Genotypic Features Of Cpe Isolates
A total of 930 phenotypically confirmed CRE isolates were screened for carbapenemase genes, 922 isolates were detected positive including 780 KPC-2 isolates, 127 NDM isolates, 7 IMP-4 isolates and 8 KPC-2 and NDM co-producing isolates (Table 1). MLST and wzi assigned 785 carbapenemase-producing KP (CPKP) isolates to 26 STs and 32 wzi alleles (wzis), respectively. Among them, KPC-2 KP accounted for the largest proportion (92.87%, 729/785) with ST11-wzi209 being the most prevalent type (60.63%, 442/729), followed by ST11-wzi141 (21.95%, 160/729) (Table 2). The 96 carbapenemase-positive E.coli isolates contained 36 KPC-2 isolates belonging to 9 distinct STs with a predominance of ST43 (55.56%, 20/36) and 60 NDM isolates belonging to 20 unique STs (13 isolates unidentified) dominated by ST167 (36.17%, 17/47).
Table 1
Distribution of carbapenemase-producing Enterobacteriaceae isolates and their carbapenemase genes
Organisms
|
Carbapenemase genes
|
No. of clinical isolates
in our hospital
|
No. of isolates from fecal specimens in our hospital
|
No. of clinical isolates in other hospitals
|
Klebsiella pneumoniae
|
|
|
|
|
KPC-2
|
517
|
90
|
122
|
NDM-1
|
11
|
|
24
|
NDM-5
|
6
|
2
|
2
|
IMP-4
|
2
|
|
2
|
KPC-2 and NDM-1 or 5
|
6
|
|
1
|
Escherichia coli
|
KPC-2
|
30
|
6
|
|
NDM-1
|
9
|
1
|
2
|
NDM-5
|
23
|
1
|
20
|
NDM-7
|
1
|
|
|
NDM-9
|
2
|
|
1
|
Enterobacter cloacae
|
NDM-1
|
7
|
|
2
|
Klebsiella oxytoca
|
KPC-2
|
3
|
1
|
|
NDM-1
|
4
|
1
|
|
IMP-4
|
|
|
3
|
Other species
|
KPC-2
|
10
|
1
|
|
NDM-1
|
5
|
|
3
|
NDM-5 and KPC-2
|
1
|
|
|
Table 2
Distribution of common STs and wzi alleles in carbapenemase-producing Klebsiella pneumoniae
Sequence types
|
wzi alleles
|
Carbapenemase genes
|
No. of clinical isolates
in our hospital*
|
No. of isolates from fecal specimens in our hospital
|
No. of clinical isolates in other hospitals**
|
ST11
|
wzi209
|
KPC-2
|
277
|
58
|
107
|
ST11
|
wzi141
|
KPC-2
|
149
|
8
|
1
|
ST11
|
wzi141
|
KPC-2 and NDM-5
|
2
|
|
|
ST11
|
wzi64
|
KPC-2
|
17
|
|
1
|
ST11
|
wzi89
|
KPC-2
|
3
|
1
|
1
|
ST11
|
wzi133
|
KPC-2
|
1
|
|
2
|
ST11
|
-a
|
KPC-2
|
2
|
2
|
2
|
ST15
|
wzi384
|
KPC-2
|
18
|
5
|
7
|
ST15
|
wzi173
|
KPC-2
|
|
|
2
|
ST15
|
wzi19
|
KPC-2
|
2
|
|
1
|
ST17
|
wzi141
|
NDM-5
|
3
|
2
|
|
ST20
|
wzi84
|
NDM-1 or 5
|
1
|
|
10
|
ST23
|
wzi1
|
KPC-2 or NDM-1
|
13
|
|
|
ST437
|
wzi109
|
KPC-2
|
21
|
6
|
|
ST617
|
wzi162
|
KPC-2
|
6
|
6
|
|
ST2068
|
wzi381
|
NDM-1
|
1
|
|
3
|
ST307
|
wzi173
|
IMP-4
|
|
|
2
|
ST48
|
wzi62
|
KPC-2 or NDM-1
|
3
|
1
|
|
ST896
|
wzi59
|
KPC-2
|
3
|
2
|
|
ST101
|
wzi137
|
KPC-2
|
|
1
|
|
ST101
|
wzi29
|
KPC-2
|
1
|
|
|
ST37
|
wzi64
|
NDM-1
|
1
|
|
|
ST37
|
wzi50
|
IMP-4 or NDM-1
|
1
|
|
2
|
ST37
|
wzi14
|
NDM-1
|
|
|
1
|
a : No detected, *No. of clinical isolates in our hospital include: Another 17 isolates belonging to 8 STs and 8 wzis, **No. of clinical isolates in other hospitals include: Another 9 isolates belonging to 5 STs and 5 wzis |
