Spread of Carbapenem-Resistant Enterobacteriaceae in a Region, China: How to Control?

Background: The increasing number of carbapenem-resistant Enterobacteriaceae (CRE) has become a serious problem globally. This study aimed to elucidate their geographically epidemiological characteristics and explore evidence-based infection control measures. Methods: Carbapenem-resistant genes were identied by polymerase chain reaction (PCR) and sequencing. Bacterial genotyping was studied using multilocus sequence typing (MLST) and wzi typing. The transferability of carbapenemase genes was determined by a broth mating method. The relationships between the rates of antimicrobial consumption and the prevalence of CRE were performed by Pearson's or Spearman's correlation analyses. The elucidation of transmission and the evaluation of control measures involved in electronic medical record review, environmental cultures, and outbreak evolution. Results: A total of 930 phenotypically conrmed CRE isolates collected from 19 hospitals were genotypically characterised. K.pneumoniae (KP) and E.coli isolates were 787 (85.17%) and 96 (10.39%) among 924 carbapenemase-producing Enterobacteriaceae (CPE) isolates. Two major carbapenemase genes KPC-2 and NDM in CPE isolates accounted for 84.63% (n = 782) and 13.74% (n = 127). ST11 comprised 86.32% (631/731) of KPC-2 KP isolates. Wzi typing could discriminate ST11 KP clones and precisely track their transmission. Conjugation assays demonstrated that Some KPC-2- and NDM-bearing plasmids could be conjugatively transferred. The transferability was inuenced by different STs and different wzis. CRE patients were becoming increasingly younger due to nosocomial CRE acquisition. The average length of hospitalization of these patients showed a downward trend mainly due to signicant increases in voluntarily discharged rates and mortality rates. The frequent transfers of CRE patients between intra- and inter-hospitals were the main driving factors for the CRE increase. No associations between the rates of antibiotics consumption and CRE prevalence were observed. Evidence-based measures could effectively reduce the prevalence of ST11-wzi209 clone but failed to control the dissemination of ST11-wzi141 KP clone. Conclusions: Continued vigilance for the importations should be maintained. Coordinated regional interventions are urgently needed to reduce CRE threat.


Introduction
Owing to the consequences of unreasonably excessive and frequently unnecessary use of antibiotics in humans and animals, bacterial strains that harbored novel and transmissible antibiotic resistance genes were continually emerging [1]. Of these, carbapenemase-producing Enterobacteriaceae (CPE) strains were of particular concern as they possessed an extremely high potential for transmission and had been associated with increased mortality, longer hospital stays and higher hospital costs [2]. This scenario had resulted in the introduction of tigecycline, colistin and ceftazidime-avibactam as the novel therapeutic agents for these infections. However, the extensive use of these antibiotics in clinical settings was rapidly followed by the emergence of new resistance to these drugs [3][4][5], leaving treatment options even fewer or non-existent. Given the paucity of novel or clinically proven effective antibiotics, infection control and prevention must be given an urgent priority.
In response, numerous international, national and regional guidelines and strategies developed to address the escalating threat of carbapenem-resistant Enterobacteriaceae (CRE) continued to increase worldwide [6,7]. At present, there was still no consensus on the optimally effective interventions or the best combination of interventions to curtail the spread of CRE. Interventions in healthcare settings most often had involved bundled control measures, which varied by institution and effect [8]. These discrepancies might re ect differences in regional epidemiology that required distinct prevention strategies.
In China, since the rst Klebsiella pneumoniae (KP) producing KPC-2 was reported in 2007 [9], the prevalence of carbapenem-resistant KP (CRKP), especially the KPC-2 ST11 clones, had increased. Recent data from the CHINET surveillance system showed that imipenem-resistance rate of KP increased from 3.0% in 2005 to 25% in 2018, with resistance rate reaching over 45% in some hospitals [10]. The annual upward trend was also obtained from the China Antimicrobial Resistance Surveillance System (http://www.carss.cn/), which further showed that the annual isolation rate of CRKP in 2014-2019 was for geriatric patients over the age of 65 years 7.4/8.7/9.9/10.2/11.3/12.2 and for adult patients 5.3/6.3/7.4/7.8/8.9/9.7 (%), respectively. However, very few detailed epidemiological data are available to uncover the underlying mechanisms for these increases.
In this context, we sought to determine the transmission modes of CRE in a region by performing a series of integrated analysis of clinical, epidemiologic, microbiologic and molecular data; and to assess the effectiveness of control measures by describing the course and management of CPE-associated outbreaks.

