Monophasic Salmonella Typhimurium, formally Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:-, is one of the leading causes of foodborne gastroenteritis and hospitalization worldwide. Unlike the classic biphasic Typhimurium serovar, the monophasic variant lacks the second-phase flagellar antigen, a difference generally attributed to deletion of the fljB gene or to sequence disruptions in its promoter region. The ST34 sequence type has emerged as the predominant pandemic lineage of this variant, spreading from its first identification in Spain in the 1990s across Europe, the Americas, Asia, and Africa. In March 2022, the World Health Organization reported a multinational outbreak of ST34 monophasic Salmonella Typhimurium linked to contaminated chocolate products, underscoring the capacity of this clone to spread through unusual food vehicles and its growing prevalence in China.

The clinical context of this isolation is significant. The patient presented with acute abdominal pain and severe diarrhea without obvious predisposing factors, and fecal samples were processed according to standard clinical microbiology protocols. Colonies with colorless or translucent edges, black centers, and smooth, moist surfaces were picked from Salmonella-Shigella agar and purified by repeated streaking. Identification was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with a confidence score exceeding 90 percent, followed by biochemical profiling on the VITEK 2 compact system and slide agglutination serotyping using the White-Kauffman-Le Minor antigenic scheme. A colloidal gold immunochromatographic assay screened for six carbapenemase types and returned a positive result for NDM-type carbapenemase, which whole-genome sequencing subsequently refined to the bla_NDM-5 variant.

Antimicrobial susceptibility testing against 25 agents spanning 11 classes revealed a stark picture. SYA001 was resistant to seven antibiotic classes, including aminopenicillins, beta-lactamase inhibitor combinations, cephalosporins, carbapenems, aminoglycosides, tetracyclines, and sulfonamides. Minimum inhibitory concentrations for imipenem, meropenem, and ertapenem all exceeded 16, 16, and 8 micrograms per milliliter respectively, confirming complete carbapenem resistance. The strain remained susceptible only to aztreonam, amikacin, tigecycline, and colistin, with intermediate resistance to levofloxacin and ciprofloxacin. This pattern fully satisfies the definition of multidrug resistance and leaves a narrow therapeutic window for clinicians managing infections caused by similar isolates.

Whole-genome sequencing was performed on the DNBSEQ-T7 platform, generating 150-base-pair paired-end reads with a Q30 ratio of 89.37 percent. Assembly produced 89 contigs totaling 5,223,782 base pairs with an N50 of 222,771 base pairs and a GC content of 51.86 percent. Annotation identified 4,923 protein-coding sequences, 76 transfer RNAs, and 3 ribosomal RNA operons. Ribosomal multilocus sequence typing assigned the genome to Salmonella enterica, and seven-gene MLST confirmed ST34. Comparative analysis against the reference strain Salmonella Typhimurium LT2 revealed 99.85 percent average nucleotide identity and a digital DNA-DNA hybridization value of 99.6 percent, both far exceeding species delineation thresholds. Only 1,275 single-nucleotide polymorphisms distinguished the two genomes, and the 654 gene clusters unique to SYA001 consisted primarily of acquired resistance genes, prophage regions, and plasmid-associated mobile elements.

The resistance determinants fell into three categories: 17 acquired genes directly mediating drug inactivation, target protection, target replacement, or efflux; 18 intrinsic efflux pump components encoded on the core chromosome; and 7 transcriptional regulators modulating resistance expression. Beyond the central bla_NDM-5 carbapenemase gene, the genome carried bla_OXA-10 for cephalosporin resistance, qnrS1 for quinolone protection, tet(A) for tetracycline efflux, sul2 and sul3 for sulfonamide resistance, and multiple aminoglycoside-inactivating enzymes including AAC(6′)-Iaa, APH(3′)-Ia, and AAC(3)-IVa. Mutations in the elongation factor EF-Tu and the UhpT hexose phosphate transporter were also detected, associated with resistance to elfamycins and fosfomycin respectively. The ST34 lineage typically carries a chromosomal ASSuT resistance island conferring ampicillin-streptomycin-sulfonamide-tetracycline resistance, and in SYA001 this background has been further augmented by plasmid-borne carbapenem resistance.