Among the 533 CRE patients, 528 cases were confirmed to have CPE colonization and/ or infection in which 127 consecutive cases with clinical infections underwent stool screening, 96 of whom had CPE in rectal cultures (75.59%, 96/127). Eighty-six patients infected by CPE strains also intestinally colonized with the same organisms. A total of 103 CPE isolates were recovered from the rectal screening cultures. The predominant species identified were KP (n = 92), with ST11-wzi209 KPC-2 KP being the most frequently isolated, followed by E. coli (n = 8) (Table 2).
ST11-wzi209 KPC-2 KP was established in all hospitals and some of which could be traced back to patients transfers between hospitals. ST20-wzi84 NDM-1 KP and ST167 NDM-5 E.coli were detected in 5 hospitals. Some ST11 KPC-2 subclones (wzi89, wzi133, wzi64 and wzi141) and ST15 KPC-2 subclones (wzi173, wzi19) based on capsular typing were found in two hospitals, Some STs-wzis or species (ST15-wzi384, ST2068-wzi381, IMP-4 K.oxytoca) were observed in three hospitals, and other relatively rare STs or wzis were only confined to their individual settings.
Clinical Characteristics Of Cpe Patients
The majority of CPE patients (68%) were male, but no statistical differences in clinical features between sexes were observed (Table 3). Further analysis showed that CPE patients were becoming increasingly younger, especially evident in 2016. This could be explained by the rapid increase in the number of patients under the age of 65 years (Table 3). One hundred and forty-seven (97.33%, 146/150) of these young cases were nosocomially acquired and the main reasons for primary admission were cerebral hemorrhage (41.45%), motor vehicle accident (11.18%), pulmonary infection (10.53%), fall injury (8.55%) and tumor (6.58%). More than half patients had a previous hospital admission within 1 month before the current admission prior to 2018, but this proportion decreased in 2018. As shown in Table 3, the average length of hospitalization of CPE patients showed a downward trend mainly due to significant increases in voluntarily discharged rates and mortality rates. The number of intra-hospital transfers of CPE patients increased gradually from 18 (17.82%, 101) in 2015 to 63 (30.73%, 205) in 2018 (p = 0.016). There was a statistically significant increase in the number of inter-hospital transfers of CPE patients [12 (11.88%, 101) in 2015 vs 51 (24.88%, 205) in 2018 (p = 0.008)].
Table 3 Characteristics of patients with carbapenemase-producing Enterobacteriaceae according to the years
|
2014-18
Male (n = 357) Female (n = 168)
No. (%) No. (%)
|
2014 ( n = 25 )
No. (%)
|
2015 (n = 101)
No. (%)
|
2016 (n = 104)
No. (%)
|
2017 ( n = 90)
No. (%)
|
2018 (n = 205)
No. (%)
|
Age (years)
|
72.17 ± 16.55
|
72.20 ± 15.14
|
80.36 ± 10.70
|
76.94 ± 13.60
|
71.08 ± 17.26
|
69.84 ± 16.61
|
70.47 ± 16.27
|
P value
|
0.641
|
|
0.213
|
0.034
|
0.494
|
0.738
|
Patients under 65 years (No.)
