Characterization Of blaNDM-5-Positive Escherichia coli Prevalent In A University Hospital In Eastern China
Authors Sun P, Xia W, Liu G, Huang X, Tang C, Liu C, Xu Y, Ni F, Mei Y, Pan S
Received 31 July 2019
Accepted for publication 13 September 2019
Published 24 September 2019 Volume 2019:12 Pages 3029—3038
Checked for plagiarism Yes
Review by Single anonymous peer review
Peer reviewer comments 2
Editor who approved publication: Dr Joachim Wink
Pengfei Sun,1,2,* Wenying Xia,1,2,* Genyan Liu,1,2 Xu Huang,1,2 Chenjie Tang,1,2 Chengcheng Liu,1,2 Yuqiao Xu,1,2 Fang Ni,1,2 Yaning Mei,1,2 Shiyang Pan1,2
1Department of Laboratory Medicine, Jiangsu Province Hospital and Nanjing Medical University First Affiliated Hospital, Nanjing, People’s Republic of China; 2National Key Clinical Department of Laboratory Medicine, Nanjing, People’s Republic of China
*These authors contributed equally to this work
Correspondence: Genyan Liu
Department of Laboratory Medicine, Jiangsu Province Hospital, Guangzhou Street No. 300, Nanjing 210029, People’s Republic of China
Tel +8625 6830 7332
Fax +8625 8372 4440
Email [email protected]
Purpose: The emergence and spread of carbapenem-resistant Enterobacteriaceae deserves special concern worldwide. Unlike the epidemiological characteristics reported in other studies, we found that the production of New Delhi metallo-β-lactamase 5 was the main mechanism for the resistance of Escherichia coli to carbapenems.
Methods: All carbapenem-resistant strains were collected from July 2017 to July 2018 of the First Affiliated Hospital of Nanjing Medical University. The presence of carbapenemase-encoding genes was detected using PCR and gene sequencing. Genetic relatedness of the blaNDM-5-positive E. coli strains was determined with PFGE and MLST. Susceptibility profiles were measured with broth microdilution method and E-test strips. Transferability features of blaNDM-5 gene were assessed by conjugation experiments, S1-PFGE, southern blotting and PCR-based replicon typing methods. The genetic structures surrounding blaNDM-5 were acquired by whole genome sequencing and PCR mapping.
Results: Among the 28 carbapenem-resistant E. coli strains, 18 (64%) were verified as NDM-5 producers. The 18 blaNDM-5-positive E. coli strains showed high resistance to most antibiotics, but 100% were sensitive to colistin and tigecycline. In addition, the 18 blaNDM-5-positive E. coli strains belonged to eight STs, among which ST167, ST410 and ST101 were found to cause clonal spread in the hospital. Further studies found that the blaNDM-5 gene was located on an IncX3-type plasmid, and all plasmids harbored an IS3000-ΔISAba125-IS5-blaNDM-5-bleMBL-trpF-dsbC-IS26 structure.
Conclusion: The clonal spread of blaNDM-5-positive E. coli strains and horizontal dissemination via the pNDM-MGR 194-like plasmids should draw more attention. Appropriate infection control operations should be performed to prevent the further spread of blaNDM-5.
Keywords: Escherichia coli, carbapenem-resistant Enterobacteriaceae, New Delhi metallo-β-lactamase, blaNDM-5, IncX3 type plasmid
Carbapenems were normally thought to be the last resort for treating multidrug resistant gram-negative pathogens.1 However, the situation has changed due to the emergence and spread of carbapenem-resistant Enterobacteriaceae (CRE) strains worldwide over the past twenty years, posing a great challenge to human health.2 Previous studies have demonstrated that carbapenemases such as KPC, IMP, VIM and OXA-48 were the main mechanism for bacterial resistance to carbapenems.3–5 In 2009, a newly emerged Amber class B carbapenemase, New Delhi metallo-β-lactamase 1 (NDM-1), was identified in a Klebsiella pneumoniae strain isolated from a tourist who had a hospitalization history in India.6 Because NDM-1 has high affinity for almost all β-lactamases, has coexistence with other drug-resistant genes and is located on transferable elements, NDM-1 has rapidly drawn extensive attention around the world and is even being called a “superbug” by the media. Shortly after, NDM-1 has already been discovered among multiple species in more than 40 countries.7 The Indian subcontinent, the Balkan states and the Middle East are thought to be three main reservoirs for the dissemination of NDM-1; according to many reports, NDM-1 strains have been recovered from patients that had travelled or received medical treatment in these places.8
Escherichia coli is a member of the human gut microbiota and is one of the most common gram-negative bacteria isolated from hospital specimens such as urine, fecal, sputum and bloodstream.9 In addition, Escherichia coli also plays an important role in community-acquired urinary tract infectious disease. To date, the production of ESBLs is still the main mechanism for the resistance of E. coli to third generation cephalosporins, and the detectable rate of ESBLs in E. coli was from 62% to 100% in India and almost up to 62% in China.10 Clinicians generally empirically prefer the use of carbapenems to treat infections caused by these strains.11 However, during the past 10 years, the isolation of carbapenemases, especially NDM-1-producing E. coli, has been reported at an increasing rate worldwide. Therefore, we should pay more attention to the emergence of NDM-1-producing genes (blaNDM-1) or their variants in E. coli strains to avoid encountering a situation where antibiotic options are limited. In this study, we describe a high prevalence of blaNDM-5 in carbapenem-resistant E. coli strains in a university hospital in Eastern China and further conduct molecular research to investigate the transmission features of this gene.
