Back to Journals » Infection and Drug Resistance » Volume 15

Co-Production of NDM-1 and OXA-10 β-Lactamase in Citrobacter braakii Strain Causing Urinary Tract Infection

Authors Han H, Zhao Z, Lin Y, Lin B, Xu H, Zheng B

Received 10 November 2021

Accepted for publication 11 March 2022

Published 19 March 2022 Volume 2022:15 Pages 1127—1133

DOI https://doi.org/10.2147/IDR.S347943

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Professor Suresh Antony



Huiming Han,1,* Zhi Zhao,2,* Yan Lin,1 Baihui Lin,1 Hao Xu,3 Beiwen Zheng3

1School of Basic Medical Sciences, Beihua University, Jilin, Jilin Province, People’s Republic of China; 2Department of Neonatology, Shaanxi Province People’s Hospital, Xi’an, Shaanxi Province, People’s Republic of China; 3Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, Hangzhou, Zhejiang Province, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Beiwen Zheng, State Key Laboratory for Diagnosis and Treatment of Infectious Diseases, The First Affiliated Hospital, College of Medicine, Zhejiang University, No. 79 Qingchun Road, Hangzhou, Zhejiang Province, 310003, People’s Republic of China, Tel +86 571 872 364 23, Fax +86 571 872 364 21, Email [email protected]

Abstract: In this study, we describe, for the first time, the co-existence of blaNDM-1and blaOXA-10 in a carbapenem-resistant Citrobacter braakii strain DY2019 isolated from a patient with urinary tract infection in China. We aimed to investigate the genomic context of two β-lactamase-producing plasmids and characterize the transmission mechanism of the carbapenemase-encoding gene. Whole-genome sequencing of strain DY2019 was performed with Nanopore and Illumina platforms, which revealed a chromosome sequence with the length of 4,830,928 bp, an IncC group plasmid pDY2019-OXA (size of 178,134 bp), and a novel IncHI2 group plasmid pDY2019-NDM (length 348,495 bp). A total of 16 antimicrobial resistance genes (ARGs) that confer resistance to nine different antibiotic groups were identified in strain DY2019, and 11 of them were carried by plasmid pDY2019-OXA. These data and analyses suggest that the carbapenem-resistant C. braakii strains may serve as potential reservoir of carbapenemase and highlight the need for further close surveillance of this species in clinical settings.

Keywords: Citrobacter braakii, carbapenem-resistant, NDM-1, OXA-10, whole-genome sequencing, urinary tract infection

Introduction

Genus Citrobacter is a Gram-negative, facultative aerobic, long rod-shaped Enterobacteriaceae.1,2 Citrobacter isolates are most commonly isolated from environmental habitats, including soil, water, sewage, and intestines of animals and humans.3–6 As an opportunistic pathogen, Citrobacter freundii complex (CFC) isolates are usually associated with a variety of nosocomial infections of the respiratory tract,7 urinary tract,8 blood,9 and even central nervous system.10 Citrobacter braakii, as a member of CFC, has rarely been recognized as a human pathogen.11

Infections caused by C. braakii are usually treated with cephalosporins, fluoroquinolones, and carbapenems. Due to the irrational use of broad-spectrum antibiotics, C. braakii developed resistance to a certain number of antibiotics. New Delhi Metallo-β-lactamase 1 (NDM-1) is an Ambler class B β-lactamase that spreads across different Enterobacteriaceae species, including C. braakii.12–17

This work reports a carbapenem-resistant C. braakii strain causing urinary tract infection, which showed extensive resistance to most tested antibiotics. Nanopore and Illumina sequencing revealed the co-existence of blaNDM-1-carrying and blaOXA-10-carrying plasmids in the isolate. Our study aimed to (i) describe the complete sequence of novel IncHI2 blaNDM-1-carrying and blaOXA-10-carrying plasmids and (ii) evaluate the phenotypic characteristics of the IncHI2 blaNDM-1-carrying plasmid.

Materials and Methods

Isolate DY2019 was recovered from a urine sample of a 71-years old female patient with urethritis in March 2019 in Dongyang, China. The isolate was found to harbor the blaNDM-1 gene by real-time PCR as part of ongoing routine work for surveillance of carbapenemase-producing bacteria. The carbapenemase-encoding genes were identified by PCR amplification as described previously.18 Bacterial identification was performed by both VITEK2 compact system (BioMerieux, France) and Microflex MALDI-TOF mass spectrometer (Bruker Daltonics, Bremen, Germany) as described previously.6,19 Using ANI analysis, species were determined if the genome in question had >95% ANI value with the type genome using pyani with default settings (https://github.com/widdowquinn/pyani).