Study design and de nition
This study was conducted in Hebei General Hospital, a 1830-bed tertiary care hospital located in Hebei, China. Seven secondary-and 11 other tertiary-care hospitals located in geographically separated areas in this province voluntarily participated in the study. These hospitals were requested to collect CRE isolates from clinical samples as well as the related basic clinical and epidemiological data between September 2015 and December 2017. For strains that caused outbreaks in these hospitals, only one representative strain was collected.
The demographic, clinical and epidemiological data were collected via electronic medical record review. CRE cases were de ned by the isolation of CRE in any biological sample obtained from the patients. Imported cases were de ned as patients with known CRE acquisition prior to admission or found to be positive in the rst 48 h of admission. Exported cases were de ned as known CRE patients discharged to other hospitals. Infection and /or colonization were de ned according to the criteria established by the U.S. Centers for Disease Control and Prevention (CDC) [11]. Microbiological methods Various clinically indicated samples, screening samples (stool or rectal swabs), and environmental samples were streaked onto blood agar plates and Eosin methylene blue agar (EMB) plates, and incubated aerobically for 48 h at 35°C. Bacterial isolates were identi ed to the species level by MALDI-TOF MS. The susceptibility of bacterial isolates to different antimicrobial agents were determined using the VITEK cards (bioMe´rieux) following the manufacturer's instructions and interpreted according to the criteria of the latest Clinical Laboratory Standards Institute (CLSI) document. Meropenem and imipenem susceptibilities were also carried out by the Kirby-Bauer disk diffusion method on EMB agar containing meropenem (1mg/L) for culture screening purposes or on Mueller-Hinton agar for con rmation if a discrepancy in susceptibilities between carbapenems by Vitek 2 occurred. Enterobacterial isolates resistant to at least one of the carbapenems or positive for carbapenemase genes were regarded as CRE. E. coli ATCC 25922 was used as a quality control.
Amplicons were sequenced and compared with the reported sequences from GenBank by Blast.

Intervention measures
Evidence-based guidelines on prevention and control of CRE were unavailable in China. Infection control measures for CRE employed in all participating hospitals were based primarily on the guidelines for the control of multi-drug resistant (MDR) organisms and typically included hand hygiene, contact precautions and/or isolation precautions, healthcare staff education, environmental cleaning and disinfection, aseptic procedures and antimicrobial stewardship (AMS) policies.
In our institution, infection surveillance and control were conducted by the department of preventive medicine. Microbiology laboratory, clinical departments and preventive medicine department shared daily data about MDR organisms. The basic control measures for CRE prevention were as follows: hand hygiene with alcohol-based hand rubs, patients with CRE isolates placed on contact precautions, CRE status marked in the electronic medical record and environment disinfection with a chlorine-based compound. Monthly environmental surveillance cultures were performed for relevant wards, which included air sedimentation cultures, hand swab cultures and environmental swab cultures. Since the release of the National Action Plan to Contain Antimicrobial Resistance (2016-2020) in September 2016, antimicrobial stewardship had been reinforced. Carbapenems were added in the restricted antibiotic list, requiring preauthorization of prescriptions. Monthly multidisciplinary antibiotic meeting was undertaken in the whole hospital, with a particular emphasis on rational use of antibiotics.
When an outbreak occurred, an infection control task force was established immediately and multidisciplinary meetings were frequently held to explore additional effective control measures. Investigative ndings on the basis of clinical observations, epidemiological surveys and molecular evidence were immediately fed back to guide development or re nement of control measures. Antibiotic Consumption Data on the rates of antibiotic consumption were obtained from hospital pharmacy database and expressed as the de ned daily dose (DDD)/1000 patient-days (PDs). Four main classes of antibacterial agents including carbapenems, uoroquinolones, cephalosporins, and beta-lactam/beta-lactamase inhibitor combinations were analysed in this study.

