Horizontal transmission of a multidrug-resistant IncM1 plasmid harbouring bla_OXA-48 and bla_CTX-M-14b among patient microbiotas

The growing crisis of antimicrobial resistance in Gram-negative bacteria is exacerbated by the spread of resistance genes through plasmids. Carbapenemase genes, notably bla_OXA-48-like genes, can spread both vertically and horizontally among bacteria. The bla_OXA-48 gene was first identified in a Klebsiella pneumoniae isolate in 2001, it probably originated from Shewanella sp. and transferred to Enterobacterales via IncL plasmids.¹ OXA-48 is now endemic in regions such as the South Asian subcontinent, the Middle East² and is frequently isolated in Europe, including Switzerland. The gut microbiome, rich in bacterial diversity and mobile genetic elements, plays a key role in horizontal gene transfer, driving the emergence of antibiotic-resistant pathogens. This study examines two cases of bla_OXA-48 plasmid dissemination across Enterobacterales species within the gut microbiome.

In Case 1 (2020), a 71-year-old male presented an urosepsis and secondary bacteraemia caused by OXA-48-producer Citrobacter freundii (urine and blood positive samples) and was found to be colonized by C. freundii, Escherichia coli and K. pneumoniae isolates from ano-rectal swabs, all harbouring bla_OXA-48 (easyplex SuperBug CRE, Amplex Biosystems GmbH, Giessen, Germany). These isolates were resistant to carbapenems and third-generation cephalosporins and cefepime, suggesting co-expression of ESBL (Table 1). Whole genome sequencing using long-read technology (Oxford Nanopore Technologies, Oxford, UK) revealed that all five isolates (three C. freundii, one E. coli and one K. pneumoniae) carried the bla_OXA-48 gene on a 74.7 kb IncM1 plasmid (Figure 1a), sharing 99.95% similarity, within a Tn1999.2 composite transposon (Figure 1b and Figure S1 available as Supplementary data at JAC Online). An inverted Tn1999.2 was identified in the K. pneumoniae plasmid (Figure 1b). While bla_OXA-48-harbouring IncL plasmids rarely carry other resistance genes, IncM plasmids often do.⁴ These IncM1 plasmids also carried additional resistance genes, including bla_CTX-M-14b, aminoglycoside-modifying enzymes and plasmid-mediated quinolone resistance gene qnrS1 (Figure 1c). A list of all acquired antibiotic resistance genes identified in the clinical strains in provided in Table S2.

Table 1.

Summary of resulting antibiotic susceptibility testing and interpretation according to EUCAST version 14.0 guidelines of clinical isolates

