Characterization of NDM-4-Producing Citrobacter amalonaticus Isolates from China

Introduction

Citrobacter spp. is a common zoonotic pathogen comprising 11 species.¹ Various species of Citrobacter spp. may cause infections, including diarrhea, sepsis, meningitis, or respiratory and urinary system infections in neonates, the elderly, and immunocompromised hosts.² Although the majority of cases are sporadic, mortality associated with invasive Citrobacter spp. infections is high.^3,4

A recent increase in multidrug resistance among Citrobacter spp. has been reported.⁴ Previous studies have demonstrated that Citrobacter spp., especially C. freundii, harbor carbapenemase genes and contribute to a high proportion of carbapenem-resistant Enterobacterales, representing an emerging infection control and public health challenge.^5–7 Different carbapenemase genes, such as bla_NDM-1, bla_KPC-2, bla_IMP-4, bla_VIM-2 and bla_OXA-48, have been identified in Citrobacter spp.⁵ In addition, several specific plasmids and other genetic mobile elements for carbapenemase gene carriage have been identified, such as IncN[pMLST15] for KPC-2 and TnAS3 for NDM-1.^5,8 Among plasmids harboring bla_NDM, in Enterobacteriaceae, IncX3 was predominant, followed by the IncFII type.⁷ Several studies have reported different carbapenemase genes and transmission mechanisms in C. freundii, C. koseri, and C. braakii.^5,8 However, genomic analyses of NDM-producing C. amalonaticus have been limited. In this study, the genomic characterization of two carbapenem-resistant C. amalonaticus isolates harboring bla_NDM-4 were analyzed.

Methods

Collection and Identification of Bacterial Strains

Fecal samples (1.0 g) from inpatients with diarrhea were cultured on MacConkey agar supplemented with 2 mg/L meropenem for 18–24 h at 37°C from 2016 to 2023 at The First Affiliated Hospital, Zhejiang University School of Medicine. Isolates were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) (Bruker, Bremen, Germany). Citrobacter spp. isolates were collected for further analysis. Carbapenemase-encoding genes (bla_NDM, bla_KPC, bla_IMP, bla_VIM and bla_OXA-48) were tested using PCR.⁹

Antibiotic Susceptibility Testing

The MICs of 18 antibiotics were determined according to Clinical and Laboratory Standards Institute (CLSI) recommendations. The results were interpreted in accordance with the guidelines of the Clinical and Laboratory Standards Institute (CLSI) guidelines.¹⁰

Whole Genome Sequencing (WGS) and Bioinformatics Analysis

Genomic DNA was extracted using the FastDNA SPIN Kit for Soil (MP Biomedicals, United States) and sequenced using Illumina HiSeq 2000 (Novogene Bioinformatics Technology Co., Ltd., Beijing, China) and Oxford Nanopore Technologies platform (Oxford Nanopore Technologies, Oxford, UK), Complete genome sequences were generated by performing a hybrid assembly of short-read and long-read sequences using Unicycler v0.4.8.

The Whole Genome Shotgun BioProject for the included isolates was deposited under accession number PRJNA1118925. The plasmid sequence was hosted in the corresponding BIGSdb database under accession number IncFII-116.

The antimicrobial resistance genes were identified using ResFinder version 2.1 (http://cge.cbs.dtu.dk/services/resfinder). Plasmid Finder v. 1.3 was used to identify the plasmid incompatibility type.¹¹ All sequenced genomes were annotated using Prokka.¹² The Pyani and Python3 modules calculate the average nucleotide identity (ANI) and related measures for species comparisons (https://github.com/HuttonICS/pyani). Plasmid alignment was performed and visualized using BLAST ring image generator (BRIG) software.

Results

Characteristics of the Carbapenem-Resistant Citrobacter Spp

Forty-two carbapenem-resistant Citrobacter spp. were identified, including 32 C. freundii, three C. koseri, three C. braakii, two C. amalonaticus, and two C. youngae.

Among C. freundii isolates, PCR revealed that 19 isolates carried bla_NDM, four isolates carried bla_KPC, one isolate carried bla_IMP, one isolate carried bla_VIM and one isolate carried bla_OXA-48 (Supplementary Table S1). In addition, bla_KPC coexisted with bla_NDM in three C. freundii isolates. All C. koseri isolates harbored bla_KPC and C. amalonaticus harbored bla_NDM-4.

