Emergence of an Extensive Drug Resistant Citrobacter portucalensis Clinical Strain Harboring bla_SFO-1, bla_KPC-2, and bla_NDM-1

Abstract

Background

To explore the plasmid characteristics and transfer mechanisms of an extensive drug resistant (XDR) clinical isolate, Citrobacter portucalensis L2724hy, co-producing bla_SFO-1, bla_NDM-1, and bla_KPC-2.

Methods

Species confirmation of L2724hy was achieved through 16S rRNA sequencing and Average Nucleotide Identity (ANI) analysis. Antimicrobial susceptibility testing (AST) employed the agar dilution and micro broth dilution methods. Identification of resistance genes was carried out by PCR and whole-genome sequencing (WGS). Essential resistance gene locations were verified by S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and southern hybridization experiments. Subsequent WGS data analysis delved into drug resistance genes and plasmids.

Results

The confirmation of the strain L2724hy as an extensive drug-resistant Citrobacter portucalensis, resistant to almost all antibiotics tested except polymyxin B and tigecycline, was achieved through 16S rRNA sequencing, ANI analysis and AST results. WGS and subsequent analysis revealed L2724hy carrying bla_SFO-1, bla_NDM-1, and bla_KPC-2 on plasmids of various sizes. The uncommon ESBL gene bla_SFO-1 coexists with the fosA3 gene on an IncFII plasmid, featuring the genetic environment IS26-fosA3-IS26-ampR-bla_SFO-1-IS26. The bla_NDM-1 was found on an IncX3 plasmid, coexisting with bla_SHV-12, displaying the sequence IS5-IS3000-IS3000-Tn2-bla_NDM-1-ble-trpF-dsbD-cutA-gros-groL, lacking ISAa125. The bla_KPC-2 is located on an unclassified plasmid, exhibiting the sequence Tn2-tnpR-ISKpn27-bla_KPC-2-ISKpn6-korC. Conjugation assays confirmed the transferability of both bla_NDM-1 and bla_KPC-2.

Conclusion

We discovered the coexistence of bla_SFO-1, bla_NDM-1, and bla_KPC-2 in C. portucalensis for the first time, delving into plasmid characteristics and transfer mechanisms. Our finding highlights the importance of vigilant monitoring of drug-resistance genes and insertion elements in uncommon strains.

Keywords:

• Citrobacter spp
• XDR
• bla_SFO
• bla_NDM
• bla_KPC
• IncFII

Introduction

In the vast microbial landscape, the genus Citrobacter stands as a diverse group of bacteria known for its adaptability and ecological ubiquity. Within this genus, the relatively lesser-explored C. portucalensis has garnered attention for its potential contributions to various fields, especially in human health. Since its first report in 2017, diverse occurrences of C. portucalensis in wastewater, sputum, stool, sludge, poultry and, sea turtles across multiple countries have been discovered.^1–6 In 2021, Chinese researcher Cao et al isolated C. portucalensis carrying bla_NDM-1 from clinical specimens for the first time.² Subsequently, in 2022, the coexistence of bla_KPC-2 and bla_NDM-1 was reported in an extensively drug-resistant C. portucalensis obtained from a hospital in China.⁷ Additionally, in 2023, researchers isolated C. portucalensis carrying bla_NDM-1 from an endangered marine animal that died from sepsis.⁸ This growing body of evidence underscores the increasing clinical and extra-clinical dissemination of drug-resistant C. portucalensis, warranting heightened attention to address the associated public health concerns.

In 2017, the World Health Organization (WHO) designated carbapenem-resistant and extended spectrum beta-lactamases (ESBLs)-producing Enterobacteriaceae as “extremely important” on its list of antimicrobial resistant “priority pathogens”. Numerous antimicrobial resistance genes (ARGs) encoding carbapenemases and ESBLs were discovered. Among these, the bla_SFO-1, an uncommon ESBLs gene exhibiting the ability to hydrolyze beta-lactams excluding cephamycins and carbapenems, was first identified on a self-transferring plasmid from Enterobacter cloacae isolated from Japan in 1999.⁹ The bla_SFO-1 later manifested in an outbreak of Enterobacter cloacae in Spain.¹⁰ Recently, its emergence has been documented in Enterobacter hormaechei, Escherichia coli and Klebsiella pneumoniae isolated from China.^11–13 Further investigations are crucial to the understanding of the epidemiology and clinical impact of bla_SFO-1. Among the carbapenemase encoding genes, bla_KPC stands out as the predominant in Enterobacteriaceae bacteria globally.¹⁴ In China, the epidemic of bla_KPC-2-carrying bacteria strains is of current concern.¹⁵ Besides bla_KPC, the rapid global spread of bla_NDM since its discovery in 2008 has posed significant public health challenges as well.