Conjugation Experiments
To determine the transferability of the carbapenemase genes, a conjugation experiment was carried out in mixed broth cultures at 37°C [16]. Carbapenemase positive donor strains from various clinical samples were randomly selected according to the colonial morphotypes (size, color, viscosity, etc.). Azide-resistant E. coli J53 was used as recipient. Transconjugants were selected on EMB containing azide (200µg/ml) and meropenem (1 mg/L) and inspected by PCRs.

Statistical analysis
Data were reported as mean ± SD, number, percentage or frequency according to data distribution. Qualitative variables were compared by Student's t tests or Mann-Whitney U tests, while categorical variables were compared by Chi-squared or Fisher's exact tests. Pearson's or Spearman's correlation coe cient was used to determine the associations between variables. All tests were two-tailed and P values of < 0.05 were considered statistically signi cant. All analyses were conducted using SPSS software.

Genotypic characterization of adult CRE isolates
A total of 930 phenotypically con rmed CRE isolates (643 from various clinical diagnostic specimens and 103 from stool specimens in our hospital as well as 184 from other 18 hospitals) during the period from 2013 through 2018 were screened for carbapenemase genes, 924 isolates were detected positive including 782 KPC-2 isolates, 127 NDM isolates and 7 IMP-4 isolates ( Table 1). MLST and wzi assigned 787 carbapenemase-producing KP (CPKP) isolates to 27 STs and 32 wzi alleles (wzis), respectively.

Clinical characteristics of adult CRE patients
The majority of CRE patients (78.5%) were male, but no statistical differences in clinical features between sexes were observed (Table 3). Further analysis showed that CRE 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. One hundred and forty-seven (96.08%, 147/153) 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. The average length of hospitalization of CRE patients showed a downward trend mainly due to signi cant increases in voluntarily discharged rates and mortality rates. The increased importation and exportation of CRE patients as well as frequent transfers of CRE patients between wards presented a rising trend, indicating that CRE patients were expected to further increase in the future . P values were comparisons between sexes or comparisons between adjacent two years. *: P value was the comparison between 2018 and 2015. Patient importation: CRE patients were recruited from the community (n = 17), tertiary care hospitals (n = 29), secondary care hospitals (n = 16), community care hospitals (n = 5), and other regional hospitals (n = 2). Patient exportation: CRE patients were transferred to secondary care hospitals (n = 19), community care hospitals (n = 5), and other regional hospitals (n = 8).  , and other regional hospitals (n = 2). Patient exportation: CRE patients were transferred to secondary care hospitals (n = 19), community care hospitals (n = 5), and other regional hospitals (n = 8).

Description of ST11 clones outbreaks and infection control measures
ST11-wzi209 clone was introduced into ICU in June 2014 and the numbers subsequently increased, causing an outbreak involving 12 cases during March 15-April 2015. Environmental screenings for room surfaces, equipment, and staff hands did not yield any CRE isolates except some carbapenem-resistant Acinetobacter baumannii isolates. But one isolate of ST11-wzi209 was recovered from the bed sheet after ultraviolet light disinfection, suggesting an important model of transmission via the hands of healthcare workers from contaminated bed linens to new patients and the inadequate disinfection. Additional control measures were then developed and implemented: 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 regular training with special emphasis on hand hygiene and contact precautions. Afterward, a substantial decrease was observed in the number of new cases (Fig. 1). However, the strains were still detected in various wards, mainly due to the continuous introductions from other hospitals and the community as well as the transfer of CRE patients between wards.
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 above measures implemented, the widespread transmissions of the clone to various wards were not contained, and it had evolved into an endemic situation in the hospital (Fig. 1). No signi cant associations of annual CRE prevalence were found with yearly consumption rates of carbapenems (r = -0.13, P = 0.806), uoroquinolones (r = -0.301, P = 0.562), rstgeneration 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) (Fig. 2).

