|
65821
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, CZO, ATM, CIP, LVX, SXT, GEN, TOB, AMK, CSL
|
XDR
|
2
|
61977
|
>32
|
0.25
|
Pos
|
MER, SAM, CTT, CRO, TZP, CAZ, CXM, LVX, SXT, GEN, TOB, CXA
|
MDR
|
3
|
62811
|
2
|
16
|
Neg
|
MER, IMP, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, CSL, ETP, LVX, SXT, TOB, AMK, CXA
|
XDR
|
4
|
61940
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CIP, LVX, NIT, SXT, GEN, TOB, AMK
|
XDR
|
5
|
54559
|
32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, CSL, ETP, LVX, SXT, GEN, TOB, AMK, CXA
|
XDR
|
6
|
55822
|
32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CSL, CIP, LVX, NIT, SXT, GEN, TOB
|
XDR
|
7
|
58123
|
2
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CSL, CIP, LVX, SXT, GEN, TOB, AMK
|
XDR
|
8
|
66974
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, CZO, ATM, CSL, CIP, LVX, SXT, GEN, TOB, AMK
|
XDR
|
9
|
65852
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CIP, LVX, NIT, SXT, GEN, TOB, AMK
|
XDR
|
10
|
61921
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CIP, LVX, NIT, SXT, GEN, TOB, AMK
|
XDR
|
11
|
57802
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, CSL, ETP, LVX, SXT, GEN, TOB, AMK, CXA
|
XDR
|
12
|
67835
|
8
|
2
|
Neg
|
MER, CRO, TZP, CAZ, FEP, TIC, PIP, CXM, CZO, ATM, CFM, CTX, NAL, OFX, CIP, LVX, TCY, MNO, NIT, SXT, GEN, TOB, CSL
|
XDR
|
13
|
65640
|
2
|
8
|
Neg
|
MER, IMP, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, ETP, LVX, MNO, SXT, TOB, AMK, CXA,CSL
|
XDR
|
14
|
62080
|
0.0625
|
4
|
Pos
|
MER, IMP, SAM, CRO, AMP, CZO, NIT, LVX, SXT, CXA
|
MDR
|
15
|
65977
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CIP, LVX, NIT, MNO, SXT, GEN, TOB, AMK, CSL
|
XDR
|
16
|
59572
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, ATM, CSL, CIP, LVX, NIT, SXT, GEN, TOB, AMK
|
XDR
|
17
|
57773
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, ATM, CSL, CIP, LVX, NIT, SXT, GEN, TOB, AMK
|
XDR
|
18
|
60485
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CXM, FOX, CSL, ETP, LVX, NIT, SXT, GEN, TOB, AMK, CXA
|
XDR
|
19
|
67859
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMP, CZO, ATM, CIP, LVX, NIT, SXT, GEN, TOB
|
XDR
|
20
|
66798
|
32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, CSL, ETP, LVX, SXT, GEN, TOB, AMK, CXA
|
XDR
|
21
|
64415
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, CAZ, FEP, CZO, ATM, CIP, LVX, SXT, GEN, TOB, AMK, CSL
|
XDR
|
22
|
59579
|
>32
|
16
|
Neg
|
MER, IMP, SAM, CTT, CRO, TZP, CAZ, FEP, AMC, CXM, FOX, CSL, ETP, LVX, SXT, GEN, TOB, AMK, CXA
|
XDR
|
23
|
55182
|
0.0625
|
4
|
Pos
|
MER, IMP, CRO, CXM, ETP, LVX, SXT, CXA
|
MDR
|
MEM, Meropenem; IMP, Imipenem; ETP, Ertapenem; ESBL, Extended spectrum β-lactamase; SAM, Ampicillin/Sulbactam; CTT, Cefotetan; CRO, Ceftriaxone; TZP, Piperacillin/tazobactam; CAZ, ceftazidime ; FEP, cefepime; AMP, Ampicillin; AMC, Amoxicillin/Clavulanic Acid; TIC, Ticarcillin; PIP, Piperacillin; CZO, Cefazolin; CXM, Cefuroxime; ATM, Aztreonam; FOX, Cefoxitin; CFM, Cefixime; CTX, Cefotaxime; CSL, Cefoperazone/Sulbactam; NAL, Nalidixic acid; OFX, Ofloxacin; CIP, Ciprofloxacin; LVX, Levofloxacin; NIT, Nitrofurantoin; TCY, Tetracycline; MNO, Minocycline; SXT, Compound trimethoprim; GEN, Gentamicin; TOB, Tobramycin; AMK, Amikacin; CXA, Cefuroxime Axetil; .Neg Negative, Pos Positive, ND Not detected.