|
103 (28.85)
|
47 (27.98)
|
2 (8)
|
13 (12.87)
|
38(36.54)
|
34 (37.78)
|
63 (30.73)
|
P value
|
0.836
|
|
0.743
|
< 0.001
|
0.859
|
0.236
|
Previous hospitalization within last one month
|
209 (58.54)
|
88(52.38)
|
19 (76)
|
71 (70.3)
|
65 (62.5)
|
49 (54.44)
|
93 (45.37)
|
P value
|
0.184
|
|
0.572
|
0.238
|
0.256
|
0.151
|
Hospital length of stay (days)
|
42.20 ± 37.56
|
42.35 ± 41.08
|
53.96 ± 37.69
|
50.11 ± 41.64
|
45.05± 44.62
|
41.99 ± 33.05
|
34.19 ± 28.61
|
P value
|
0.882
|
|
0.748
|
0.093
|
0.793
|
0.069
|
Outcome
|
|
|
|
|
|
|
|
Improvement
|
191 (53.5)
|
92 (54.76)
|
8 (33.33)
|
52 (54.74)
|
68 (65.38)
|
59 (65.56)
|
96 (46.83)
|
Voluntary discharge
|
61(17.09)
|
36 (21.43)
|
3 (12.5)
|
17 (17.89)
|
15 (14.42)
|
15 (16.67)
|
47 (22.93)
|
Death
|
82(22.97)
|
32 (19.05)
|
13 (54.17)
|
26 (27.37)
|
20 (19.23)
|
13 (14.44)
|
42 (20.49)
|
P value
|
0.375
|
|
0.051
|
0.256
|
0.677
|
0.047
|
Patient transfer between wards
|
105(29.41)
|
36 (21.43)
|
8 (32)
|
18 (17.82)
|
27 (25.96)
|
25 (27.78)
|
63 (30.73)
|
P value
|
0.054
|
|
0.117
|
0.159
|
0.776
|
0.610
|
Patient transfer between hospitals
|
74(20.73)
|
24 (14.29)
|
7 (28)
|
12(11.88)
|
13(12.5)
|
15 (16.67)
|
51 (24.88)
|
P value
|
0.077
|
|
0.088
|
0.892
|
0.41
|
0.119
|
P values were comparisons between sexes or comparisons between adjacent two years
Epidemiological Evolution Of St11 Kp Clones
The ST11-wzi209 clone was first detected in February 2014 in a patient transferred to the ICU in our hospital, and its number had increased since August 2014. The clone caused an outbreak involving 12 patients in the ICU during March 15-April 2015. A multidisciplinary infection control team was then formed in the ICU in May 2015 to identify problems regarding nosocomial infection control and develop practical way to limit the transmission of the clone. Some basic control measures were enhanced immediately, including twice-weekly training of medical staff regarding CRE, with special emphasis on hand hygiene and contact precautions, partial restrictions on ward admission, and extensive environmental screenings for room surfaces, equipment, and staff hands. In multiple environmental samples, only one isolate of ST11-wzi209 was recovered from a bed sheet after ultraviolet light disinfection, suggesting an important model of transmission via the hands of healthcare workers from the contaminated bed linens to new patients and the inadequate disinfection. Evidence-based control measures were then developed and gradually implemented in the ICU since July: patient wiping with chlorhexidine once per day, disinfection of bed linens with an ozone sterilizer twice a week, reinforcement of patients’ environmental disinfection with chlorine-based compound twice-daily, and inspection of strict adherence to hand hygiene and compliance with contact precautions. Afterward, a substantial decrease was observed in the number of new cases in the ICU (Fig. 1). In March 2016, considering the rapid increase of CRE in our hospital and the continuing risk of importation pressures, the above measures were gradually taken in most wards of our hospitals, and we observed a significant drop in the incidence of ST11-wzi209 KP acquisition (Fig. 2). Notably, the number of new patients who were colonized and infected with ST11-wzi209 KP in the ICU and the emergency wards increased slightly in 2018, suggesting that infection control measures still needed to be further improved. However, stringent implementation of these measures had been maintained to avoid further transmission of the clone in our hospital.