Materials And Methods
Bacterial Collection, Species Identification And Antimicrobial Susceptibility Testing
Isolates were collected from July 2017 to July 2018 in the clinical microbiology laboratory of the First Affiliated Hospital of Nanjing Medical University, a large medical center with more than three thousand beds in Jiangsu, China. All isolates were first identified using the VITEK2 compact system (bioMérieux, France) and then confirmed by the MALDI-TOF MS apparatus (Bruker Microflex LT, Germany). Antimicrobial susceptibilities were determined by measuring the minimum inhibitory concentrations (MICs) with the broth microdilution method (for piperacillin-tazobactam, ceftazidime, aztreonam, amikacin, gentamicin, levofloxacin, imipenem, ertapenem, meropenem, and colistin), followed by E-test strips (for tigecycline). The MIC results were interpreted according to the Clinical and Laboratory Standards Institute guidelines (M100-S26),12 except for colistin and tigecycline, for which the 2016 European Committee on Antimicrobial Susceptibility Testing breakpoints was used (http://www.eucast.org/clinical_breakpoints/). E. coli ATCC 25922, K. pneumoniae ATCC 1705 and ATCC 1706 served as the quality control strains.
Detection Of Carbapenemase Producing Isolates And Genes
Carbapenemase production was screened by the modified Hodge test and imipenem-EDTA double-disc synergy test. The presence of common carbapenemase-encoding genes (blaKPC, blaNDM, blaIMP, blaVIM, blaOXA-48), extended-spectrum β-lactamase (ESBL) genes (blaCTX-M, blaSHV, blaTEM, blaOXA-1), 16S rRNA methylase genes (armA, rmtB) and plasmid-mediated quinolone resistance (PMQR) genes (qnrA, qnrB, qnrS, aac(6′)-Ib-cr) were detected using PCR methods as described previously.13–15 Positive amplicons were determined by nucleotide sequencing.
The XbaI-digested genomic DNA from blaNDM-positive E. coli strains was used to investigate the genetic relationship of the strains by pulse field gel electrophoresis (PFGE). DNA fragments were separated on a CHEF Mapper apparatus (Bio-Rad, USA) according to the following parameters: voltage of 6 V/cm, pulse angel of 120°, pulse time from 6.75 to 35.38 s and run for 20 h at 14°C. Salmonella enterica H9812 was used as the reference marker. The PFGE patterns were analyzed with BioNumerics software (Applied Maths, Belgium) using the Dice similarity coefficient. Clusters were defined as DNA patterns sharing >85% similarity. Multilocus sequence typing (MLST) was also performed using the given primers to amplify seven housekeeping genes in the E. coli strains according to the protocol available on website (http://mlst.warwick.ac.uk/mlst/dbs/Ecoli). PCR products were sequenced and compared, and then a number was given to each allele. Sequence types (STs) were assigned according to the combination of seven allele numbers listed on the online database.
Conjugation experiments were performed using a parent mating conjugation assay with blaNDM-5-positive isolates acting as donor strains and azide-resistant E. coli J53 acting as a recipient strain After overnight shacking culture in Luria-Bertani (LB) broth, the donor strain and E. coli J53 were mixed at a ratio of 1:1 and inoculated on Mueller-Hinton (MH) agar for 18 h at 37 °C. Subsequently, the mixture was streaked directly on MH agar supplement with sodium azide (150 µg/mL) and meropenem (2 µg/mL). Successful transconjugants were the colonies that survived on the selective MH plates with a positive blaNDM-5 gene. Other drug-resistant genes and antibiotic susceptibilities were also investigated as mentioned above.