|
Table 4. Virulence characteristics of 23 highly virulent Klebsiella pneumoniae
|
No.
|
Isolates
|
Age (years)
|
HD
|
Infection source
|
String Test
|
Virulence genes
|
Capsule
|
ST
|
1
|
65821
|
89
|
EMW
|
Blood
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
2
|
61977
|
75
|
EMW
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
3
|
62811
|
83
|
GA
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH, prmpA
|
K1
|
ST11
|
4
|
61940
|
81
|
GS
|
Secretions
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
5
|
54559
|
79
|
GS
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
6
|
55822
|
82
|
GS
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
7
|
58123
|
84
|
GS
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
8
|
66974
|
90
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH, prmpA
|
K1
|
ST11
|
9
|
65852
|
104
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
10
|
61921
|
76
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
11
|
57802
|
85
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
12
|
67835
|
79
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
13
|
65640
|
79
|
ICU
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
14
|
62080
|
94
|
ICU
|
Sputum
|
Pos
|
iucA, ybtS, mrkD, fimH
|
ND
|
ST11
|
15
|
65977
|
85
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
16
|
59572
|
64
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
17
|
57773
|
81
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
18
|
60485
|
83
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST690
|
19
|
67859
|
85
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
K1
|
ST11
|
20
|
66798
|
86
|
ICU
|
Urine
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
21
|
64415
|
91
|
PCCM
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
22
|
59579
|
75
|
PCCM
|
Sputum
|
Pos
|
iucA, iutA, fepA, entB, iroN, ybtS, mrkD, fimH
|
ND
|
ST11
|
23
|
55182
|
86
|
PCCM
|
Sputum
|
Pos
|
entB, ybtS, mrkD, fimH
|
ND
|
ST1108
|
HD, Hospital Department; AR, antimicrobial resistance; PCCM, Respiratory Medicine; EMW, Emergency Medicine Ward; GS, General Surgery; GA, Gastroenterology; The related genes iucA, iutA and iroN are typically plasmid-encoded, which associated with Iron uptake; The rmpA gene-encoded HVKP gene is hypermucoviscous. The fimH and mrkD mediate adhesion by encoding KP type 1 and type 3 fimbriae, and mrkD may promote the development of biofilms. Neg Negative, Pos Positive, ND Not detected.
|
Mouse survival assay
SPF Kunming mice were injected intraperitoneally with various titres of the 200 µL CR-HVKP strains to assess the survival rates. Figure 1 shows that the survival rates (%) of the mice were 33.33% vs 0% (NTUH-K2044), 50.00% vs 0% (KP66974), 83.33% vs 0% (KP62811), and 100.00% vs 0% (KP65640) when the mice were challenged with 2 × 103 to 5 × 103 CFU and 3 × 105 to 5 × 105 CFU, respectively. The survival rate of mice was 100% when challenged with KP55182 and HS11286 at different concentrations ranging from 2 × 103 to 5 × 103 CFU, 3 × 105 to 5 × 105 CFU and 3 × 107 to 6 × 107 CFU.
