Outbreak description
From January 29 to March 11, 2019, 6 patients admitted to EICU were included in this analysis. The age of patients ranged from 54 to 87 years and the patients were admitted to EICU due to respiratory failure, severe pneumonia, and septic shock. They all showed the typical symptoms of pulmonary edema, excessive sputum, pleural effusion, and shortness of breath. All 6 patients received traumatic and antibiotic treatment during their hospitalization in the EICU (Fig. 1). The antibiotics used mainly included carbapenems, cefoperazone/sulbactam, piperacillin/tazobactam, quinolones, and tigecycline. In addition, patient 2 was treated with voriconazole. The first CRKp was isolated from the sputum of patient 4, the first patient to be admitted to EICU. He had the longest survival time after CRKp isolation. The survival time of patient 3 was the shortest after CRKp isolation, which was only 3 days. Finally, only one patient improved and was discharged, four patients died during treatment in hospital, one gave up treatment and finally died on March 4, 2019 at home. The mortality rate was 83.3%.
In this study, 32 CRKp isolates were obtained, of which 11 isolates were from clinical samples of the 6 patients, including sputum, bronchoalveolar lavage fluid, and blood (Fig. 1). During the period of this study, the hospital environment was sampled on February 10 and February 20, 2019. 21 isolates were obtained from the patients' environment in the hospital, including bed sheets, pillows, sphygmomanometers, ventilators, tables, mice, keyboards, and the hands of medical staff.
In order to control the outbreak, each patient was placed in a single room. The infected patients' rooms were thoroughly disinfected with glutaraldehyde. The staffs of EICU were trained and educated in environmental disinfection and carrying out hand hygiene in a timely and effective manner. Eventually, the epidemic situation was effectively controlled. Except for these 6 patients, no other infected patients have been found in EICU to date.
Drug resistance and resistance genes of CRKp isolates
The antimicrobial susceptibility profiles of the 32 CRKp isolates are listed in Table 1. All isolates exhibited high resistance to aztreonam, cephalosporin, β-lactam/β-lactamase inhibitor combinations, carbapenems, quinolones, and aminoglycosides. Seven isolates (21.88%, 7/32) were resistant to trimethoprim-sulfamethoxazole. All isolates were susceptible to colistin, tigecycline, and ceftazidime/averbatan (100%). All strains were positive for MCIM test and negative for ECIM test.
Table 1
The antibiotic susceptibility of 32 CR-hvKP isolates (µg/ml).
isolate No.
|
KZ
|
CXM
|
CRO
|
CAZ
|
FEP
|
FOX
|
ATM
|
SAM
|
TZP
|
ETP
|
IPM
|
MEM
|
CN
|
AK
|
CIP
|
LEV
|
SXT
|
TGC
|
COL
|
CZA*
|
Kp1
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
0.5
|
23
|
Kp2
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
24
|
Kp3
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
24
|
Kp4
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
0.5
|
23
|
Kp5
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.5
|
0.5
|
23
|
Kp6
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
1
|
24
|
Kp7
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
25
|
Kp8
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
26
|
Kp9
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
1
|
25
|
Kp10
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
1
|
0.5
|
26
|
Kp11
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
1
|
0.5
|
26
|
Kp12
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
1
|
0.5
|
23
|
Kp13
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
24
|
Kp14
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
23
|
Kp15
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
1
|
24
|
Kp16
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
26
|
Kp17
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
24
|
Kp18
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
0.5
|
24
|
Kp19
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
25
|
Kp20
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
0.5
|
23
|
Kp21
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.5
|
1
|
23
|
Kp22
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.5
|
0.5
|
25
|
Kp23
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.5
|
0.5
|
26
|
Kp24
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
1
|
0.5
|
23
|
Kp25
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
1
|
0.5
|
24
|
Kp26
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
1
|
0.5
|
24
|
Kp27
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
25
|
Kp28
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
1
|
25
|
Kp29
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.25
|
0.5
|
25