Molecular Analysis Of Transferrable Plasmids
Molecular sizes of transferable blaNDM-5-harboring plasmids were evaluated by S1-PFGE and the Southern blotting method.16 The whole genomic DNA of the blaNDM-5-positive strains was digested by S1 nuclease, separated by the PFGE method and then transferred horizontally to a positively charged nylon membrane. The membranes were hybridized with a digoxigenin-labeled blaNDM probe and detected using a nitro-blue tetrazolium/5-bromo-4-chloro-3ʹ-indolylphosphate color detection kit (Roche, Germany). The incompatibility groups of these plasmids were determined according to PCR-based replicon typing methods.
To investigate the genetic context around the blaNDM-5 gene, we first chose the plasmid pNDM-CREC-8 which was extracted from transconjugant CREC-8-J and harbored a blaNDM-5 gene for sequencing. The whole plasmid sequence was obtained on the HiSeq 2500 platform generating 150 bp paired-end reads. Then, the derived reads were trimmed and assembled using GLC Genomics Workbench 5 (CLC Bio, Aarhus, Denmark), and gaps were closed through PCR and Sanger sequencing. Sequence analysis was conducted using the ORF Finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and BLAST functions (http://blast.ncbi.nlm.nih.gov/Blast.cgi). Additionally, the genetic context around blaNDM-5 in the other 14 transconjugants was investigated by the PCR mapping method with primers designed according to the sequence of pNDM-CREC-8.
Clinical Characteristics Of The blaNDM-5 Strains
During the study period from July 2017 to July 2018, 197 strains with decreased sensitivity to imipenem (MIC ≥ 4 µg/mL) or meropenem (MIC ≥ 4 µg/mL) were identified as CRE, including 144 (73%) K. pneumoniae, 28 (14%) E. coli and 25 (13%) other isolates. Among the carbapenem-resistant E. coli strains, 18 (64%) were verified as NDM-5 producers, and the rest were KPC-2. Clinical characteristics associated with the 18 blaNDM-5-positive E. coli strains are summarized in Table 1. The strains were isolated from urine (n=7), pus (n=4), sputum (n=3), blood (n=3) and cerebrospinal fluid (n=1). All patients carrying blaNDM-5-positive strains were initially admitted to the hospital with diverse underlying diseases, and none of them had a travel history to NDM endemic countries, e.g., India and the Balkans. Medical records revealed that most of the patients had received different kinds of antimicrobial treatment during their hospital stay. However, unfortunately, only 10 of them recovered, and the rest either had poor prognosis or died. Further analysis the reasons for the poor outcomes of the patients, we found that not all the patients with poor prognosis are due to NDM-5-positive CREC infections. There are also some patients whose deaths are attributed to underlying diseases and abandonment of treatment. For example, patient 2 and 17 were finally died with blood culture negative after effectively antimicrobial treatment.
Table 1 Clinical Characteristics Of blaNDM-5-Positive E. Coli Strains
Antimicrobial susceptibilities were performed on all blaNDM-5-positive E. coli strains, and their susceptibility profiles are listed in Table 2. All blaNDM-5-positive E. coli strains exhibited 100% resistance to ceftazidime, piperacillin/tazobactam, imipenem, meropenem, ertapenem and levofloxacin. In addition, the resistance rates of aztreonam and gentamicin were both 72.2%. In contrast, amikacin, colistin and tigecycline showed high susceptibility rates, which were 66.7%, 100% and 100%, respectively, suggesting their potential use for fighting against blaNDM-5-positive strains infections.
Table 2 Antimicrobial Susceptibilities Of Additional Drug-Resistant Genes Of blaNDM-5-Positive E. Coli Strains
Detection Of Carbapenemase Producing Isolates And Genes
The modified Hodge test and imipenem-EDTA double-disc synergy test were used to screen all blaNDM-5-positive strains. Apart from blaNDM-5, the other four carbapenemase-encoding genes were not discovered in any of the strains. The results of nucleotide sequencing of other drug resistance genes are listed in Table 2. ESBL genes existed in most strains (n=16, 88.9%), among which blaCTX-M groups were the main types and accounted for 12 strains. In addition, 3 strains cocarried 2 groups of blaCTX-M genes, including 2 with a newly emerged blaCTX-M-64 gene that evolved by homologous recombination between the blaCTX-M-15-like and blaCTX-M-14 genes.17 The rmtB and aac(6′)-Ib-cr genes were identified in 4 and 7 strains, respectively.