Genotypic prevalence and multilocus sequence typing
The MLST and PCR amplification results of the carbapenem-ESBL-quinolones-aminoglycosides-sulfonamides-resistance genes of 23 CR-HVKP strains are presented in Table 4 and Figure 2. MLST revealed an overwhelming prevalence of ST11 (91.30%, 21/23) among the CR-HVKP strains, followed by ST690 (4.35%, 1/23) and ST1108 (4.35%, 1/23). Eighteen CR-HVKP strains were resistant to meropenem (17 strains had MICs ≥ 32 mg/L), three strains had intermediate MICs, and two strains were susceptible. Twenty-one CR-HVKP strains were resistant to imipenem (18 strains had an MIC =16 mg/L), one strain was an intermediate strain, and one strain was susceptible. The distribution of carbapenem target genes among CR-HVKP strains was as follows: blaKPC (91.30%, n = 21/23), blaVIM (86.96%, n = 20/23), blaSPM (39.13%, n = 9/23), blaGES (34.78%, n = 8/23) and blaNDM (8.70%, n = 2/23). blaIMP and blaOXA-48 were not detected in any of the isolates. Three strains of CR-HVKP produced ESBL. The distribution of ESBL genes among CR-HVKP strains was as follows: blaTEM (100%, n = 23/23), blaCTX-M9 (95.65%, n= 22/23), blaSHV (82.61%, n = 19/23), blaCTX-M2 (56.52%, n= 13/23) and blaCTX-M1 (13.04%, n= 3/23). blaCTX-M8 was not detected in any of the isolates. Twenty-two strains of CR-HVKP are resistant to aminoglycosides. The distribution of aminoglycoside target genes among CR-HVKP strains was as follows: rmtB (95.65%, n= 22/23), ant(3'')-I (86.96%, n=20/23), armA (65.22%, n= 15/23), aac(6')-II (21.74%, n = 5/23), and aac(3')-II (13.04%, n = 3/23). aac(6')-Ib and aph(3')-VI were not detected in any of the isolates. All the CR-HVKP strains were resistant to quinolone. The distribution of quinolone target genes among CR-HVKP strains was as follows: aac (6')-Ib-cr (100%, n = 23/23), QnrS (86.96%, n= 20/23), OqxA (86.21%, n=19/23), QnrB (60.87%, n = 14/23), QnrD (43.48%, n= 10/23), Qep (30.43%, n= 7/23), QnrA (17.39%, n = 4/23), QnrC (8.70%, n=2/23) and OqxB (8.70%, n= 2/23). All the CR-HVKP strains were resistant to sulfonamide. The distribution of sulfonamide target genes among CR-HVKP strains was as follows: sul2 (100%, n = 23/23) and sul1 (73.91%, n = 17/23).
Conjugation Assay
The iucA, iutA, iroN, prmpA, blaKPC, blaNDM, blaVIM, blaSHV, blaTEM, rmtB, QnrA, QnrB, and QnrS genes were successfully transferred from KP 66974 to E. coli J53, and the conjugative transfer frequencies ranged from 1.3×10-11 to 2.6×10-5. The genes fepA, entB, ybtS, mrkD, fimH, blaCTX-M2, blaCTX-M9, aac (6')-Ib-cr, ant(3'')-I, and OqxA were not transferred to E. coli J53. Compared with those of the recipient E. coli J53, the MICs of meropenem, imipenem, levofloxacin, and amikacin increased 64-fold (from 0.5 to 32 mg/L), >16-fold (from <1 to 16 mg/L), 128-fold (from <0.0625 to 8 mg/L), and 64-fold (from <1 to 64 mg/L), respectively. The genes iucA, iutA, iroN, blaKPC, blaGES, blaVIM, blaSHV, blaTEM, rmtB, QnrB, QnrD and QnrS were successfully transferred from KP65640 to E. coli J53, and conjugative transfer frequencies ranged from 4.2 ☓10-12 to 3.8☓10-4. Compared with those of the recipient E. coli J53, the MICs of meropenem, imipenem, levofloxacin and amikacin in the transconjugants increased by 4-128 times (Table 5).