In our study, we identified an extensively drug-resistant clinical isolate of C. portucalensis featuring a rare coexistence of bla_SFO-1, bla_KPC-2, and bla_NDM-1 genes. This report makes the first documented instance of bla_SFO-1 in C. portucalensis. Additionally, we investigated the plasmid characteristics and transfer mechanisms associated with drug-resistant genes in this particular strain.

Materials and Methods

Strain Isolation and Identification

In September 2020, we obtained a clinical isolate (L2724hy) from the faeces of a 56-year-old outpatient male patient with acute diarrhea at the First Affiliated Hospital of Zhejiang University. Initially, this isolate was mistakenly identified as C. freundii using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS).¹⁶ To confirm its accurate species, we uploaded the 16s rRNA sequence of this strain to BioEZCloud (https://www.ezbiocloud.net/) and analyzed ANI, and the result indicated it to be C. portucalensis.^17,18

Antimicrobial Susceptibility Testing

The minimum inhibitory concentration (MIC) of L2724hy, transconjugant L2724hy-KPC-EC600, transconjugant L2724hy-NDM-EC600 and E. coli EC600 were determined by agar dilution method and micro-broth dilution method.¹⁹ AST results were interpreted according to the Clinical and Laboratory Standards Institute (CLSI) 2023 (https://clsi.org), while the breakpoints for tigecycline and colistin were interpreted based on EUCAST (https://www.eucast.org/).

S1-PFGE-Southern Hybridization and Conjugation Assays

S1-PFGE was used to identify the size and number of plasmids. The location of the bla_SFO-1, bla_NDM-1, and bla_KPC-2 were determined by S1-PFGE and hybridization experiments. Conjugation assays were conducted using E. coli EC600 as recipient and L2724hy as donor.²⁰ Subsequently, individual colonies of donor and recipient bacteria were carefully selected and suspended LB liquid culture medium at 37 °C for 6 hours. Afterwards, the two suspensions were mixed in a 1:1 ratio (donor: recipient), introduced into 3 mL LB liquid medium, and incubated at 37 °C for 20 hours. An appropriate amount of bacterial solution was spread onto Mueller–Hinton agar (MHA) plates containing rifampicin (200 ug/mL) and meropenem (2 ug/mL). After the incubation at 37°C overnight, a single colony was selected for further cultivation. Moreover, the strain was identified through MALDI-TOF/MS and the presence of drug-resistant genes was confirmed through PCR.

Whole-Genome Sequencing and Data Analysis

We utilized a Qiagen DNA purification kit in the extraction of the whole genomic DNA of strain L2724hy, which was then sequenced via Oxford Nanopore technology to generate raw data SRA. Then, we used Unicyler 0.4.9 to assemble the SRA, resulting in the acquisition of the whole-genome sequence.²¹ The prokka v1.14.6 annotation tool was used to annotate the whole-genome sequence.²² To predict drug-resistant genes, the data were submitted to ResFinder (https://cge.food.dtu.dk/services/ResFinder/), and the identification of insertion sequence and transposons was performed using ISFinder (https://www-is.biotoul.fr/). Plasmid types were confirmed through PlasmidFinder (https://cge.food.dtu.dk/services/PlasmidFinder/), and the MLST of the strains was determined via Pubmlst (https://pubmlst.org/). The discovery of transfer-related components was conducted using OriTfinder (https://bioinfo-mml.sjtu.edu.cn/oriTfinder/). The genetic environment surrounding bla_SFO-1, bla_NDM-1, and bla_KPC-2 on the plasmid was presented using Easyfig 2.2.3.²³ Additionally, the comparison of multiple plasmids was carried out using the BLAST Ring Image Generator (BRIG) software.²⁴

Results

Strain Isolation and Identification

L2724hy was isolated from the fecal sample of a patient with acute diarrhea and initially identified as C. freundii using MALDI-TOF/MS. Subsequent sequencing of the 16S rRNA lead to the reclassification of L2724hy as C. portucalensis (99.93% similarity for C. portucalensis and 99.86% similarity for C. freundii). Further ANI analysis confirmed L2724hy as C. portucalensis, showing a 98.73% ANI to C. portucalensis ATCC (CP044098) and a 94.52% ANI to C. freundii ATCC (CP033744).

