NDM-1–producing Strains, Family Enterobacteriaceae, in Hospital, Beijing, China

To the Editor: The prevalence of New Delhi metallo-β-lactamase-1 (NDM-1)–producing strains (family Enterobacteriaceae) in China remains unclear. Recently, to clarify the prevalence of blaNDM-1 in Enterobacteriaceae strains, we carried out retrospective surveillance for blaNDM-1 among carbapenem-resistant enterobacterial strains isolated from patients at the Chinese PLA General Hospital in Beijing. This tertiary teaching hospital has 4,000 beds and 12,000 daily outpatient visits. More than 50% of patients admitted to the hospital are from areas outside Beijing. During January 2009–June 2013, a total of 8,586 enterobacterial isolates were obtained from routine clinical samples that had been passively sent to the microbiology department. Of these, 242 (2.8%) strains exhibited resistance to carbapenems. 
 
In this study, we used PCR amplification to screen the carbapenem-resistant strains for the blaNDM-1 gene and other common resistance determinants. The MICs of various antimicrobial drugs were measured by E-test (AB bioMerieux, Solna, Sweden). S1 nuclease pulsed-field gel electrophoresis and Southern blot analysis were used to identify the sizes of blaNDM-1-carrying plasmids. The incompatibility (Inc) groups of the plasmids were detected by several multiplex and simplex PCRs. Multilocus sequence typing (MLST) was carried out for Klebsiella pneumoniae and Escherichia coli isolates, according to protocols provided on MLST websites (www.pasteur.fr/recherche/genopole/PF8/mlst/Kpneumoniae.html and http://mlst.ucc.ie/mlst/dbs/Ecoli). The transferability of plasmids was identified by conjugation experiments. 
 
Five blaNDM-1-positive enterobacterial isolates of the following species were identified: E. coli (1 isolate in October2010), K. pneumoniae (1 isolate in August 2012), Providencia rettgeri (1 isolate in October 2012), Enterobacter cloacae (1 isolate in November 2012), and Raoultella ornithinolytica (1 isolate in March 2013). According to the 2013 Clinical and Laboratory Standards Institute performance standard M100-S23 (www.clsi.org/), the NDM-1-producing K. pneumoniae (IR5047) isolate exhibited low-level resistance to imipenem and meropenem, whereas other isolates showed high-level resistance to carbapenems. Only E. coli and Providencia rettgeri, which carry 16S rRNA methylase genes, exhibited high-level resistance to amikacin (Table). S1 nuclease pulsed-field gel electrophoresis and Southern blot analysis showed that the blaNDM-1 gene was located on plasmids of various sizes belonging to different Inc groups. The K. pneumoniae isolate was defined as a novel ST1240 with the allelic profile 2–1-1–1-1–3-24, and the E. coli isolate was identified as ST167. 
 
 
 
Table 
 
Phenotype and molecular characteristics of NDM-1–producing strains isolated from Chinese PLA General Hospital, Beijing, China, 2009–2013* 
 
 
 
In China, various blaNDM-1-carrying strains of the Enterobacteriaceae have been sporadically identified, including K. pneumoniae, K. oxytoca, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter freundii (1–4). We identified a P. rettgeri isolate and an R. ornithinolytica isolate that produced NDM-1. The blaNDM-1-positive P. rettgeri isolates have also been identified in Pakistan, India, Canada, and Mexico, whereas the NDM-1-producing R. ornithinolytica strain has only been detected in India (5–9). In this study, all 5 NDM-1–producing strains were isolated only once, and no dissemination of NDM-1–producing strains of Enterobacteriaceae has been found. Two strains (K. pneumoniae and Enterobacter cloacae) were isolated within 48 hours of the patient’s hospital admission, indicating the infections were imported (from Shandong and Hebei Provinces, respectively). Escherichia coli, P. rettgeri, and R. ornithinolytica were isolated 48 hours after admission of patients (from Henan and Hebei Provinces). Therefore, the patients might have acquired the NDM-1–producing Enterobacteriaceae strains at the hospital. 
 