The VITEK 2 system with panel AST-GN-16 (bioMérieux, Marcy-l’Étoile, France) was employed for antimicrobial susceptibility testing (AST) of isolate DY2019, with Escherichia coli ATCC 25922 as control. AST results were interpreted following the standards of the Clinical and Laboratory Standards Institute.20 Minimum inhibitory concentration (MIC) of strain DY2019 showed that DY2019 resistant to a various type of antibiotics, including cefotaxime (MIC =64 μg/mL), ceftazidime (MIC >128 μg/mL), cefpirome (MIC =16 μg/mL), piperacillin/tazobactam (MIC =128 μg/mL), ciprofloxacin (MIC =16 μg/mL), imipenem (MIC =4 μg/mL), and only susceptible to tigecycline (MIC =0.06 μg/mL) and colistin (MIC =1 μg/mL).

According to the manufacturer’s instructions, total DNA (chromosomal and extrachromosomal) was extracted and purified using the QIAamp DNA Mini Kit (Qiagen, Germany). One microgram of the genomic DNA extract was subjected to whole-genome sequencing (WGS) using the Nanopore platform (Oxford Nanopore, Oxford, UK) and Illumina Novaseq-600 sequencer (Illumina, San Diego, United States) as described before.18,21 Protein-coding genes were initially identified and annotated using RAST (https://rast.nmpdr.org/), while insertion elements (IS) were determined using ISFinder (https://www-is.biotoul.fr/). Queries were generated using the ResFinder 3.1 database22 to identify acquired antibiotic resistance genes. PlasmidFinder and pMLST were used to identify plasmid incompatibility types.23

S1-PFGE characterized the number and size of the plasmid of the DY2019. The location of blaNDM-1 gene was confirmed by Southern blot and hybridization with a digoxigenin-labeled blaNDM-1 probe using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics). Conjugation transfer experiments were conducted to explore the transferability of plasmids with rifampicin-resistant E. coli 600 as the recipient strain as recipients, as described previously.24 After that, Mueller-Hinton agar (OXOID, Hampshire, United Kingdom) plates contained both 200 μg/mL rifampicin (Meilunbio, Dalian, China) and two μg/mL meropenem were used to select blaNDM-1 carrying transconjugants. Plasmid typing was performed by using PlasmidFinder.25

Results and Discussion

Strain DY2019 was finally identified as C. braakii by ANIb analysis. A chromosome of 4,830,928 bp in length with a GC content of 52.2%, a plasmid (pDY2019-NDM) of 348,495 bp in length with a GC content of 47.9%, and a plasmid (pDY2019-OXA) of 178,134 bp in length with a GC content of 52.5%, were assembled from the Illumina and Nanopore sequencing reads with a hybrid strategy utilizing Unicycler program.26 Whole-genome sequencing showed that strain DY2019 harboured blaNDM-1, blaOXA-10, aac(6’)-Ib-cr, aac(6’)-Ib3, qnrB4, aadA1, sul1, sul2, dfrA14, tet(D), ARR-2, mph(A), cmlA1, floR, blaDHA-1, and blaCMY-93 (Table 1), which is consistent with the phenotypic results. It is worthy to note that one AmpC-type beta-lactamase encoding gene, blaCMY-93, was found in the chromosome of strain DY2019 using ResFinder 3.1. This result further supported the previous hypothesis that blaCMY genes were ubiquitous in CFC isolates.19