Four plasmid replicons were identified: IncHI2, IncHI2A, IncQ1, and p0111. The bla_NDM-5 gene was located on contig000040, while the IncHI2 replicon was predicted on contig000011, a physical separation likely attributable to the limitations of short-read assembly, which fragments large conjugative plasmids containing long repeats and insertion elements. Alignment against the reference plasmid CP162903.1, a 246,818-base-pair IncHI2 plasmid carrying bla_NDM-5 from a clinical ST34 strain in Zhuhai, China, showed backbone coverage of 96.1 percent, supporting the conclusion that bla_NDM-5 resides on an IncHI2-type plasmid. The gene sits within a Tn125-type composite transposon cassette flanked by IS3000, ISAba125, and IS5, immediately downstream of the conserved ble_MBL bleomycin resistance gene, a structure that facilitates stable inheritance and expression. The coexistence of multiple conjugative plasmid replicons suggests potential for horizontal transmission, though this requires confirmation by long-read sequencing and conjugation experiments.

Fifteen prophage regions were predicted across the genome, classified as eight intact, five incomplete, and two questionable, encoding 393 prophage-associated genes including capsid, tail, portal, integrase, and terminase proteins. Intact prophages possess lysogenic conversion and lytic capabilities and can mediate horizontal transfer of virulence and resistance genes into the bacterial chromosome. The abundance of prophage sequences suggests high genomic plasticity, enabling rapid acquisition of exogenous DNA under antibiotic pressure. In parallel, SPIFinder confirmed a complete set of Salmonella pathogenicity islands including SPI-1 through SPI-5, SPI-9, SPI-13, and SPI-14, along with the C63PI iron uptake island and the CS54 island. SPI-1 encodes a type III secretion system mediating invasion of intestinal epithelial cells through genes such as invA, prgH, prgI, prgJ, hilD, and sipD, while SPI-2 carries the ssrA/ssrB two-component regulatory system essential for intracellular survival within host macrophages.

Protein-protein interaction network analysis using the STRING database identified AcrA as the central hub of the efflux-mediated resistance regulatory network. High-confidence interactions linked AcrA to AcrB with a combined score of 0.999, supported by genomic co-localization, phylogenetic co-occurrence, experimental validation, and literature mining. Together with TolC, AcrD, and AcrF, these proteins form the core scaffold of multiple resistance-nodulation-cell division efflux systems, including AcrAB-TolC, AcrEF-TolC, and EmrAB-TolC. Transcriptional regulators such as MarA, BaeR, and CRP form a hierarchical regulatory layer above the efflux genes, with MarA acting as a classic activator of the acrAB-tolC operon while simultaneously downregulating the outer membrane porin OmpF. This architecture suggests a dual strategy of enzymatic hydrolysis through NDM-5 and active drug efflux through RND pumps, though the predicted synergy requires experimental validation.

The isolation of this strain from a rectal cancer patient highlights the vulnerability of immunocompromised hosts, whose impaired intestinal mucosal barrier and chemotherapy-induced immunosuppression heighten susceptibility to foodborne pathogens. Carbapenem-resistant infections in such patients carry high treatment failure rates and mortality. The authors emphasize that clinicians should strengthen screening for carbapenemase-producing Enterobacteriaceae in immunocompromised cancer patients presenting with diarrhea, and that public health authorities should expand surveillance of bla_NDM-producing monophasic Salmonella Typhimurium in food, animal, and clinical samples. The study’s limitations include its single-center, single-isolate design, the inability of short-read sequencing to resolve complete circular plasmid structures, and the absence of in vitro and in vivo virulence experiments. Future work will pursue nationwide genomic epidemiology, conjugation assays, cell models, and animal infection studies to clarify the transmission dynamics and pathogenic mechanisms of this high-risk clone.

Subject of Research: Genomic characterization of a carbapenem-resistant monophasic Salmonella Typhimurium ST34 strain carrying blaNDM-5 isolated from a rectal cancer patient in Hainan, China

Article Title: Multidrug-resistant monophasic Salmonella Typhimurium ST34 strain producing blaNDM-5 carbapenemase isolated from a patient with rectal cancer in Hainan, China: comprehensive whole genome dissection of antibiotic resistance and virulence determinants

Article References: Zhong, S., Xing, L., Wang, L., Wang, C., Fu, X., Xu, L., Li, Y., Zhu, H., He, L., Zhu, X., & Li, H. (2026). Multidrug-resistant monophasic Salmonella Typhimurium ST34 strain producing blaNDM-5 carbapenemase isolated from a patient with rectal cancer in Hainan, China: comprehensive whole genome dissection of antibiotic resistance and virulence determinants. Frontiers in Cellular and Infection Microbiology, 16, Article 1942157. https://doi.org/10.3389/fcimb.2026.1942157

Image Credits: AI Generated

DOI: 10.3389/fcimb.2026.1942157

Keywords: Salmonella Typhimurium, ST34, blaNDM-5, carbapenem resistance, monophasic Salmonella, whole-genome sequencing, multidrug resistance, IncHI2 plasmid, prophage, pathogenicity islands, efflux pump, rectal cancer