Clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients in South China

ABSTRACT Carbapenem-resistant Enterobacteriaceae (CRE) are a substantial health burden. Existing management guidelines are primarily based on clonal evidence. Therefore, epidemiological data are required. We evaluated the risk factors for CRE colonization and described the molecular and clinical characteristics of CRE colonization in pediatric inpatients in Guangzhou, China. Pediatric inpatients from three hospitals in Guangzhou, China, between July 2019 and January 2021 were included. Fecal samples were collected and screened for CRE. Microbiological cultures were grown on MacConkey agar plates, and bacteria were identified with a mass spectrometry microbial identification system. Antimicrobial susceptibility was tested with a VITEK2 system, and carbapenemase genes were amplified. A case-control study was conducted to evaluate the risk factors for fecal CRE colonization in pediatric inpatients. Of 4,033 patients screened, 158 (3.92%) were positive for CRE, including Escherichia coli (51.27%), Klebsiella pneumoniae (37.97%), and Enterobacter cloacae (6.96%). The most common carbapenemase genes were blaNDM-5 (51.89%), blaNDM-1 (15.19%), and blaIMP-4 (7.60%) and blaKPC-2 (3.80%). Hematological malignancies, respiratory diseases, otolaryngological diseases, nervous system diseases, oral administration of third-generation cephalosporins, and the combined use of two or more antibiotics were independently associated with CRE colonization. The most prevalent carbapenem-resistant K. pneumoniae sequence types (STs) were ST11 (8.06%), ST37 (8.06%), and ST76 (8.06%), and the most prevalent carbapenem-resistant E. coli STs were ST10 (9.88%), ST48 (13.58%), and ST58 (8.64%). Pediatric inpatients were colonized by a diversity of CRE strains. Infection control and prevention measures should be implemented to reduce CRE colonization. IMPORTANCE This study assessed the clinical and molecular epidemiology of carbapenem-resistant Enterobacteriaceae in pediatric inpatients at three hospitals in South China by means of screening stool samples for carbapenem-resistant genes and a nested case-control study to determine risk factors for carriage of carbapenem-resistant Enterobacteriaceae. Of 4,033 fecal samples screened, 158 (3.92%) were positive for CRE, including Escherichia coli (51.27 %), Klebsiella pneumoniae (37.97%), and Enterobacter cloacae (6.96%). The most common carbapenemase genes harbored by gastrointestinal CRE strains were blaNDM-5, blaNDM-1, and blaIMP-4. Hematological malignancies, respiratory diseases, otolaryngological diseases, nervous system diseases, oral administration of third-generation cephalosporins, and the combined use of two or more antibiotics were independently associated with CRE colonization.

(CRE) have emerged due to the widespread use of carbapenems over the past few decades and represent a threat to public health (2).Since the first identification of CR Klebsiella pneumoniae (CRKP) in 1996 in the United States, CRE has rapidly spread worldwide (3)(4)(5).In China, Klebsiella pneumoniae resistance to meropenem and imipenem grew from 2.9% and 3.0% in 2005 to 26.3% and 25% in 2018, an increase of more than the eightfold (6,7).In Europe, CRKP is prevalent in the Balkan and Mediterra nean countries with a prevalence of 30% in Romania, 40% in Italy, and 60% in Greece in 2014 (8).In addition, CRE infections are difficult to treat and are associated with higher mortality rates in vulnerable population (9).Therefore, surveillance and prevention of CRE are required.
There are three major resistance mechanisms in CRE: production of carbapenemases capable of hydrolyzing carbapenems, modification of the function of porins and other membrane-associated proteins, and activation of drug efflux pumps (10,11).Carbapene mases found in CRE include Ambler class A β-lactamases such as blaKPC and blaSIM; Ambler class B metallo-β-lactamases, such as blaNDM, blaIMP, and blaVIM; and Ambler class D oxacillinases (OXAs), such as blaOXA-232 and blaOXA-48 (12).A previous study showed that the most prevalent genotypes in clinical isolates from China were blaKPC in K. pneumoniae and blaNDM in Escherichia coli (13).
CRE disseminates through the gastrointestinal tract and plays a crucial role in the transmission of antibiotic resistance (14).Gastrointestinal CRE colonization can result in life-threatening bloodstream infections, especially children with immunodeficiency or hematological diseases (15).Existing CRE management guidelines are generally based on clonal evidence, although the epidemiology of CRE colonization and clinical risk factors for CRE colonization in pediatric inpatients are not well characterized in South China.Therefore, we aimed to investigate the molecular and clinical epidemiology, evaluate the risk factors for the acquisition of gastrointestinal CRE colonization, and detect carbape nemases, extended-spectrum β-lactamases (ESBLs), and AmpC cephalosporinase (AmpC) in CRE strains isolated from fecal samples of pediatric inpatients.