Table 1 Characteristics of ARGs Carried by DY2019

S1-PFGE and Southern blot demonstrated that DY2019 encoded two plasmids (Figure 1), and blaNDM-1 was located on a ~348 kb novel IncHI2 plasmid (Figure 2). Interestingly, the principal NDM variant found in CFC was blaNDM−1, which is usually located on the self-transferable IncX3 plasmid.1,27 However, despite repeated efforts, no transconjugants carrying blaNDM-1 were obtained. This could explain by the fact that pDY2019-NDM lacks a set of transfer (tra) genes (Figure 2). Resfinder Identified four antimicrobial resistance genes (ARGs) in pDY2019-NDM. These genes were associated with β-lactams resistance (blaNDM-1), aminoglycoside resistance (aac(6’)-Ib-cr and aac(6’)-Ib3) and tetracycline resistance (tet(D)) phenotypes of strain DY2019. Interestingly, plasmid comparison based on full plasmid BLAST query revealed that pDY2019-NDM exhibited very low similarity to other plasmids (Figure 2A), which suggested that it is a novel blaNDM-carrying plasmid. Genetic context characterization revealed that blaNDM-1 was located in an ISCR1 complex class 1 integron with a conserved structure of ISAba125-blaNDM-1-bleMBL-trpF-dsbD. This conserved structure is normal among elements carrying blaNDM-1 and is commonly found in various blaNDM-1-carrying plasmids in Enterobacteriaceae. It is noteworthy that blaNDM-1-carrying IncHI2 plasmids were mainly Identified in the Enterobacter cloacae complex in China.28,29 This study further suggests that blaNDM-positive IncHI2 plasmids might circulate in Citrobacter species.

Figure 1 Plasmid profiles and Southern blot-hybridization of C. braakii DY2019. Southern blot-hybridization of S1-nuclease digested DNA using a specific probe (blaNDM). M: XbaI digested total DNA of Salmonella enterica serotype Braenderup H9812 as a size marker and Enterobacter cloacae DY1901 as the control.

Figure 2 Major structural features and comparison of beta-lactamase-encoding plasmids. (A) Schematic illustration showing the structural features of pDY2019-NDM. (B) Linear maps of five NDM-1 encoding plasmids, pDY2019-NDM, pCC1-1/2b (MT559998), pKC3-1/2b (MT560001), pPmi70746_1 (CP023274), and AR_0156 plasmid unitig_1 (CP021853). ORFs are portrayed by arrows and colored according to their putative functions. The alignment of the plasmids was performed and visualized by BLAST ring image generator (BRIG) software.

Plasmidfinder also indicated that pDY2019-OXA was an IncC type plasmid. A total of 11 ARGs were Identified in pDY2019-OXA by ResFinder. These ARGs enabled pDY2019-OXA to exhibit resistance to different types of antimicrobial agents, including β-lactams (blaDHA-1 and blaOXA-10), fluoroquinolone (qnrB4), sulphonamide (sul and sul2), chloramphenicol (cmlA1), aminoglycoside (aadA1), macrolide (mph(A)), tetracycline (tet(D)), rifampin (ARR-2), and trimethoprim (dfrA14). We further compared the sequence of pDY2019-OXA against the NCBI database by Blastn. The result showed that pDY2019-OXA shares the 99.95% identity (92% coverage) with plasmid pCC1-1/2b (MT559998), and 99.84% identity (99% coverage) with plasmid pKC3-1/2b (MT560001). It is worth noting that plasmids pKC3-1/2b and pCC1-1/2b were classified as type 1/2b IncC plasmid. The plasmid pCC1-1/2b was carried by a C. freundii isolate. In contrast, pKC3-1/2b was carried by a Klebsiella pneumoniae strain, and both of them were identified in food-producing animals originating from China. These data indicated that blaOXA-10-carrying plasmid Identified in this study might disseminate from food-producing animals to humans, and the proliferation of IncC plasmids represents a potential public health risk.

It is reported that CFC represents approximately 29% of all opportunistic nosocomial infections.30 However, there is little information about carbapenemase-producing CFC. In this work, we report an adult case of urinary tract infection caused by multi-antibiotic resistant C. braakii, although a single case limits this work. Furthermore, we describe the structure of pDY2019-NDM, a 348-kb IncHI2 plasmid carrying the blaNDM-1 gene, and pDY2019-OXA, a 178-kb IncC plasmid carrying the blaOXA-10 gene.

Conclusion

To the best of our knowledge, co-production of NDM-1 and OXA-10 in the same isolate has never been reported. The results of this study improved our understanding of the genetic context of C. braakii strains and their antibiotic resistance phenotypes and genotypes. In general, we highlighted the emergence of NDM-1-producing C. braakii as worrisome and emphasized the need for close surveys for controlling potential dissemination.

Nucleotide sequence accession numbers. The nucleotide sequences of the chromosome and the plasmids of C. freundii strain DY2019 have been deposited into DDBJ/EMBL/GenBank under accession numbers CP080539, CP080540, and CP080541, respectively.