|
Kp30
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
> 2/38
|
0.5
|
0.5
|
26
|
Kp31
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.5
|
0.5
|
23
|
Kp32
|
> 64
|
> 16
|
> 32
|
> 16
|
> 64
|
> 16
|
> 16
|
> 16/8
|
> 64
|
> 32
|
> 32
|
> 32
|
> 8
|
> 32
|
> 2
|
> 4
|
<=2/38
|
0.25
|
0.5
|
23
|
MIC:Minimum inhibitory concentration; KZ:cefazolin; CXM:cefuroxime; CRO:ceftriaxone; CAZ, ceftazidime; FEP, cefepime; FOX,cefoxitin; ATM, aztreonam; SAM, ampicillin sulbactam; TZP, piperacillin/tazobactam; ETP, ertapenem; IPM, imipenem; MEM, meropenem; CN:gentamicin; AK:amikacin; CIP:ciprofloxacin; LEV:levofloxacin; SXT: trimethoprim-sulfamethoxazole; TGC: tigecycline; CO: colisin; CZA: ceftazidime/avibatan. *: diameter of bacteriostatic zone. |
For the resistance to β-lactams, all 32 isolates carried the blaKPC−2, blaSHV−11, and blaTEM−1 genes (Table 2). In addition, 27 isolates carried blaCTX−14 gene, and 3 isolates (Kp15, Kp16, and Kp17) from the hospital environment of patient 3 carried blaCTX−14 and blaCTX−15 simultaneously. For aminoglycoside resistance, all isolates carried rmtB, and three isolates also carried armA. In addition, all 32 isolates carried qnrD, which may have led to quinolone resistance.
Table 2
The drug-resistance genes and virulence genes of 32 CR-hvKP isolates
Isolates no.
|
Drug-resistance genes
|
Virulence genes
|
β-lactamases
|
Aminoglycoside
|
Quinolone
|
Capsule
|
Fimbriae
|
Iron acquistion
|
blaKPC-2
|
blaSHV-11
|
blaTEM-1
|
blaCTX-M14
|
blaCTX-M15
|
rmtB
|
armA
|
qnrD
|
rmpA
|
wabG
|
uge
|
fimH
|
mrkD
|
iucB
|
Kp1
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp2
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp3
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp4
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp5
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp6
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp7
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp8
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp9
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp10
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp11
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp12
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp13
|
★
|
★
|
★
|
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp14
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp15
|
★
|
★
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp16
|
★
|
★
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp17
|
★
|
★
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp18
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp19
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp20
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp21
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp22
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp23
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp24
|
★
|
★
|
★
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp25
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp26
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp27
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp28
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp29
|
★
|
★
|
★
|
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp30
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp31
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
Kp32
|
★
|
★
|
★
|
★
|
|
★
|
|
★
|
★
|
★
|
★
|
★
|
★
|
★
|
★: positive; blank: negative |
Serotype and virulence of CRKp isolates
The string test of 32 isolates showed negative results. The serotype of all 32 isolates was K57. In the virulence gene analysis, all 32 isolates were found to contain 6 virulence genes, including fimH, iucB, mrkD, rmpA, uge, and wabG (Table 2).
We infected G. mellonella larvae with 11 CRKp isolates obtained from clinical specimens of the 6 patients. Kp1 and Kp11 from patient 1 were the least toxic, with 20% of larvae surviving after 96 hours. All larvae infected by the other 9 strains died within 96 hours. Kp3 from patient 3 was the most toxic. The 24-hour and 48-hour survival rates of larvae infected with Kp3 were 40% and 20% respectively, and all larvae died within 72 hours. The results are shown in Fig. 2.
MLST and PFGE analysis of CRKp isolates
MLST analysis revealed that all 32 CRKp isolates were of the ST11 type, which is the most common type of CRKp found in China. The results of PFGE showed that the homology of 32 isolates was more than 80%, indicating that they were a cluster. With the exception of Kp5, Kp21, Kp22, and Kp30, the homology of other 28 isolates was higher than 91.8%. Furthermore, three isolates (Kp5, Kp21, and Kp22) from patient 5 and four isolates (Kp1, Kp12, Kp13, and Kp14) from patient 1 showed complete homology (Fig. 3).