Antimicrobial Susceptibility Testing

As presented in Table 1, the results of antimicrobial susceptibility testing indicated that C. portucalensis exhibited resistance to almost all antibiotics, with the exception of tigecycline and polymyxin B. For both transconjugant L2724hy-KPC-EC600 and L2724hy-NDM-EC600, we observed a decrease in the MIC values of carbapenems (imipenem, meropenem), levofloxacin, ciprofloxacin, amikacin, gentamicin, fosfomycin, chloramphenicol, and trimethoprim/sulfamethoxazole (Table 1). Notably, the transconjugant L2724hy-NDM-EC600 was highly resistant to ceftazidime-avibactam (MIC=64 mg/l), as was L2724hy, emphasizing the hydrolysis of ceftazidime-avibactam by NDM-1.

Table 1 The MIC Values of L2724hy, L2724hy-KPC-EC600, L2724hy-NDM-EC600 and EC600

Antimicrobials	MIC Values (mg/L)			EC600
	L2724hy	L2724hy-KPC-EC600	L2724hy-NDM-EC600
Aztreonam	>128 (R)	>128 (R)	>128 (R)	0.125 (S)
Imipenem	16 (R)	4 (R)	4 (R)	0.5 (S)
Meropenem	>32 (R)	4 (R)	4 (R)	0.06 (S)
Ceftriaxone	>128 (R)	64 (R)	128 (R)	0.06 (S)
Cefotaxime	>128 (R)	16 (R)	128 (R)	0.03 (S)
Ceftazidime	>128 (R)	64 (R)	>128 (R)	0.5 (S)
Levofloxacin	>64 (R)	0.5 (S)	0.25 (S)	1 (I)
Ciprofloxacin	>64 (R)	0.25 (S)	0.25 (S)	0.5 (S)
Amikacin	>128 (R)	1 (S)	2 (S)	2 (S)
Gentamicin	>128 (R)	1 (S)	1 (S)	2 (S)
Piperacillin	>128 (R)	>128 (R)	128 (R)	1 (S)
Fosfomycin	>512 (R)	4 (S)	2 (S)	8 (S)
Chloramphenicol	>128 (R)	8 (S)	8 (S)	8 (S)
Trimethoprim/Sulfamethoxazole	>8 (R)	≤0.125 (S)	≤0.125 (S)	≤2.375 (S)
Amoxicillin/Clavulanic acid	>128 (R)	128 (R)	128 (R)	4 (S)
Cefepime	128 (R)	16 (R)	16 (R)	<0.008 (S)
Ceftazidime-avibactam	64 (R)	1 (S)	64 (R)	0.5 (S)
Tigecycline	0.06 (S)	0.06 (S)	≤0.03 (S)	<0.05 (S)
Polymyxin B	2 (I)	2 (I)	2 (I)	0.5 (I)

Note: The quality control strains were E. coli ATCC 25922 and K. pneumoniae ATCC 700603.

Abbreviations: S, susceptible; R, resistant; I, intermediate.

S1-PFGE-Southern Hybridization and Conjugation Experiment

S1-PFGE profiles revealed that L2724hy harbored one chromosome and four plasmids (Figure 1). However, due to overlap or small size, not all plasmids were discernible on the electrophoresis profiles. The presence of bla_SFO-1, bla_NDM-1, and bla_KPC-2 genes on plasmids of different sizes was verified through Southern blotting hybridization. Specifically, the bla_SFO-1 gene was identified on a ~130 kb plasmid, the bla_NDM-1 gene was found on a ~54 kb plasmid, and the bla_KPC-2 gene was located on a ~41 kb plasmid (Figure 1). These results are consistent with the data obtained from whole-genome sequencing.

