Shared Plasmids Drive Spread of Resistant CTX-M-65 Bacteria in Hospitals

Antimicrobial resistance remains one of the most consequential threats in modern medicine, and the extended-spectrum beta-lactamase enzymes known as CTX-M proteins sit at its center. These enzymes hydrolyze the third-generation cephalosporins that anchor empirical therapy for countless infections, and among the more than two hundred CTX-M variants described to date, CTX-M-65 occupies a particularly interesting position. Unlike the globally dominant CTX-M-15 or the epidemic CTX-M-27, the bla_CTX-M-65 gene has a more patchy and less well-mapped distribution, appearing across multiple bacterial species and plasmid types in ways that have resisted simple categorization. A new genomic epidemiology study published in BMC Genomics by Xiankai Chen, Xiwei Zhang, Jingjie Song, Shihan Zeng, Yulan Huang and Xiaoyan Li now provides a detailed look at how this gene circulates in clinical settings, combining whole-genome sequencing of resistant isolates with a large-scale comparison of plasmids drawn from public databases around the world.

The research team began with a large and clinically grounded collection: 373 Enterobacteriaceae isolates that had been recovered from routine diagnostic specimens and found to be resistant to third-generation cephalosporins. Because these isolates came from the clinical microbiology laboratory rather than from a targeted screening program, they represent the resistant organisms that clinicians actually encountered in practice. Every isolate was subjected to antimicrobial susceptibility testing and to polymerase chain reaction confirmation of bla_CTX-M-65, a deliberate step that guards against the false positives that can arise when sequence reads are assigned to closely related CTX-M alleles. Whole-genome sequencing followed, allowing the investigators to place each confirmed isolate within its species phylogeny, to characterize its full resistome and to extract and compare the plasmids carrying the resistance gene.

The headline finding from the local collection was one of relative rarity paired with striking breadth. Only 20 of the 373 isolates, or 5.4 percent, carried bla_CTX-M-65, confirming that this allele is a minority player among cephalosporin-resistant Enterobacteriaceae. Yet those 20 isolates were distributed across three distinct species: 14 Escherichia coli, 5 Salmonella enterica serovar Typhimurium and a single Klebsiella pneumoniae. That spread across genera is itself informative, because it suggests that bla_CTX-M-65 is not locked into a single bacterial lineage. Instead, the gene appears able to establish itself in genetically very different hosts, consistent with the idea that horizontal gene transfer, rather than clonal expansion alone, drives its dissemination.

The resistance phenotypes of these isolates added a sobering clinical dimension. All 20 were resistant to cefuroxime, an expected consequence of possessing an extended-spectrum beta-lactamase, but the co-resistance extended well beyond the beta-lactam class. Seventy percent were resistant to levofloxacin, a fluoroquinolone that is frequently used when cephalosporin therapy fails or when oral treatment is preferred. Multidrug resistance of this kind narrows therapeutic options significantly, particularly for pathogens such as Salmonella Typhimurium, where fluoroquinolones are a mainstay for invasive disease. The combination of a cephalosporin-hydrolyzing enzyme and fluoroquinolone resistance on the same bacterial background is a recurring pattern in Enterobacteriaceae, and this study confirms that bla_CTX-M-65-positive isolates fit squarely within it.

Phylogenetic analysis of the sequenced genomes revealed two episodes of likely within-hospital transmission hiding inside the otherwise diverse collection. Among the E. coli isolates, a pair belonging to sequence type ST155 differed by only 9 single nucleotide polymorphisms across their core genomes, despite having been recovered from different hospital departments. In genomic epidemiology, core-genome differences of that magnitude are generally interpreted as evidence of a very recent common ancestor, pointing to direct or indirect transmission of a single clone between wards. Similarly, the five Salmonella Typhimurium isolates formed a tight cluster belonging to ST34, with fewer than 20 core-genome SNPs separating members of the group, and the affected patients included children treated in pediatrics. Together these findings illustrate how genomic resolution can uncover transmission chains that routine susceptibility testing would never flag.

Perhaps the most clinically alarming single isolate was the K. pneumoniae strain, which belonged to ST11 and carried both bla_CTX-M-65 and bla_KPC-2. The ST11 lineage is already notorious as one of the principal vehicles for carbapenem resistance in K. pneumoniae worldwide, and bla_KPC-2 encodes a carbapenemase that inactivates the last-line beta-lactam antibiotics used against multidrug-resistant Gram-negative infections. The co-occurrence of a carbapenemase and an extended-spectrum cephalosporinase in the same genome means the organism carries enzymatic firepower against nearly the entire beta-lactam arsenal. Whether the two genes reside on the same plasmid or on separate elements was not the central question of the study, but their coexistence in a single ST11 background underscores how resistance determinants accumulate on successful lineages, creating organisms that are progressively harder to treat.