Sample collection and microbiological culture
We collected unique fecal samples from 4,033 inpatients aged ≤18 years between July 2019 and January 2021 from three hospitals in Guangzhou, China (Yuexiu District, Tianhe District, and Zengcheng District), as previously described (16).The fecal samples were individually streaked onto selective MacConkey agar plates with 1.5 µg/mL meropenem added (Macklin, China).After 48 h of incubation at 5% CO 2 at 35°C, suspected CRE colonies from each MacConkey agar plate were isolated and streaked on blood agar plates for further confirmation.All positive strains were maintained at −80°C for further investigation.

Clinical data collection
We conducted a case-control study to identify risk factors for gastrointestinal CRE colonization in pediatric inpatients.Among the 4,033 patients investigated, review of medical record was completed for 3,486 patients, including all 158 CRE carriers and 3,328 non-CRE carriers.We were unable to obtain the medical records of 547 non-CRE carriers and, therefore, excluded these patients from the case-control study.Data on general characteristics, length of hospitalization, and previous use of antibiotics (particularly carbapenems, β-lactam + β-lactamase inhibitors, and extended-spectrum cephalospor ins) were collected from the electronic patient management system for all 3,486 patients.The non-CRE carriers were used as the control group.

Statistical analysis
SPSS software 26.0 (IBM Corp., Armonk, NY, USA) was used for statistical analysis.Chi-square tests were performed to identify risk factors for CRE colonization.Factors showing P < 0.05 were considered as candidate predictors that were significantly related to CRE isolation Multivariable logistic regression was performed including these factors in the model to estimate odds ratios (ORs) with 95% confidence intervals (CIs).

Antimicrobial resistance genes
Among the 158 CRE-colonized patients, 145 were tested positive using the mCIM assay.Carbapenemase genes were detected in 134 of 158 CRE isolates using PCR and