Figure 1 S1-PFGE profiles and southern blotting hybridization for L2724hy. S1-PFGE determines the number and size of plasmids in the strain. Southern blotting hybridization indicates the location of resistance genes bla_SFO-1, bla_NDM-1, and bla_KPC-2.

The transconjugants were identified as E. coli by MALDI-TOF mass spectrometry. PCR confirmed that the transconjugants, namely L2724hy-KPC-EC600 and L2724hy-NDM-EC600, carried the bla_KPC-2 and bla_NDM-1 genes, respectively. This indicates the transferability of bla_NDM-1 and bla_KPC-2 to E. coli EC600.

Genomic Characteristics

The MLST analysis showed that strain L2724hy belongs to ST85. Strain L2724hy has a circular chromosome (5,031,620 bp) and eleven plasmids of various sizes (Table 2). The chromosome contains 5316 coding sequences and 121 RNAs, including 86 tRNAs, 25 rRNAs, and 10 ncRNAs. Its average G+C content is 51.9%. ResFinder identified ten ARGs in strain L2724hy, with bla_CMY-46 and qnrB18 located on the chromosome, while the remaining eight were situated on plasmids. The plasmid type of p2-L2724hy-SFO-1 is IncFII, carrying the bla_SFO-1 and fosA3 genes. Meanwhile, p3-L2724hy-NDM-1 carries two ARGs, bla_NDM-1 and bla_SHV-12, and is classified as IncX3. p4-L2724hy is an IncR plasmid with mph (A), whereas bla_KPC-2 is on an unclassified p5-L2724hy-KPC-2. Another unclassified plasmid, p6-L2724hy, contains two resistance genes, including aac(6’)-Ib-cr and aac(6’)-Ib-Hangzhou.

Table 2 The Genomic Characteristics of L2724hy

L2724hy	Sizes	Type	G+C%	ARGs
Chromosome	5,031,620 bp	ST85	51.9	blaCMY-46, qnrB18,
p1-L2724hy	365,162 bp	IncH12/lncH12A	46.5
p2-L2724hy-SFO-1	131,226 bp	IncFII	53.7	blaSFO-1, fosA3
p3-L2724hy-NDM-1	54,000 bp	IncX3	49.2	blaSHV-12, blaNDM-1
p4-L2724hy	45,944 bp	IncR	51.9	mph(A)
p5-L2724hy-KPC-2	41,244 bp	Undefined	49.1	blaKPC-2
p6-L2724hy	5,933 bp	Undefined	51.1	aac(6’)-Ib-cr, aac(6’)-Ib-Hangzhou
p7-L2724hy	5,234 bp	Undefined	50.7
p8-L2724hy	5,040 bp	Undefined	50.0
p9-L2724hy	2,182 bp	Undefined	50.1
p10-L2724hy	2,152 bp	Undefined	49.9
p11-L2724hy	2,013 bp	Undefined	49.3

Our analysis revealed that these distinct drug-resistance genes are consistent with antibiotic phenotypes in L2724hy, which included resistance to carbapenems (bla_NDM-1, bla_KPC-2), cephalosporins (bla_CMY-46, bla_SFO-1, bla_SHV-12, bla_NDM-1, bla_KPC-2), 4-quinolones (qnrB18), fosfomycin (fosA3), and aminoglycosides (aac(6’)-Ib-cr, aac(6’)-Ib-Hangzhou).