To place these local findings in a global context, the investigators compiled and analyzed 1,421 bla_CTX-M-65-positive plasmids from public sequence databases. This comparative step is where the study’s architecture becomes most distinctive. Rather than treating the plasmid as an accessory detail, the team asked which replicon types dominate the plasmids that carry this particular gene and how conserved their backbones are across geography and time. The answer was a small set of recurrent architectures. IncHI2 plasmids, large low-copy-number elements with a well-documented role in carrying multiple resistance genes, were prevalent, as were members of the IncF family, specifically IncFIA/IB combinations and IncFII/R plasmids. The prominence of IncF-type plasmids aligns with their established reputation as the workhorses of CTX-M dissemination in E. coli, while the frequent appearance of IncHI2 elements highlights that versatile, broad-host-range plasmids also serve as major carriers of this allele.

Backbone comparison delivered the study’s most geographically expansive insight. Representative plasmids sequenced in this study showed high backbone similarity to bla_CTX-M-65-carrying plasmids previously reported from China and South Korea, indicating that the same underlying plasmid frameworks, rather than merely the same gene, are circulating across the region. In other words, the dissemination of CTX-M-65 appears to follow a two-track model: diverse bacterial clones pick up the gene, but they pick it up on a limited set of shared plasmid scaffolds. This pattern matters for surveillance, because tracking the plasmid backbones, and not just the bacterial lineages, can reveal importation events and cross-border spread that host-based analysis alone would miss. It also matters mechanistically, since a conserved backbone implies conserved fitness properties, copy number and transfer machinery that may explain why these particular plasmids succeed.

The analysis of the DNA immediately surrounding bla_CTX-M-65 added a third layer to the picture. Insertion sequences are short mobile DNA elements that can shuttle genes between chromosomes and plasmids, and certain ones are known accomplices of CTX-M genes. ISEcp1 in particular is famous for its role in mobilizing bla_CTX-M alleles by providing promoter sequences and acting as a transposition vehicle, and IS26 has become synonymous with the assembly of multidrug-resistance regions through a replicative transposition mechanism. In this study, ISEcp1, IS26 and IS903 were the mobile elements most commonly identified within the bla_CTX-M-65-associated genetic contexts, painting a consistent picture of a gene that has been repeatedly captured, re-packaged and redeployed by the standard genetic toolkit of the Enterobacteriaceae resistome.

Taken together, the study delivers a message that is more nuanced than a simple alarm. On one hand, bla_CTX-M-65 is uncommon, appearing in only 5.4 percent of cephalosporin-resistant isolates in this collection, and its detection should not overshadow the dominant CTX-M lineages. On the other hand, its behavior is instructive: it colonizes multiple species, rides on a small number of successful plasmid backbones that span international boundaries, hitches its mobility to well-known insertion sequences and occasionally lands in the most dangerous possible hosts, such as carbapenemase-producing ST11 K. pneumoniae. The identification of a clonally related E. coli pair spanning hospital departments and an ST34 Salmonella cluster within pediatrics demonstrates that genomic surveillance can catch transmission events that conventional methods overlook. The authors’ conclusion, that the diversity of bacterial hosts combined with related plasmid backbones demands continued genomic monitoring, is a fitting one. As sequencing becomes cheaper and databases grow, the ability to distinguish a gene hitchhiking on a shared plasmid from an independently acquired allele will increasingly determine how effectively hospitals and public health agencies can interrupt the silent traffic in resistance genes that moves between wards, species and countries.

Subject of Research: Genomic epidemiology of CTX-M-65-producing multidrug-resistant Enterobacteriaceae and the plasmid backbones carrying the resistance gene

Article Title: Genomic epidemiology of blaCTX−M−65-positive clinical Enterobacteriaceae reveals clonal diversity and shared plasmid backbones

Article References: Genomic epidemiology of blaCTX−M−65-positive clinical Enterobacteriaceae reveals clonal diversity and shared plasmid backbones. (n.d.). https://doi.org/10.1186/s12864-026-13446-8

Image Credits: AI Generated

DOI: 10.1186/s12864-026-13446-8

Keywords: CTX-M-65, Enterobacteriaceae, antimicrobial resistance, extended-spectrum beta-lactamases, plasmids, whole-genome sequencing, Escherichia coli, Salmonella, Klebsiella pneumoniae, genomic epidemiology, mobile genetic elements, hospital transmission