Acknowledgments

This work was supported by research grants from the National Key Research and Development Program of China (No.2016YFD0501105), the National Natural Science Foundation of China (No. 82072314), the Research Project of Jinan Microecological Biomedicine Shandong Laboratory (JNL-2022011B), the Jilin Province’s Training Program of Innovation and Entrepreneurship for Undergraduates (S202110201067), the Training Program of Innovation and Entrepreneurship for Undergraduates of Beihua University (202110201172), and Innovation funding project for undergraduate students of Beihua University (202110201252).

Disclosure

The authors report no conflicts of interest in this work.

References

1. Zhang T, Lin Y, Li P, et al. Characterization of plasmid co-harboring NDM-1 and SHV-12 from a multidrug-resistant Citrobacter freundii strain ZT01-0079 in China. Infect Drug Resist. 2021;14:947–952. doi:10.2147/IDR.S301736

2. Yang L, Li P, Liang B, et al. Multidrug-resistant Citrobacter freundii ST139 co-producing NDM-1 and CMY-152 from China. Sci Rep. 2018;8(1):10653. doi:10.1038/s41598-018-28879-9

3. Braak H. Onderzoekingen over Vergisting van Glycerine (thesis). Delft, Netherlands 166: WD Meinema-Utitgeuer; 1928.

4. Li M, Li F, Mi Z, et al. Comparative genomics analysis of pTEM-2262, an MDR plasmid from Citrobacter freundii, harboring two unclassified replicons. Future Microbiol. 2018;13(15):1657–1668. doi:10.2217/fmb-2018-0243

5. Osieka V, Grobbel M, Schmoger S, et al. Complete draft genome sequence of an extended-spectrum β-lactamase-producing Citrobacter freundii strain recovered from the intestine of a house sparrow (Passer domesticus) in Germany, 2017. Genome Announc. 2018;6(26):e00599–18. doi:10.1128/genomeA.00599-18

6. Xu H, Wang X, Yu X, et al. First detection and genomics analysis of KPC-2-producing Citrobacter isolates from river sediments. Environ Pollut. 2018;235:931–937. doi:10.1016/j.envpol.2017.12.084

7. Lockhart SR, Abramson MA, Beekmann SE, et al. Antimicrobial resistance among Gram-negative bacilli causing infections in intensive care unit patients in the United States between 1993 and 2004. J Clin Microbiol. 2007;45(10):3352–3359. doi:10.1128/JCM.01284-07

8. Ouyang J, Sun F, Zhou D, et al. Comparative genomics of five different resistance plasmids coexisting in a clinical multi-drug resistant Citrobacter freundii isolate. Infect Drug Resist. 2018;11:1447–1460. doi:10.2147/IDR.S165818

9. Anderson MT, Brown AN, Pirani A, et al. Replication dynamics for six gram-negative bacterial species during bloodstream infection. mBio. 2021;12(4):e0111421. doi:10.1128/mBio.01114-21

10. Plakkal N, Soraisham AS, Amin H. Citrobacter freundii brain abscess in a preterm infant: a case report and literature review. Pediatr Neonatol. 2013;54(2):137–140. doi:10.1016/j.pedneo.2012.10.004

11. Dong D, Mi Z, Li D, et al. Novel IncR/IncP6 hybrid plasmid pCRE3-KPC recovered from a clinical KPC-2-producing Citrobacter braakii isolate. mSphere. 2020;5:e00891–19.

12. Loqman S, Soraa N, Diene SM, Rolain JM. Dissemination of Carbapenemases (OXA-48, NDM and VIM) producing Enterobacteriaceae isolated from the Mohamed VI University Hospital in Marrakech, Morocco. Antibiotics. 2021;10(5). doi:10.3390/antibiotics10050492

13. Ahmad N, Khalid S, Ali SM, Khan AU. Occurrence of blaNDM variants among Enterobacteriaceae from a neonatal intensive care unit in a Northern India Hospital. Front Microbiol. 2018;9:407. doi:10.3389/fmicb.2018.00407

14. Arana DM, Ortega A, Gonzalez-Barbera E, et al.; Spanish Antibiotic Resistance Surveillance Programme Collaborating G. Carbapenem-resistant Citrobacter spp. isolated in Spain from 2013 to 2015 produced a variety of carbapenemases including VIM-1, OXA-48, KPC-2, NDM-1 and VIM-2. J Antimicrob Chemother. 2017;72(12):3283–3287. doi:10.1093/jac/dkx325

15. Parvez S, Khan AU. Hospital sewage water: a reservoir for variants of New Delhi metallo-beta-lactamase (NDM)- and extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae. Int J Antimicrob Agents. 2018;51(1):82–88. doi:10.1016/j.ijantimicag.2017.08.032