However, the source of the blaNDM-1 determinant remains unclear. The possibility that the strains were imported cannot be excluded for the several reasons. First, examination to determine the infectious agent had not been performed for a considerable number of patients within 48 hours of their admission. Second, NDM-1–producing Enterobacteriaceae species have not spread in this hospital. Third, the blaNDM-1-carrying plasmids in the same Inc group and of similar size exhibited substantial differences in resistance determinants (Table), which suggests a different evolutionary origin for these isolates. In an additional survey of clinical data, we found no epidemiologic relationship between the patients who were infected by NDM-1–producing pathogens. These data suggest a sporadic pattern of NDM-1–producing enterobacteria in the hospital. 
 
Sequencing analysis (data not shown) indicated that the blaNDM-1-carrying plasmid carried by K. pneumoniae (≈50 kb, IncX3) was different from the plasmid found in the Acinetobacter pitti isolate that was disseminated in an intensive care unit of the Chinese PLA General Hospital in 2008 (10). This finding suggests that the 2 plasmids had a different evolutionary origin. The IncX3 plasmid that we found was highly homologous (>99%) to the plasmid pNDM-HN380 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"JX104760","term_id":"402914504","term_text":"JX104760"}}JX104760), which has been identified in several Enterobacteriaceae strains isolated from patients in southern China (1). This finding showed that the IncX3 plasmid that was 50 kb in size acted as the main factor mediating the transmission of the blaNDM-1 gene across China. IncA/C plasmids are the leading group of blaNDM-1-carrying plasmids and have been detected in E. coli isolated from China (1). In this study, E. coli (IR5028) and P. rettgeri (IR5337) carried IncA/C plasmids. However, these 2 strains exhibited diverse resistant determinants on these plasmids (Table). This observation suggested that the 2 plasmids have different integrating processes. For R. ornithinolytica, the blaNDM-1 gene was located on an IncN plasmid of ≈70 kb, which is very different from other plasmids. 
 
In conclusion, we identified various NDM-1–producing enterobacterial isolates at the Chinese PLA Hospital in Beijing and the emergence of novel blaNDM-1-carrying clones among common species of Enterobacteriaceae, such as K. pneumoniae ST1240 and E. coli ST167. There is an urgent need for monitoring and surveillance of epidemiologic and genotypic profiles of NDM-1–producing Enterobacteriaceae species in China.