	Case 1						Case 2
Species	C. freundii		E. coli		K. pneumoniae		M. morganii		S. marcescens		E. coli		C. farmeri		K. grimontii
Isolate	RS1255,RS1254,RS1252a		RS1251		RS1253		RS1614		RS1615		RS1616		RS1617		RS1618
ST type	396		3014		1454		n.a.		n.a.		69		n.a.		n.a.
Specimen	blood/urine/rectal swab		rectal swab		rectal swab		rectal swab		rectal swab		rectal swab		rectal swab		rectal swab
Antibiotic (AST)	MICb	Int.c	MICb	Int.c	MICb	Int.c	MICb	Int.c	MICb	Int.c	MICb	Int.c	MICb	Int.c	MICb	Int.c
Amoxicillin	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Amoxicillin-clavulanic acid	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Piperacillin-tazobactam	>32	R	>32	R	32	R	>32	R	>32	R	>32	R	>32	R	>32	R
Cefuroxime	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Ceftriaxone	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Ceftazidime	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Cefepime	d	R	d	R	d	R	d	R	d	R	d	R	d	R	d	R
Ceftazidime-avibactam	≤1	S	≤1	S	≤1	S	≤1	S	≤1	S	≤1	S	4	S	≤1	S
Ceftolozane-tazobactam	16	R	4	R	4	R	16	R	>16	R	4	R	>16	R	>16	R
Cefiderocol	0.5	S	0.06	S	0.12	S	0.25	S	4	R	0.25	S	0.12	S	0.5	S
Imipenem	2	S	1	S	2	S	8	R	>8	R	2	S	>8	R	4	I
Meropenem	1	S	0.5	S	1	S	1	S	8	I	1	S	>16	R	4	I
Ertapenem	2	R	1	R	2	R	0.5	S	>2	R	>2	R	>2	R	>2	R
Imipenem-relebactam	2	S	0.5	S	1	S	8	R	>8	R	2	S	>8	R	4	R
Meropenem-vaborbactam	1	S	≤0.25	S	0.5	S	1	S	8	S	0.5	S	>16	R	4	S
Aztreonam	16	R	8	R	8	R	>32	R	>32	R	8	R	>32	R	32	R
Amikacin	>32	I	>32	I	>32	I	>32	I	>32	I	>32	I	>32	I	>32	I
Gentamicin	d	S	d	R	d	S	d	S	d	S	d	R	d	S	d	S
Ciprofloxacin	>1	R	>1	R	>1	R	≤0.25	S	1	R	≤0.25	S	>1	R	1	R
Levofloxacin	>2	R	2	R	2	R	≤0.25	S	1	I	0.5	S	>2	R	1	I
Furane	d	S	d	S	d	S	d	R	d	R	d	S	d	S	d	S
Colistin	1	S	0.5	S	1	S	>8	R	>8	R	0.5	S	0.5	S	0.5	S
Fosfomycin + G-6-P	≤8	S	≤8	S	16	S	>64	R	64	R	64	R	16	S	64	R
Tigecyclin	2	R	0.5	S	1	R	2	R	>2	R	0.5	S	1	R	0.5	S
Trimethroprim-sulfamethoxazole	>8	R	>8	R	≤1	S	≤1	S	≤1	S	≤1	S	≤1	S	≤1	S

Transconjugant ASTs are shown in Table S1. n.a., not applicable.

^aThree C. freundii isolates presenting the same antibiogram results were merged into a single column.

^bMinimum inhibitory concentration (mg/L) by broth microdilution.

^cInterpretation. S, susceptible; R, resistant; I, Increased dosage susceptible.

^dFor these antimicrobials, AST was obtained by the disc diffusion method, MIC value was not determined.

Figure 1.

Analysis of bla_OXA-48-haboring IncM1 plasmids. (a) Circular representation of a 74.7 kb IncM1 plasmid carrying bla_OXA-48 and bla_CTX-M-14b isolated from C. freundii in a bloodstream infection; figure created with Proksee.ca. (b) Detailed annotation of the Tn1999 composite transposons disseminating bla_OXA-48 and comparison of the characterized Tn1999.2 (presence of an IS1R transposase), the Case 1 K. pneumoniae carrying a Tn1999.2 inversion (invTn1999.2) and Case 2 plasmid harbouring the novel transposon type Tn1999.8; figure created with BioRender.com. (c) Whole plasmid alignment of the pOXA-48 IncL plasmid (JN626286)³ bearing bla_OXA-48 with an IncM1 plasmid bearing bla_OXA-48 from the Netherlands (CP068320) and two IncM1 plasmids from this study; antibiotic resistance genes are shown in red, lysR within Tn1999 in green, IS1999 in dark grey, insertion sequences in blue and conjugation machinery in orange.

In Case 2 (2022), a 58-year-old female was colonized by five Enterobacterales species (E. coli, Morganella morganii, K. grimontii, Serratia marcescens and C. farmeri) isolated from ano-rectal swabs, all isolates harbouring bla_OXA-48. Long-read whole genome sequencing identified high similarity among the bla_OXA-48-containing IncM1 plasmids, also 74.7 kb in size. The same resistance genes identified in Case 1 plasmids were also found in Case 2 plasmids. Interestingly, an IS26 element was identified within Tn1999, resulting in a novel Tn1999.8 transposon structure in Case 2 plasmids, as shown for M. morganii plasmid (Figures 1b and 1c). These plasmids also share 99.95% similarity (Figure S1). This insertion is located within the bla_OXA-48 promoter region, potentially modifying carbapenemases expression, as shown for some Tn1999 variants.⁵

