Fecal carriage and molecular epidemiology of carbapenem-resistant Enterobacteriaceae from outpatient children in Shanghai

Carbapenems such as imipenem and meropenem are often recommended as antimicrobial agents of the last resort, especially in cases where extended-spectrum β-Lactamase (ESBL) producing organisms involved [1]. Since the KPC1 carbapenem- resistant Klebsiella pneumoniae was first isolated in the United States of America [2], the prevalence of carbapenem-resistant Enterobacteriaceae (CRE) has emerged as a public health problem and given rise to epidemic outbreaks in hospital surroundings all around the world [3‐5]. According to the CHINET surveillance of bacterial resistance data in China, CRE strains showed high levels of resistance to most antibiotics and the rates of carbapenem resistance in E. coli and K. pneumoniae had increased from 0 and 2.9% in 2005 to 1 and 13.4% in 2014, respectively [6]. In general, the mechanism of resistance to carbapenems is mainly associated with the production of carbapenemases.

Enterobacteriaceae are a family of bacteria that mainly colonize in normal human intestines. Gastrointestinal tract colonization with CRE strains can be a risk factor for bacterial translocation resulting in subsequent endogenous infections in immunocompromised children [7]. As Das et al. reported that neonates with Gram negative bacilli in the gut had a higher incidence of clinical sepsis than those without these bacteria [8]. The acquisition of mobile genetic elements may be involved in the horizontal transmission. Several studies have been conducted to investigate the fecal carriage characteristics of CRE strains among inpatients [9‐11], however, there are few data regarding the prevalence of CRE colonization in community settings, especially in China. Therefore, this research retrospectively aimed to conduct a surveillance of molecular epidemiology of CRE strains colonized in stool samples of outpatient children in a tertiary pediatric hospital of Shanghai, China.

Investigation of the fecal carriage prevalence of CRE among outpatients from the community setting can help us to better understand the origin of CRE isolates responsible for outbreak events and contribute to control CRE dissemination. Our study displayed that the rate of colonization with CRE in fecal sample collected from children in outpatient visits was 3.6%, which was lower than what had been reported in patients from community setting in other countries [10, 19]. Several reasons contribute to this phenomenon of the colonization of CRE isolates in rectal gastrointestinal tract. Firstly, further analysis of patient information of CRE carriers showed that 28.1% of them had a history of prior hospitalization in recent 3 months and also had received therapy with antibiotics, but only 4.5% of non-carriers had. Previous studies reported that exposure to hospital setting or/and antimicrobial agents might increase the risk of colonization and it would be easier to acquire CRE isolates, which may also explain the occurrence in community-onset cases [20, 21]. However, despite the previous studies showed an increased risk of CRE carriage with hospital exposure, there is no significance between the min, max and mean times of hospitalization for the CRE and non-CRE groups with recent hospital exposure in this study. What is more, many outpatients receive antimicrobial treatment without the advice or prescription of a physician or other trained health care provider. Last but not least, CRE strains could spread via physical contact with other people and have the propensity to acquire genetic materials mostly in the form of plasmids and transposons through horizontal gene transfer [22]. Colonization with CRE strain plays an important role in transmission of the orgnism through horizontal gene transfer in health care settings [22].

The surveillance definition for CRE are designed that Enterobacteriaceae are resistant to imipenem, meropenem, doripenem, or ertapenem or is document that the isolate possess a carbapenemase and CRE are reported to be commonly identified in K.pneumoniae, E.coli, and E.cloacae [23, 24]. The major resistance mechanism of CRE is the production of carbapenemases such as NDM, KPC, and IMP, which were also observed in this study. The NDM type, including NDM-1 and NDM-5, is found to be the key carbapenemase responsible for mediating development of the carbapenem resistance phenotypes in children [3, 25]. NDM-1 and its minor variants, a class B carbapenemase first clinically isolated from a patient at a hospital in New Delhi, India, has since been identified all over the world and only detected in E.coli and K.pneumoniae [26‐29]. Consistent with the above findings, most of the E.coli and K.pneumoniae colonized CREs in the fecal samples in the present study harbored the bla_NDM gene and a previous study in our hospital also has reported the outbreak of CRE strains caused by NDM-1 producing K.pneumoniae among neonates [3]. NDM-1 producing K.pneumoniae are highly resistant pathogens with no effective beta-lactams, including recent ones such as ceftolozane tazobactam and ceftazidime avibactam and the only one that works is aztreonam [30]. Additionally, only three K.pneumoniae strains carried bla_KPC-2 gene, which was mostly prevalent in adults population [31], were found in this study. IMP type carbapenemases (IMP-4 and IMP-26) were observed in E.cloacae and K.pneumoniae, but not in E.coli. However, it is remarkable that no carbapenemase resistance genes were detected in 9.4% (3/32) of the CRE strains. This finding suggests that a new mechanism might contribute to the resistance to carbapenems and further studies into this mechanism should be conducted.

Molecular analysis revealed a high level of genetic diversity, including ST101, ST11, ST37, and ST48, and several STs are clearly related to specific bacteria, and are prevalent all over the world. The most common STs of CRE in K.pneumoniae were ST11 and ST37. Interestingly, the carbapenem-resistant K.pneumoniae harboring bla_KPC-2 belonged to ST11, which exhibited signs of multi-clonal dissemination and caused a series of outbreaks in China [32, 33]. Additionally, another carbapenem- resistant K.pneumoniae harboring bla_NDM-1 belonged to ST37 and this ST had been identified previously during an outbreak of this phenotype in our hospital [3]. Clonally diverse carbapenem-resistant E.coli (ST405, ST131, ST156 and ST101) have been reported globally, and ST101 was the predominant ST isolated in this study. The ST101 type of E.coli produced NDM-1 carbapenemase, a phenotype that has been linked to nosocomial transmission in Korea [34].

Importantly, children with CRE strains in fecal samples are considered as a high risk group by World Health Organization (WHO), which can spread CRE by intimate contact and travel [35]. The origin of CRE isolates in these children remains unknown and it is the main limitation that we don’t know if the source of the described CRE carriage in these children is the result of transmission in the community or in the hospital. Screening for CRE at discharge may provide an explanation for this hypothesis. Exposure to antimicrobial agents in both the hospital setting and environments is always the key to acquisition. Also we know that children are at high risk for multi-drug organism carriage and further studies should be carried out to evaluate the phenomenon of the original source of CRE strains.

In summary, the current data reveal the prevalence of CRE colonization in fecal sample from pediatric outpatients and strategies to control the dissemination of antimicrobial resistant isolates from stool to other sterilized sites should be developed. Children who had hospitalization exposure should be screened for CRE at discharge, which may help to definitively establish where acquisition of CRE is truly occurring and decrease the occurrence and transmission of CRE in children group.