Plasmid Characteristics of bla_KPC-2, bla_NDM-1, and bla_SFO-1

L2724hy carries plasmids of various types, including IncHI2/IncHI2A, IncFII, IncX3, IncR, and unclassified plasmids, as identified by PlasmidFinder. Among these, the IncFII plasmid, designated as p2-L2724hy-SFO-1, has a length of 131,226 bp and carries the resistance genes bla_SFO-1 and fosA3. Comparative genomics with three closely related plasmid genomes (CP044029 with 95% coverage and 100% identity, CP042483 with 93% coverage and 100% identity, and CP042519 with 93% coverage and 100% identity) revealed primary variations, most notably the absence of the resistance gene bla_SFO-1 and the transcriptional activator ampR (Figure 2a). Additionally, we also identified two strains of K. pneumoniae (Genebank: CP114855, and JQ724541) that closely resembled the genetic environment (IS26-ampR-bla_SFO-1-IS26) surrounding bla_SFO-1 in L2724hy (Figure 3a). However, these two strains lack the fosA3 gene, which is upstream of the IS26-ampR-bla_SFO-1-IS26 sequence in L2724hy. In our study, IS26, which is a critical mobile element, is present on both sides of fosA3. Based on the OriTfinder results, p2-L2724hy-SFO-1 possesses mobile genetic elements associated with transfer, including an oriT region, type IV coupling protein (T4CP), and various type IV secretion system components (T4SS), which includes traI, traD, traQ, traN, traC, traV, traA, traJ, and traM (Figures 2a and 4).

Figure 2 Comparative analysis of plasmids p2-L2724hy-SFO-1, p4-L2724hy-NDM-1, and p5-L2724hy-KPC-2 in L2724hy. The BRIG circle maps show plasmid resistance genes and mobile genetic elements (MGEs). (a) plasmid p2-L2724hy-SFO-1 carrying bla_SFO-1 and fosA3. (b) plasmid p4-L2724hy-NDM-1 harboring bla_NDM-1 and bla_SHV-12. (c) plasmid p5-L2724hy-KPC-2 bearing bla_KPC-2.

Figure 3 Genetic environment of bla_SFO-1, bla_NDM-1, and bla_KPC-2 in L2724hy. (a) bla_SFO-1 (b) bla_NDM-1 (c) bla_KPC-2. Red arrows denote drug resistance genes, green arrows denote transposons, pink arrows denote other genes, and yellow arrows represent hypothetical proteins.

Figure 4 A conjugative plasmid p2-L2724hy-SFO-1. ORF1-57,fosA3; ORF1-60, bla_SFO-1.

Abbreviation: AR (ARGs), acquired antibiotic resistance determinant genes ;ORF, Open Reading Frame

p4-L2724hy-NDM-1 was classified as an IncX3 plasmid, with a length of 54,000 bp and carried two resistance genes, bla_NDM-1 and bla_SHV-12 (Figure 2b). The NCBI BLAST search revealed 29 highly similar plasmids to p4-L2724hy-NDM-1, all exhibiting 100% coverage and 100% identity. Specifically, plasmids KU314941, MH234941, and KP987216, originating from distinct strains (K. pneumoniae, E. coli, and C. freundii) were selected for a comparative analysis of plasmid characteristics. The analysis revealed that these plasmids share similar backbones (Figure 2b), underscoring the widespread of bla_NDM-1 across different species. Upon analyzing the genetic environment of bla_NDM-1 in L2724hy (Figure 3b), a conserved sequence (ble-trpF-dsbD-cutA-gros-groL) downstream of bla_NDM-1 on the plasmid was observed. The upstream of bla_NDM-1 comprises an IS5-IS3000-IS3000-Tn2 sequence, with the notable deletion of ISAa125 deviating from the typical bla_NDM-1 structure. Additionally, an IS26 insertion element surrounding bla_SHV-12 was noted suggesting a potential role in facilitating the dissemination of bla_SHV-12.

The plasmid p5-L2724hy-KPC-2, carrying bla_KPC-2 gene, is currently unclassified and 41,244 bp in length. As shown in Figure 2c, the plasmid contains multiple-insertion elements (ISKpn27, ISKpn6), transposons (Tn2, tnpR, TnAs1), and transfer-related type IV secretion systems (virB2, virB4, virB9, virB10, and virB11). The plasmids CP110915, CP126883, and KP726894, are the most similar to p5-L2724hy-KPC-2, all displaying 100% coverage and 100% identity. Upon examination of these highly similar plasmids, it was observed that CP110915 and CP126883 originated from C. freundii. As illustrated in Figure 3c, a comparison of the genetic context of the two most similar C. freundii plasmids (CP110915, CP126883) with p5-L2724hy-KPC-2 revealed a similar genetic environment for bla_KPC-2 (Tn2-tnpR-ISKpn27-bla_KPC-2-ISKpn6-KorC) which is entirely consistent with CP110915 from the same region but exhibits a small gap with CP126883 from a different region.