This IncM1-OXA-48 plasmid contains the tra genes involved in the conjugation machinery and was confirmed to be transferable by liquid conjugation assays from K. pneumoniae and C. freundii donors into azide-resistant E. coli J53 at a frequency of 1.54 × 10⁻⁵ and 1.16 × 10⁻⁵, respectively. Long-read sequencing of the E. coli transconjugants confirmed the successful transfer of the intact 74.7 kb plasmid. This IncM1 plasmid showed conjugation frequency comparable to the original IncL-OXA-48 63 kb plasmid (8.0 × 10⁻⁵).³ Similar to the IncL plasmid containing bla_OXA-48, these IncM1 plasmids have disrupted tir genes, which enhance transmissibility.⁶ A core plasmid analysis revealed a close genetic relationship among the IncM1 plasmids from seven Enterobacterales species across both cases presented here (Figure S2), suggesting that they could serve as highly efficient vectors for spreading resistance within the microbiota. The IncM1 plasmid’s ability to transfer between multiple Enterobacterales species within a single host, as observed in the gut microbiome, suggests efficient within-patient plasmid dissemination.

IncL and IncM plasmids, previously classified under the IncL/M group, have closely related backbones (Figure 1c and Figure S2) but are now recognized as separate branches. While bla_OXA-48 has been predominantly associated with the IncL branch,⁴ here we identified an emerging IncM1 multidrug-resistance plasmid harbouring bla_OXA-48, bla_CTX-M-14b, aminoglycoside and quinolone resistance genes (Figure 1a). This contrasts with the original IncL plasmid (pOXA-48a) description,³ which only carries bla_OXA-48. Homology searches revealed similarities between the IncM1 plasmids described here and 10 IncM1 plasmids reported by the Dutch national surveillance programme,⁷ which ranged from 30 to 89 kb in size and carried bla_OXA-48 and bla_CTX-M-14b. A whole plasmid alignment, including eight plasmids from 65 to 68 kb in K. pneumoniae analysed by Hendrickx et al.,⁷ showed these plasmids lacked the trbABC and trbN genes, as well as IS1999 upstream of the bla_OXA-48 gene. They had also acquired different aminoglycoside resistance genes. These findings suggest that parallel evolutionary events gave rise to these IncM1-OXA-48 plasmids (Figure 1c and Figure S1).

The co-expression of bla_OXA-48 and bla_CTX-M-14b on this plasmid broadens the β-lactam resistance spectrum, providing bacteria with a significant survival advantage under antibiotic pressure. This study alongside reports from Switzerland,⁸ the Netherlands,⁷ UK⁹ and Spain¹⁰ highlights the increasing prevalence of IncM1 plasmids carrying bla_OXA-48. The extensive within-patient dissemination of bla_OXA-48-harbouring IncM1 plasmids may facilitate the establishment of long-term bla_OXA-48 carriers, as shown for IncL plasmids.¹¹ Given its broad resistance profile and its high conjugation efficiency among Enterobacterales within patient microbiotas, this emergent plasmid poses a significant risk for the rapid dissemination of carbapenemase-producing Enterobacterales. Therefore, close monitoring is ongoing at our institution to determine the prevalence of this plasmid and identify potential healthcare-associated transmission events.

Funding

This study was funded by the Hôpitaux Universitaires de Genève (Research and Development Program scheme PRD 3-2022-II) and Fondation Ernst et Lucie Schmidheiny.

Transparency declarations

All authors declare no financial or non-financial competing interests.

Author contributions

R.S. was involved in the design and conceptualization of the study, performed experiments, analysis, visualization of results and writing of the original draft. M.R. performed experiments and formal analysis. J.P. did the computational analysis and data visualization. A.N. and S.H. worked on clinical profiling and manuscript editing. A.C. and G.R. carried out laboratory procedures. J.S. and N.V. edited the manuscript. D.O.A. was involved in the design and conceptualization, analysis and writing the original draft. All authors revised the manuscript.

Data availability

Data are available under BioProject No. PRJNA1095224.

Supplementary data

Figures S1 and S2 and Tables S1 and S2 are available as Supplementary data at JAC  Online.