Discussion
In this study, KPC-2 was the most frequently detected carbapenemase gene, and ST11 KPC-2 KP clones were the most prevalent isolates. The wzi typing method could further subdivide KP strains of the same STs into different subgroups and accurately track their transmission. The frequent inter-and intrahospital transfers of CRE patients were the main driving factors for the continued KPC-2 increase. CRE patients were becoming increasingly younger, largely due to nosocomial CRE acquisition. Although basic control measures had been strictly implemented throughout the study period, they were fundamentally inadequate to curb their transmission. The implementation of evidence-based measures had proven to be effective for some ST11 clones.
Molecular typing methods were especially essential in transmission surveillance and outbreak investigation and management. Pulsed eld gel electrophoresis (PFGE) and enterobacterial repetitive intergenic consensus sequence-based PCR (ERIC-PCR) were the most important molecular typing tools for the analysis of bacterial strain clonality. However, these methods were associated with poor reproducibility and often tended to produce subjective biases, making direct comparison of data across different laboratories an immense challenge [17,18]. Moreover, these approaches were not suitable for precisely tracking CRKP epidemics due to their high clonality [19]. In this study, we also found that ST11 KP clones were closely related and not accurately discriminated by ERIC-PCR (data not shown). So we tried to use the wzi typing method for typing the isolates of KP. Our ndings showed that it was su cient to distinguish isolates showing the same ST types. Furthermore, this method could identify the extent of the transmission, evaluate the trends and allow for direct comparisons among laboratories. So it should be applicable to the ST11 clone group as this group was the most predominant clone group and continued to rise in China.
Conjugation results showed that the carbapenemase genes could reside on transferable plasmids. As a result of frequent plasmid horizontal transfers, newly emerging clones of different STs and/or wzis, and species diversity tended to increase over time. We found that the number of STs in CPKP were positively associated with the prevalence of CRE (Fig. 3), suggesting that horizontal transfer events would be rapidly increased if rigorous infection control measures were not implemented to disrupt their clonal spread. Factors such as temperature, substrate, plasmid content, and donor and recipient strain identity in uenced conjugation rate [20]. In the study, conjugation e ciency was in uenced by different STs and different wzis. Further exploring the underlying mechanisms in uencing horizontal plasmid transfer might help design effective ways to block them.
Unnecessary and excessive use of antibiotics usually led to the emergence, colonization, clonal expansion and plasmid transmission of CRE strains [21,22]. There exsisted a close correlation between the carbapenem consumption and the rate of carbapenem-resistant gram negative bacilli [23]. However, there were studies suggesting no effect of change in antimicrobial use in reducing CRKP infections [24,25]. The current work did not nd any association between antimicrobial consumption and CRE prevalence, the possible reason might be that the rapid spread of resistant bacteria and subsequent outbreaks masked these relationships.
The implementation of evidence-based control measures reduced the number of ST11-wzi209 clone during the study period, but insu cient to control the prevalence of ST11-wzi141 clone after its introduction to the hospital. This clone had spread to all wards and become the main clone, and it still continued to accrue despite these control measures taken, suggesting a higher transmission capacity or greater epidemic potential than ST11-wzi209 clone. The spread of CRE across hospitals due to the mobility of patients was increasingly recognized as an important mode of transmission [29][30][31]. Enterobacterial strains that producing KPC-2 or NDM-5 had already spread throughout the region and most hospitals would experience an increasing frequency of CRE introduction without any inter-hospital CRE status communication. In China, patients usually chose hospitals completely according to their individual preference without any restriction from regional or local health authorities. This situation might provide great opportunities for the movement of asymptomatic carriers to transmit CRE. Currently, almost all infection control efforts focused exclusively on individual institutions, while regional or national guidelines for controlling the dissemination of CRE across health care settings were still de cient. The values of regional/national strategies had been demonstrated by experiences in Israel [32] and others [33,34] Conclusions CRE were still evolving and spreading rapidly, and they would become a persistent problem in the future, mainly due to the continuing risk of importation pressures and de cits in regional strategies. Hence, continued vigilance and comprehensive epidemiology studies speci c for institutions or regions were urgently needed to develop effective control measures to combat the ever-changing threat.

Availability of data and materials
The datasets used and analysed during the current study are available from the corresponding author on reasonable request.
Ethics approval and consent to participate Protocols for collection of samples as well as the experiment plan and all methods were performed in accordance with the guidelines and regulations of all participating hospitals and approved by the institutional ethical committees. Individual informed consent was waived because all samples collected from patients were as part of routine management and surveillance.

Consent for publication
All authors read and approved the nal manuscript and gave consent for publication.  (caption in gure le)