Discussion

In recent years, the coexistence of ESBLs and carbapenemase genes in Enterobacteriaceae, posing challenges to clinical antibiotic treatment.²⁵ To the best of our knowledge, our study is the first to report the coexistence of bla_SFO-1 with bla_NDM-1 and bla_KPC-2. Furthermore, there are no reports of the ESBL gene bla_SFO-1 in C. portucalensis.

Since its discovery in 2017, C. portucalensis has been found in humans, animals, and environment. However, the misidentification of C. portucalensis as C. freundii has led to an underestimation of its prevalence in clinical settings.^7,26 In this study, L2724hy C. portucalensis was initially misidentified as C. freundii using MALDI-TOF/MS, and was later identified as C. portucalensis by a combination of 16s rRNA sequencing and ANI analysis. Thus, it is essential to enhance the whole-genome sequencing of less common strains.

Data analysis reveals that the plasmids carrying bla_SFO-1 include IncA/C, IncHI2, unclassified types.^12,13,27,28 In our study, plasmidFinder revealed that stain L2724hy carries the bla_SFO-1 gene on an IncFII plasmid that has not been reported before. According to the genetic environment of bla_SFO-1 (IS26-ampR-bla_SFO-1-IS26), the presence of IS26 on both sides of bla_SFO-1 suggests a potential correlation with plasmid-mediated horizontal transmission of bla_SFO-1, which is consistent with prior research findings.²⁹ Notably, we also observed that the fosA3, is located upstream of IS26-ampR-bla_SFO-1-IS26 and shares the same IS26 with ampR-bla_SFO-1. The fosA3 has been reported to be closely associated with the transmission of ESBL gene bla_CTX-M by the insertion element IS26.³⁰ Therefore, in our study, we believe that IS26 is a vital insertion element that gradually integrates with fosA3 and bla_SFO-1 on the IncFII plasmid, forming a multidrug resistance region, which plays a crucial role in mediating the spread of drug resistance genes. Importantly, this study marks the first instance of bla_SFO-1 and fosA3 coexisting on a plasmid.

NDM-1, a carbapenemase, can hydrolyze nearly all beta-lactam antibiotics, including carbapenemase.³¹ The emergence of carbapenemase-producing strains of NDM-1 represents a significant risk to global public health, necessitating a high level of vigilance. In our study, we observed an absence of ISAba125 from IS5 to IS3000 when comparing it to classical bacteria. According to previous studies, ISAba125 is typically involved in the formation of Tn125, a complex transposon associated with the plasmid-mediated spread of bla_NDM-1.^32,33 Our findings suggest that the lack of ISAba125 does not impede the horizontal transfer of bla_NDM-1. In the conjugation experiment, we successfully transferred bla_NDM-1 and bla_KPC-2 into EC600 for expression, affirming the transferability of the plasmids in our study. As mentioned above, the genetic environment of bla_KPC-2 in this study aligns entirely that of C. freundii (Genebank: CP110915) from the same region, strongly suggesting that bla_KPC-2 was transmitted from CP110915.

Conclusion

In conclusion, our study reported for the first time the coexistence of bla_SFO-1, bla_NDM-1, and bla_KPC-2 in a clinical isolated C. portucalensis strain, presenting challenges in multidrug resistance. IS26 is an essential insertion element that facilitates the propagation of drug-resistant genes on IncFII plasmids. These findings emphasize the urgency of continued monitoring and understanding evolving drug resistance dynamics in uncommon strains.

Nucleotide Sequence Accession Numbers

The nucleotide sequences in L2724hy containing a circular chromosome and eleven plasmids have been submitted to GenBank and assigned accession numbers CP136601-136612, respectively.

Ethical Approval

This study was approved by the clinical research ethics committee of the First Affiliated Hospital, Zhejiang University School of Medicine [number 2020-IIT-591], and was conducted according to the ethical principles of the Declaration of Helsinki. Parental written consent was obtained.

Disclosure

The authors report no conflicts of interest in this work.

Additional information

Funding

This work was supported by the National Key R&D Program of China (2020YFE0204300), National Natural Science Foundation of China (grant no. 82072314), and Natural Science Foundation of Zhejiang Province (grant no. LY23H270006).