NDM-1-producing
Strains, Family Enterobacteriaceae, in Hospital, Beijing, China To the Editor: The prevalence of New Delhi metallo-β-lactamase-1 (NDM-1)-producing strains (family Enterobacteriaceae) in China remains unclear. Recently, to clarify the prevalence of bla NDM-1 in Enterobacteriaceae strains, we carried out retrospective surveillance for bla NDM-1 among carbapenem-resistant enterobacterial strains isolated from patients at the Chinese PLA General Hospital in Beijing. This tertiary teaching hospital has 4,000 beds and 12,000 daily outpatient visits. More than 50% of patients admitted to the hospital are from areas outside Beijing. During January 2009-June 2013, a total of 8,586 enterobacterial isolates were obtained from routine clinical samples that had been passively sent to the microbiology department. Of these, 242 (2.8%) strains exhibited resistance to carbapenems.
In this study, we used PCR amplification to screen the carbapenemresistant strains for the bla NDM-1 gene and other common resistance determinants. The MICs of various antimicrobial drugs were measured by E-test (AB bioMérieux, Solna, Sweden). S1 nuclease pulsed-field gel electrophoresis and Southern blot analysis were used to identify the sizes of bla NDM-1 -carrying plasmids. The incompatibility (Inc) groups of the plasmids were detected by several multiplex and simplex PCRs. Multilocus sequence typing (MLST) was carried out for Klebsiella pneumoniae and Escherichia coli isolates, according to protocols provided on MLST websites (www.pasteur.fr/recherche/ genopole/PF8/mlst/Kpneumoniae.html and http://mlst.ucc.ie/mlst/dbs/Ecoli). The transferability of plasmids was identified by conjugation experiments.
Five bla NDM-1 -positive enterobacterial isolates of the following species were identified: E. coli (1 isolate in Oc-tober2010), K. pneumoniae (1 isolate in August 2012), Providencia rettgeri (1 isolate in October 2012), Enterobacter cloacae (1 isolate in November 2012), and Raoultella ornithinolytica (1 isolate in March 2013). According to the 2013 Clinical and Laboratory Standards Institute performance standard M100-S23 (www.clsi.org/), the NDM-1-producing K. pneumoniae (IR5047) isolate exhibited low-level resistance to imipenem and meropenem, whereas other isolates showed high-level resistance to carbapenems. Only E. coli and Providencia rettgeri, which carry 16S rRNA methylase genes, exhibited high-level resistance to amikacin (Table). S1 nuclease pulsed-field gel electrophoresis and Southern blot analysis showed that the bla NDM-1 gene was located on plasmids of various sizes belonging to different Inc groups. The K. pneumoniae isolate was defined as a novel ST1240 with the allelic profile 2-1-1-1-1-3-24, and the E. coli isolate was identified as ST167.
In China, various bla NDM-1 -carrying strains of the Enterobacteriaceae have been sporadically identified, including K. pneumoniae, K. oxytoca, Escherichia coli, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter freundii (1)(2)(3)(4). We identified a P. rettgeri isolate and an R. ornithinolytica isolate that produced NDM-1. The bla NDM-1 -positive P. rettgeri isolates have also been identified in Pakistan, India, Canada, and Mexico, whereas the NDM-1-producing R. ornithinolytica strain has only been detected in India (5)(6)(7)(8)(9). In this study, all 5 NDM-1-producing strains were isolated only once, and no dissemination of NDM-1producing strains of Enterobacteriaceae has been found. Two strains (K. pneumoniae and Enterobacter cloacae) were isolated within 48 hours of the patient's hospital admission, Search past issues of EID at wwwnc.cdc.gov/eid indicating the infections were imported (from Shandong and Hebei Provinces, respectively). Escherichia coli, P. rettgeri, and R. ornithinolytica were isolated 48 hours after admission of patients (from Henan and Hebei Provinces). Therefore, the patients might have acquired the NDM-1-producing Enterobacteriaceae strains at the hospital.
However, the source of the bla NDM-1 determinant remains unclear. The possibility that the strains were imported cannot be excluded for the several reasons. First, examination to determine the infectious agent had not been performed for a considerable number of patients within 48 hours of their admission. Second, NDM-1-producing Enterobacteriaceae species have not spread in this hospital. Third, the bla NDM-1 -carrying plasmids in the same Inc group and of similar size exhibited substantial differences in resistance determinants (Table), which suggests a different evolutionary origin for these isolates. In an additional survey of clinical data, we found no epidemiologic relationship between the patients who were infected by NDM-1-producing pathogens. These data suggest a sporadic pattern of NDM-1-producing enterobacteria in the hospital.
Sequencing analysis (data not shown) indicated that the bla NDM-1 -carrying plasmid carried by K. pneumoniae (≈50 kb, IncX3) was different from the plasmid found in the Acinetobacter pitti isolate that was disseminated in an intensive care unit of the Chinese PLA General Hospital in 2008 (10). This finding suggests that the 2 plasmids had a different evolutionary origin. The IncX3 plasmid that we found was highly homologous (>99%) to the plasmid pNDM-HN380 (GenBank accession no. JX104760), which has been identified in several Enterobacteriaceae strains isolated from patients in southern China (1). This finding showed that the IncX3 plasmid that was 50 kb in size acted as the main factor mediating the transmission of the bla NDM-1 gene across China. IncA/C plasmids are the leading group of bla NDM-1 -carrying plasmids and have been detected in E. coli isolated from China (1). In this study, E. coli (IR5028) and P. rettgeri (IR5337) carried IncA/C plasmids. However, these 2 strains exhibited diverse resistant determinants on these plasmids (Table). This observation suggested that the 2 plasmids have different integrating processes. For R. ornithinolytica, the bla NDM-1 gene was located on an IncN plasmid of ≈70 kb, which is very different from other plasmids.
In conclusion, we identified various NDM-1-producing enterobacterial isolates at the Chinese PLA Hospital in Beijing and the emergence of novel bla NDM-1 -carrying clones among common species of Enterobacteriaceae, such as K. pneumoniae ST1240 and E. coli ST167. There is an urgent need for monitoring and surveillance of epidemiologic and genotypic profiles of NDM-1-producing Enterobacteriaceae species in China.