Antimicrobial Susceptibility

Two NDM-4-producing C. amalonaticus isolates (L5091 and L5094) were resistant to multiple antibiotics (Table 1).

Table 1 Antimicrobial Drug Susceptibility Profiles

Genetic Characteristics of NDM-4-Producing C. amalonaticus

Strains L5091hy and L5094hy had the highest ANI values compared to the type strain of C. amalonaticus (GCA_001558935.2) (Figure S1). A circular chromosome and several plasmids were confirmed in two C. amalonaticus isolates (L5091hy and L5094hy) by whole-genome sequencing. Plasmids pL5091_bla_NDM-4 and pL5094_bla_NDM-4 are novel IncFII-116 plasmids. The lengths of the two plasmids were 87124 bp and 87952 bp, respectively. The average GC content of the two plasmids was 53.6% and 53.6%, respectively.

The genetic structures of pL5091_bla_NDM-4 and pL5094_bla_NDM-4 are similar (Figure 1). The genetic environment for bla_NDM-4 in the two C. amalonaticus isolates was trpF-ble-bla_NDM-4-IS15. However, it is of note that a large fragment of recombination in IS15 were identified downstream of bla_NDM-4 in pL5094_bla_NDM-4.

Figure 1 Major structural features and comparison of NDM-encoding plasmids. Genomic map of the blaNDM-4 producing pL5091_blaNDM-4/pL5094_blaNDM-4 plasmids with three closely related plasmids (CP043383.1, ON209143.1, ON964513.1). (A) pL5091_blaNDM-4; (B) pL5094_blaNDM-4. 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.

Discussion

The continued increase in the prevalence of multidrug-resistant Citrobacter spp. poses a global health security threat.^3,5 The present and previous studies have demonstrated C. freundii is the predominant epidemic isolate.^5,6 The bla_NDM gene is commonly detected in carbapenem-resistant Citrobacter spp. In addition, bla_NDM-4 in two C. amalonaticus isolates were first identified in this study.

A previous study reported C. freundii accounted for 83% of Citrobacter spp., which was higher than our result (76.2%).^5,13 This difference may be because the strains in our study were isolated only from fecal samples. As previously described for C. freundii, the transmission mechanisms of bla_NDM-1-, bla_VIM-1-, and bla_OXA-48-carrying plasmids were IncF, IncN, and IncM1, respectively.¹⁴ Notably, bla_NDM was the most frequent carbapenemase detected in C. freundii, which differs from other strains in France.¹³ Both local and global carbapenem-resistant Citrobacter spp. are genetically diverse.⁶ In addition, several earlier studies have reported the co-production of two or three carbapenemases, such as KPC-2 plus NDM-1, KPC-2 plus VIM-2, VIM-1 plus OXA-48, or KPC and NDM plus SHV, in Citrobacter spp. isolates.^5,8,15,16

C. amalonaticus is seldom isolated from fecal or urine samples. A study described the coexistence of bla_NDM-1 and mcr-1-like genes in C. amalonaticus.¹⁷ To the best of our knowledge, this is the first report of C. amalonaticus harboring bla_NDM-4, significantly extends our understanding of the structural diversification of plasmids. These results highlight the necessity of continuously monitoring the dissemination of carbapenem-resistant Citrobacter spp. in the clinical setting.

The most prevalent plasmids carrying bla_NDM are IncX3, IncFIB, IncFII, and IncC types.¹⁸ Here, we report two carbapenem-resistant C. amalonaticus isolates in China, carrying a novel IncFII-116 plasmid containing bla_NDM-4, which is relatively close to IncFII. Therefore, the comprehensive resistance surveillance of Citrobacter spp. should be further strengthened.

Conclusion

Here, we characterized the first IncFII-116 plasmid carrying bla_NDM-4 in two C. amalonaticus isolates from China. The genetic environment of NDM-4 has been elucidated, highlighting the need to monitor the dissemination of carbapenemase-encoding genes and plasmids.