Genetic relatedness of the blaNDM-5-positive E. coli strains isolated from diverse departments was investigated by PFGE and MLST. Among the blaNDM-5-positive E. coli strains, ST167 (n=7) was the most prevalent, followed by ST410 (n=4) and ST101 (n=2). In addition, analysis of PFGE profiles revealed that strains in either ST167 (except CREC-8), ST410 or ST101 each shared very similar patterns (>85%), suggesting that clonal spread of blaNDM-5-positive E. coli strains occurred in our hospital. Notably, when taking the hospitalization time overlap and admission units into consideration, we thought that an outbreak (CREC-10, CREC-11, CREC-12) had occurred in the department of nephrology during September 2017. The remaining strains belonged to the other 5 STs and displayed clearly different PFGE patterns, suggesting that these E. coli strains were not genetically related to other strains even though some of them were recovered from the same unit (Figure 1).
Figure 1 Dendrogram derived from the PFGE patterns of 18 blaNDM-5-positive E. coli strains.
Transferability Of The blaNDM-5 Gene
Our study showed that the blaNDM-5 genes from 15 E. coli strains were successfully transferred to E. coli J53, whereas CREC-19, CREC-26 and CREC-28 failed after multiple tries. Antimicrobial susceptibilities of the transconjugants showed they were resistant to all β-lactams except aztreonam. In addition to blaNDM-5, the blaCTX-M group, rmtB and aac(6′)-Ib-cr genes could also be cotransferred in several donor strains, resulting in resistance to aztreonam and aminoglycosides in their transconjugants.
S1-PFGE found that the transconjugants all possessed nearly the same plasmids approximately 50 kb in size, and Southern blotting with a blaNDM probe confirmed that all the plasmids contained blaNDM-5 (Figure 2). Subsequently, the plasmid incompatibility classification methods assigned them to IncX3. Considering that the blaNDM-5-harbored transferrable plasmids that were of the same size and incompatibility group, we further selected the plasmid pNDM-CREC-8 as a reference for whole genome sequencing to better understand the genetic environment surrounding blaNDM-5. The completed sequence length of pNDM-CREC-8 was 46,263 bp with an average GC content of 47%. When submitted to the GenBank database for sequence alignment, it showed more than 99% nucleotide identity to the previously reported IncX3 pNDM-MGR 194 plasmid (GenBank accession number KF220657) in India.18 The flanking genetic structure of blaNDM-5, which was composed of an IS3000 and incomplete ISAba125 interrupted by IS5 located upstream, and the genes bleMBL, trpF, dsbC and IS26 downstream (IS3000-ΔISAba125-IS5-blaNDM-5-bleMBL-trpF-dsbC-IS26) in plasmid pNDM-MGR 194 were also identified in pNDM-EC-133. Furthermore, the other 14 strains were detected by PCR mapping and sequencing methods.
Figure 2 S1-PFGE and Southern blotting of the transferable plasmids in 15 conjugants. The arrows represent the plasmids where the blaNDM-5 genes located on.
NDM-5 was first identified in a multidrug resistant Escherichia coli strain from a patient in United Kingdom through routine swabbing.19 Compared to the amino acid sequences, NDM-5 differs from NDM-1 at positions 88 (Val→Leu) and 154 (Met→Leu), and NDM-5 has an enhanced hydrolytic activity against carbapenems. Since then, NDM-5-producing strains have been reported in Singapore, India, Algeria, Japan, China, the Netherlands, Denmark, Australia, South Korea, Spain, Egypt and America.20 Obviously, NDM-5 seems to have become a secondary polular NDM variant of the NDM-1 reported in the world. In addition, the increasing prevalence of NDM-5-producing Enterobacteriaceae should be of deep concern because these bacteria are usually combined with other drug-resistant mechanisms, such as cephalosporins, quinolones, and aminoglycosides. On the other hand, NDM-5-producing Enterobacteriaceae have already been isolated from pets, food animals and the environment, which serve as important reservoirs in the community.21–23
In this study, homology analysis indicated that ST167, ST410 and ST101 caused clonal expansion in our hospital, which revealed endemic potential and deserved extensive surveillance. Noticeably, ST167 and ST410 E. coli were both previously identified as successful pandemic clones for the dissemination of ESBL genes in humans and animals.24–27 In addition, the transmission of blaNDM-1 and other blaNDM variants, e.g., blaNDM-4 and blaNDM-7, in E. coli also had a strong association with these two sequence types worldwide.28–30 These findings revealed that further spread of these strains may lead to significant clinical and public health concerns. In particular, ST167, which was continuously isolated from multiple specimens in different departments during the study period, had been found