DISCUSSION
In this multicenter analysis of CRE in pediatric inpatients hospitalized in Guangzhou, we determined the molecular epidemiology and identified risk factors for CRE colonization.The carriage rate of CRE among pediatric patients varies in different geographic areas (23).In this study, the fecal CRE carriage rate in pediatric inpatients was 3.92%, which is lower than that of three other hospitals in Hunan (8.5%), Fujian (6.6%), and Shanghai (8.6%) (23)(24)(25).There are several possible reasons that could contribute to this phenom enon.First, inpatients from neonatology (15.63%, 545/3486) comprised a large part of the inpatient population in our study.The fecal CRE colonization rate in the newborn population is usually lower, due to less exposure to antibiotics.Second, seasonal and temperature variations can affect CRE detection in fecal samples (26).Although the CRE colonization rate is dynamic owing to seasonal variations, geographical region, and health state, further research is needed to better identify the causes of these disparities.
Globally, as in China, carbapenemase production is the primary mechanism used by Enterobacteriaceae to resist carbapenems (27).Our research confirmed this, as 145 (91.77%) of our CRE isolates were positive in the mCIM assay.In a 5-year study on CRKP conducted in a pediatric hospital in China, blaIMP-4 was the most common carbapene mase from 2010 to 2012, whereas blaNDM-1 and blaNDM-5 were the most common carbapenemases in 2018 (7,28), which is consistent with our findings.Notably, blaKPC-2 gene was only detected in four isolates: three CRKP strains and one CR E. coli strain.blaKPC-2 is the most prevalent carbapenemase in the adult population (29).These results suggest that anti-CRE treatment strategies for children should differ from those used in adults.In addition, we found that two K. pneumoniae strains and one E. coli strain carried blaIMP-4 and blaNDM-5, and one E. coli strain carried blaNDM-5 and blaKPC-2 simultaneously, suggesting that these carbapenemase genes are readily acquired.
All CRE isolates in our study were multidrug resistant and showed high resistance to carbapenems and cephalosporins.The prevalence of resistance to amikacin, tobramy cin, colistin, and tigecycline was low, which may be because of limited use of these antibiotics in children.This suggests that tigecycline or colistin, in conjunction with other antibiotics such as meropenem and imipenem, may be useful for the treatment of complex CRE infections.In recent years, avibactam-ceftazidime, a novel cephalosporin/βlactamase inhibitor combination, has been used to inhibit the activities of carbapene mase (30) but it is less effective at inhibiting in Ambler class B carbapenemases such as blaNDM (NDM) (31).The use of avibactam-ceftazidime in our region's medical center for children should be carefully considered.
The case-control analysis revealed that several serious diseases are associated with CRE colonization, including hematological malignancies, respiratory disease, otolaryngo logical disease, and nervous system disease.Among patients with hematological malignancies, respiratory disease, and nervous system disease, CRE colonization was associated with immunosuppression and high use of antibiotics.It is noteworthy that otolaryngological diseases are a risk factor for CRE colonization at our medical center, as previous studies have not identified otolaryngological disease as a risk factor for CRE colonization.Therefore, it is important to understand the prevalence of CRE in the otolaryngology departments.Additionally, physicians should consider the possibility that a patient may have CRE if risk indicators are present.Our study also showed that oral administration of third-generation cephalosporins and the combined use of two or more antibiotics were risk factors for CRE colonization, which is consistent with the findings of other studies (32)(33)(34).Surprisingly, oral administration of carbapenems and β-lactam + β-lactamase inhibitor was not a risk factor for CRE colonization in this study.This may be due to the sample size of our study or missing information during extraction of data from the electronic medical system.Therefore, the influence of varied antibiotic exposure on gastrointestinal CRE colonization requires further study.
The MLST results showed a high level of genetic diversity among K. pneumoniae and E. coli isolates, with 60 K. pneumoniae strains divided into 32 STs and 81 E. coli strains divided into 39 STs.The most prevalent K. pneumoniae STs were ST11, ST37, and ST414.Four of the five ST11 K. pneumoniae isolates carried the blaKPC-2 gene.K. pneumoniae ST11 carrying blaKPC-2 has been identified as the major type in another area of China, which suggests that clonal transmission of ST11 CRKP occurred in the hospitals in this study (35).NDM-5-producing K. pneumoniae had different clonal backgrounds; blaNDM-1 was observed in ST17, ST36, ST45, and ST180 isolates.The most prevalent E. coli STs in this study were ST48, ST10, and ST58, and blaNDM-5 was more prevalent in the E. coli group.Notably, E. coli ST48 and ST101 co-harbored blaNDM-1 and blaIMP-4, and a novel K. pneumoniae ST2010 co-harbored blaNDM-1 and blaIMP-4.
This study has some limitations.First, stool samples may not be representative because the stool samples were restricted to those submitted for routine analysis.It would be worthwhile to assess the CRE situation in the study facilities.Second, because we used only one method for molecular CRE characterization some CRE strains may have been missed.
In conclusion, the CRE carriage rate in pediatric inpatients in South China was 3.92%, with a diversity of STs found in CRKP and CR E. coli.Moreover, blaNDM-5 and blaNDM-1 were the most prevalent resistance genes in CRE isolates.Hematological malignancies, respiratory disease, otolaryngological disease, nervous system disease, oral administra tion of third-generation cephalosporins, and combined use of two or more antibiotics were independently associated with CRE colonization.Physicians should consider the possibility of a patient carrying CRE if risk indicators are present.This study provides insight into the current status of fecal carriage of CRE among pediatric inpatients in South China.Implementation of prevention and control measures in clinical settings should be encouraged based on our findings.

FIG 1
FIG1 Phylogenetic tree of multilocus sequence typing data from carbapenem-resistant Klebsiella pneumoniae isolates.

FIG 2
FIG 2 Phylogenetic tree of multilocus sequence typing data from carbapenem-resistant Escherichia coli isolates.

TABLE 1
Number of CRE strains isolated from fecal samples of pediatric inpatients

TABLE 2
Antibiotic susceptibility of CRE isolates from fecal samples of pediatric inpatients

TABLE 3
Clinical characteristics of CRE carriers

TABLE 4
Prevalence of carbapenemase genes among 158 CRE strains isolated from fecal samples of pediatric inpatients