16. Bahramian A, Khoshnood S, Hashemi N, et al. Identification of metallo-beta-lactamases and AmpC production among Escherichia coli strains isolated from hemodialysis patients with urinary tract infection. Mol Biol Rep. 2021;48(12):7883–7892. doi:10.1007/s11033-021-06814-y

17. Ahmad Farajzadeh Sheikh SK, Saki M, Shahi F, Seyed-Mohammadi S, Abdi M, Moradi M. Prevalence of carbapenemases and ESBL encoding genes among K. pneumoniae isolates obtained from an educational hospital in Ahvaz, Southwestern Iran. Gene Rep. 2021;23:101128.

18. Zheng B, Yu X, Xu H, et al. Complete genome sequencing and genomic characterization of two Escherichia coli strains co-producing MCR-1 and NDM-1 from bloodstream infection. Sci Rep. 2017;7(1):17885. doi:10.1038/s41598-017-18273-2

19. Jiang X, Cui X, Liu W, Xu H, Zheng B. Genetic characterization of a novel sequence type of multidrug-resistant Citrobacter freundii strain recovered from wastewater treatment plant. Infect Drug Resist. 2019;12:2775–2779. doi:10.2147/IDR.S213525

20. CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 27th ed. CLSI supplement M100. Wayne, PA, USA: Clinical and Laboratory Standards Institute; 2017

21. Jiang X, Liu W, Zheng B. Complete genome sequencing of Comamonas kerstersii 8943, a causative agent for peritonitis. Sci Data. 2018;5(1):180222. doi:10.1038/sdata.2018.222

22. Zankari E, Hasman H, Cosentino S, et al. Identification of acquired antimicrobial resistance genes. J Antimicrob Chemother. 2012;67(11):2640–2644. doi:10.1093/jac/dks261

23. Carattoli A, Hasman H. PlasmidFinder and in silico pMLST: identification and typing of plasmid replicons in Whole-Genome Sequencing (WGS). Methods Mol Biol. 2020;2075:285–294.

24. Zeng L, Zhan Z, Hu L, et al. Genetic characterization of a blaVIM–24-carrying IncP-7β plasmid p1160-VIM and a blaVIM–4-harboring integrative and conjugative element Tn6413 from clinical Pseudomonas aeruginosa. Front Microbiol. 2019;10:213. doi:10.3389/fmicb.2019.00213

25. Carattoli A, Zankari E, Garcìa-Fernandez A, et al. PlasmidFinder and pMLST: in silico detection and typing of plasmids. Antimicrob Agents Chemother. 2014;58. doi:10.1128/AAC.02412-14

26. Wick RR, Judd LM, Gorrie CL, Holt KE. Unicycler: resolving bacterial genome assemblies from short and long sequencing reads. PLoS Comput Biol. 2017;13(6):e1005595. doi:10.1371/journal.pcbi.1005595

27. Mouftah SF, Pal T, Darwish D, et al. Epidemic IncX3 plasmids spreading carbapenemase genes in the United Arab Emirates and worldwide. Infect Drug Resist. 2019;12:1729–1742. doi:10.2147/IDR.S210554

28. Liu C, Qin S, Xu H, et al. New Delhi Metallo-beta-Lactamase 1(NDM-1), the Dominant Carbapenemase detected in carbapenem-resistant Enterobacter cloacae from Henan Province, China. PLoS One. 2015;10(8):e0135044. doi:10.1371/journal.pone.0135044

29. Cai Y, Chen C, Zhao M, et al. High prevalence of metallo-beta-lactamase-producing Enterobacter cloacae from three tertiary hospitals in China. Front Microbiol. 2019;10:1610. doi:10.3389/fmicb.2019.01610

30. Whalen JG, Mully TW, English JC 3rd. Spontaneous Citrobacter freundii infection in an immunocompetent patient. Arch Dermatol. 2007;143(1):124–125. doi:10.1001/archderm.143.1.124

Creative Commons License © 2022 The Author(s). This work is published and licensed by Dove Medical Press Limited. The full terms of this license are available at https://www.dovepress.com/terms.php and incorporate the Creative Commons Attribution - Non Commercial (unported, v3.0) License. By accessing the work you hereby accept the Terms. Non-commercial uses of the work are permitted without any further permission from Dove Medical Press Limited, provided the work is properly attributed. For permission for commercial use of this work, please see paragraphs 4.2 and 5 of our Terms.