The ST11-wzi141 clone initially appeared in neurosurgery ward in April 2017 and spread rapidly to different wards, causing several outbreaks in the ICU and neurosurgery wards largely due to frequent patient transfers between both wards. Successive attempts failed to identify sources or reservoirs of the epidemic clone during the surveillance of the affected wards’ environment. Despite the implementation of the above measures that had greatly reduced the transmission of the ST11-wzi209 clone in our hospital, the widespread transmission of the ST11-wzi141clone to various wards were not contained, and it had evolved into an endemic situation in the hospital (Fig. 1).
The ST11-wzi64 clone was introduced into the emergency department in December 2013, resulting in 3 cases of infection. The clone was detected in 2 cases in 2014, 3 cases in 2015 and 1 case in 2016 in the emergency department of our hospital, but not in other wards. None cases with the clone were found in 2017. However, this clone re-emerged in 2018, with 7 new cases in five different wards. The ST11-wzi89 clone and ST11-wzi133 clone were found in the Department of Infectious Diseases in March 2016 and the ICU in May 2018, respectively. No signs of their transmission in the hospital were found and no special control measures were taken.
Epidemiological features of carbapenemase-producing E.coli
A total of 73 carbapenemase-producing E.coli isolates were recovered from 64 patients during the study period, including 36 KPC-2 isolates from 30 patients and 37 NDM isolates from 34 patients, of which 5 patients with NDM E.coli were transferred from other hospitals and the remaining 59 cases were all hospital acquired. The KPC-2 E.coli was first detected in our hospital in June 2016, but not in other hospitals. Due to an outbreak of ST43 E.coli in neurosurgery ward, its number increased markedly in 2017 (Fig. 3). This clone first appeared in January 2017, lasted for 6 months and disappeared since July 2017. A total of 16 patients acquired pneumonia infecion with this clone and 3 cases died. However, the clone was not detected in other wards in our hospital. The first NDM E.coli appeared in January 2015, and the number gradually increased, especially NDM-5 isolates (Fig. 3), which were distributed in different wards and had not caused any substantial outbreaks. These NDM-5 belonged to a variety of STs, with ST167 being more common.
Correlation Between Antibiotic Consumption And Cre Prevalence
Yearly consumption rate of carbapenems decreased from 38.95 in 2016 to 27.22 DDDs/1000 PDs in 2018, while the usage of third-generation cephalosporins increased from 37.98 in 2016 to 68.39 DDDs/1000 PDs in 2018 in our hospital. No significant associations of annual CRE prevalence were found with yearly consumption rates of carbapenems (r = -0.13, P = 0.806), fluoroquinolones (r = -0.301, P = 0.562), first-generation cephalosporins (r = 0.732, P = 0.098), second-generation cephalosporins ( r = -0.529, P = 0.280), third-generation cephalosporins ( r = -0.1, P = 0.851), and beta-lactam/beta-lactamase inhibitor combinations (r = -0.485, P = 0.329).
Transferability of carbapenemase genes via plasmid conjugation
A total of 210 clinical CPE isolates were tested for mobility of carbapenemase-bearing plasmids by conjugation with E.coli J53, 49 isolates were successfully transferred. High conjugation rates were found among NDM plasmids in both CPKP (54.84%, 17/31) and E.coli (46.67%, 14/30). The conjugation rates of the KPC-2 plasmids in E.coli and CPKP were 25% (2/8) and 10.08% (12/119), respectively. Nine isolates of Klebsiella oxytoca that included 3 KPC-2 isolates, 3 NDM-1 isolates and 3 IMP-4 isolates were conjugated with E.coli J53, only isolates that producing KPC-2 achieved conjugative transfer. All ST15-wzi19 KPC-2-producing KP isolates were readily transferable whereas all ST15-wzi384 KPC-2 KP were not conjugative. Among the ST11 KPC-2 KP clones, isolates of ST11-wzi209 (n = 53), ST11-wzi64 (n = 5), ST11-wzi89 (n = 3) and ST11-wzi133 (n = 2) failed to transfer, while only one strain of the 7 ST11-wzi141 isolates was able to transfer its plasmid, suggesting that different STs, different wzis and the local evironment of the host in which bacteria inhabited might influence conjugation.