carrying the blaNDM-5 gene in several cities (including Chengdu, Shanghai, Shandong, Shenyang, Chifeng and Haikou) with no obvious geographical contact across China,20,31–33 suggesting that ST167 plays an important role in the transmission of the blaNDM-5 gene in China. In addition to a possible outbreak of K. pneumoniae in Europe,34 our study reported the first blaNDM-5-related outbreak of E. coli ST410 in mainland China. Medical records showed that two ST101 E. coli isolates with very similar PFGE patterns (> 95%) were isolated from two patients by endoscopic retrograde cholangiopancreatography (ERCP) with the same duodenoscope in November 2017. It remains unclear whether the ERCP was responsible for the two infectious experiences because we were unable to culture the organism from the implicated instrument. However, a few studies in the United States have identified outbreaks of NDM-1-producing E. coli after receiving ERCP, even though the disinfection procedure of the associated endoscope was finished following standard recommendations and guidelines,35 reminding us that a potential risk of contamination exists despite high-level disinfection. The four clonally diverse STs of E. coli (ST10, ST38, ST448, ST540) were identified as NDM-5 or its variants producers identified previously. 36–39 Furthermore, ST359 was first regarded as an NDM producer.
Compared with clonal expansion, mobile elements were thought to play a major role in the dissemination of blaNDM in different species in Enterobacteriaceae. The results of the conjugation experiment in our study showed that 15 strains can transfer their blaNDM-5 gene to the recipient strain through IncX3 conjugative plasmids with the same molecular weight of approximately 50 kb. The IncX3-type plasmids were previously identified as a narrow host range and only transmitted in Enterobacteriaceae. The first emergence of the IncX3-type plasmid carrying blaNDM-1 was reported in China, and the blaNDM-1 gene was disseminated among various species worldwide. Our team recently also found a nearly same size IncX3-type plasmid responsible for the transmission of blaNDM-1 in K. pneumoniae in the Department of Neonatology.40 Whether the blaNDM-5 gene carrying an ~50 kb IncX3-type plasmid was evolved from the ~50 kb IncX3-type plasmid containing blaNDM-1 gene needs further investigation. Complete sequencing of the plasmid pNDM-EC-133 shows an almost identical sequence to pNDM-MGR 194, which was first identified as an NDM-5 producer of IncX3-type in India.18 In most studies in China, the pNDM-MGR 194-like plasmids were discovered to be associated with NDM-5 and thought to be efficient vehicles for the dissemination of blaNDM-5 across the country. Except for E. coli, the pNDM-MGR 194-like plasmids were also found in K. pneumoniae, K. michiganensis, C. freundii and P. mirabilis, which implies the potential to spread intra- or interspecies in Enterobacteriaceae.41 It is noteworthy that one study in Jiangsu Province pointed out that the pNDM-MGR 194-like plasmid played a role in the dissemination of blaNDM-5 in cows in three dairy farms distributed in scattered regions of Jiangsu Province. This finding raised the possibility of blaNDM-5 spreading from animals to humans and represented an important blaNDM-5 reservoir in the community, which may lead to enormous losses to public health and property. Therefore, there is an urgent need to implement stringent and comprehensive strategies to monitor the emergence of blaNDM-5 and avoid its further spreading.
In conclusion, our study demonstrated a high prevalence of blaNDM-5, which was combined with clonal spread and plasmid-mediated transmission of carbapenem-resistant E. coli strains in a university hospital in Jiangsu Province, China. The continuous isolation of ST167 and an outbreak of ST410 E. coli strains in our hospital underline appropriate infection control operations, such as isolating colonized or infected patients individually, cohorting nursing, and severe hand, equipment and environmental disinfection, should be taken immediately after the first isolation of blaNDM-5-positive strains. The pNDM-MGR 194-like plasmid acts as an important vector for the dissemination of blaNDM-5 in E. coli, and its emergence in diverse STs implies its potential to a further extent.
This study was supported by the Project of the Key Laboratory for Laboratory Medicine of Jiangsu Province (No. XK201749) and the innovation project for postgraduate education of Jiangsu Province (No. SJLX15_0436). We would like to thank Professor Wenen Liu (Zhongnan University) and Huimin Qian (Jiangsu Provincial Centers for Disease Control and Prevention) for kindly providing the reference strains.
The authors report no conflicts of interest in this work.
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