Table 5 Transferability of virulence genes and resistance genes
Organism name
|
KP66974
|
E.coli J53
|
Transconjugants
|
KP65640
|
E.coli J53
|
Transconjugants
|
Meropenem, MIC (mg/L)
|
>32
|
0.5
|
>32
|
2
|
0.5
|
2
|
Imipenem, MIC (mg/L)
|
16
|
<1
|
16
|
8
|
<1
|
8
|
Levofloxacin, MIC (mg/L)
|
8
|
0.0625
|
8
|
8
|
0.0625
|
8
|
Amikacin, MIC (mg/L)
|
>64
|
<1
|
>64
|
>64
|
<1
|
>64
|
Gene markers
|
iucA, iutA, iroN, prmpA, blaKPC, blaNDM, blaVIM, blaSHV, blaTEM, rmtB, QnrA, QnrB, QnrS
|
Pos
|
Neg
|
Pos
|
|
|
|
Conjugative transfer frequencies
|
|
|
1.3☓10-11 to 2.6☓10-5
|
|
|
|
Gene markers
|
fepA, entB, ybtS, mrkD, fimH, blaCTX-M2, blaCTX-M9, aac (6')-Ib-cr, ant(3'')-I, OqxA
|
Pos
|
Neg
|
Neg
|
|
|
|
Gene markers
|
iucA, iutA, iroN, blaKPC, blaGES, blaVIM, blaSHV, blaTEM, rmtB, QnrB, QnrD, QnrS
|
|
|
|
Pos
|
Neg
|
Pos
|
Conjugative transfer frequencies
|
|
|
|
|
|
4.2 ☓10-12 to 3.8☓10-4
|
Gene markers
|
fepA, entB, ybtS, mrkD, fimH, blaCTX-M2, blaCTX-M9, aac (6')-Ib-cr, ant(3'')-I, OqxA, Qep
|
|
|
|
Pos
|
Neg
|
Neg
|
Antimicrobial susceptibility of CRKP and CR-HVKP to tigecycline, colistin and fosfomycin
The antimicrobial susceptibilities of CRKP and CR-HVKP to tigecycline, colistin and fosfomycin are presented in Table 6. The susceptibility rates of CRKP to tigecycline, colistin and fosfomycin were 96.20%, 0% and 1.27%, respectively, with MICs ranging from 0.25 to 16 mg/L, MIC50 =0.5 mg/L, and MIC90 =2 mg/L for tigecycline; MICs ranging from 0.5 to 16 mg/L, MIC50 =1 mg/L, and MIC90 =2 mg/L for colistin; and MICs ranging from 64 to >512 mg/L, MIC50 =256 mg/L, and MIC90 >512 mg/L for fosfomycin. The susceptibility rates of CR-HVKP to tigecycline, colistin and fosfomycin were 100%, 0 and 0, respectively, with MICs ranging from 0.25 to 2 mg/L, MIC50 =0.5 mg/L, and MIC90 =2 mg/L for tigecycline; MICs ranging from 0.5 to 2 mg/L, MIC50 =1 mg/L, and MIC90 =2 mg/L for colistin; and MICs ranging from 128 to >512 mg/L, MIC50 =256 mg/L, and MIC90 =512 mg/L for fosfomycin.
Table 6. Antimicrobial susceptibility of CRKP to tigecycline, colistin and fosfomycin
|
Isolates
|
Antibiotic
|
R (%)
|
I (%)
|
S (%)
|
MIC50 (mg/L)
|
MIC90 (mg/L)
|
MIC range (mg/L)
|
CRKP (n=79)
|
Tigecycline
|
1.27 (1/79)
|
2.53 (2/79)
|
96.20 (76/79)
|
0.5
|
2
|
0.25 to 16
|
|
Colistin
|
1.27 (1/79)
|
98.73 (78/79)
|
0 (0/79)
|
1
|
2
|
0.5 to 16
|
|
Fosfomycin
|
87.34 (69/79)
|
11.39 (9/79)
|
1.27 (1/79)
|
256
|
>512
|
64 to >512
|
Hv-CRKP (n=23)
|
Tigecycline
|
0 (0/23)
|
0 (0/23)
|
100 (23/23)
|
0.5
|
2
|
0.25 to 2
|
|
Colistin
|
0 (0/23)
|
100 (23/23)
|
0 (0/23)
|
1
|
2
|
0.5 to 2
|
|
Fosfomycin
|
91.30 (21/23)
|
8.70 (2/23)
|
0 (0/23)
|
256
|
512
|
128 to >512
|
Tigecycline, susceptibility ≤ 2 mg/L, intermediates =4 mg/L, resistant ≥ 8 mg/L; Colistin, intermediates ≤2 mg/L, resistant ≥4 mg/L; Fosfomycin, susceptibility≤ 64 mg/L, intermediates =128 mg/L, resistant ≥ 256 mg/L.
|