Plague remains a globally important zoonotic disease maintained in natural foci, with ongoing epizootic activity and periodic human cases reported in several regions of the world. Continuous monitoring of antimicrobial susceptibility in Yersinia pestis is essential because the emergence of resistant strains could compromise the effectiveness of currently recommended therapeutic regimens. In this study, 75 Y. pestis isolates originating from natural plague foci of Kazakhstan were investigated using an integrated approach combining phenotypic susceptibility testing, targeted molecular screening, and whole-genome sequencing (WGS)-based resistome analysis. The collection included historical clinical isolates obtained during plague outbreaks as well as more recent epizootic strains recovered from animal hosts and flea vectors. Phenotypic testing demonstrated uniformly high susceptibility to clinically relevant antimicrobial agents used for plague treatment. Targeted molecular screening by real-time PCR did not detect the analyzed resistance determinants. Genome-wide analysis based on WGS data from NCBI BioProject PRJNA1249055 did not identify acquired antimicrobial resistance genes, major resistance-associated mutations in key chromosomal loci (rpsL, gyrA, and parC), or plasmid-borne resistance determinants. Regulatory loci associated with adaptive responses were highly conserved across the analyzed genomes. The complete concordance between phenotypic, molecular, and genomic findings indicates a stable antimicrobial susceptibility profile of Y. pestis circulating in natural plague foci of Kazakhstan. These results support the continued effectiveness of current therapeutic strategies for plague and highlight the value of integrating genomic surveillance into long-term monitoring programs for this pathogen.

Plague remains a significant natural focal zoonotic infection with continuing epidemiological relevance in the Republic of Kazakhstan. This study provides a comprehensive assessment of epizootological dynamics in natural plague foci during 2020–2025 through the integration of historical epidemiological data, phenotypic and molecular characterization of Yersinia pestis, and GIS-based spatial analysis. The study utilized long-term surveillance data (1920–2025), epidemiological records of human plague cases (1926–2003), phenotypic analysis of 1526 strains, and whole-genome sequencing of 75 representative isolates. Epizootological monitoring demonstrated high surveillance coverage and stable monitoring capacity, together with a marked increase in the application of molecular diagnostic methods. By 2025, both the number and isolation rate of Y. pestis strains increased substantially, while the epizootically active area expanded in 2024–2025, although the overall long-term trend in active area was not statistically significant. Despite these fluctuations, Y. pestis populations remained highly stable, with 94.9% phenotypically typical and 97.5% genotypically typical strains, and no evidence of antimicrobial resistance. Spatial autocorrelation analysis using Moran’s I revealed significant clustering of epizootics (Moran’s I = 0.1627; z = 4.39; p < 0.001), indicating non-random spatial distribution and localized zones of increased epizootic activity. No human plague cases have been recorded since 2003 during the period of sustained epidemiological surveillance and control measures. These findings highlight the potential utility of integrating spatial modeling and molecular surveillance into risk-oriented plague monitoring and control strategies.

Plague remains a significant natural-focal infection, yet the mechanisms underlying the long-term persistence of Yersinia pestis during inter-epizootic periods remain incompletely understood. This study integrated published evidence with epizootiological monitoring data from Kazakhstan (1990–2025) and detailed field and laboratory investigations conducted during 2021–2023 to develop an integrated ecological framework of plague persistence. Abiotic and biotic niches, reservoir hosts, flea vectors, and adaptive states of Y. pestis were evaluated according to the strength and type of supporting evidence. Long-term surveillance identified spatially recurrent enzootic cores and documented the continued presence of the great gerbil (Rhombomys opimus)–flea host–vector system across major desert plague territories. Field investigations covered approximately 120,000 km² and included 4586 environmental and biological samples. Bacteriological examination yielded 24 viable Y. pestis isolates belonging to the Medievalis biovar, whereas real-time PCR detected Y. pestis-specific DNA in 88 of 4586 samples (1.92%), with the highest positivity observed in ectoparasite suspensions (66/868; 7.60%). Laboratory evidence was concentrated in five investigated foci containing recurrent enzootic core areas, while no positive findings were obtained in three comparison foci. These findings support an evidence-weighted ecological framework in which established host–vector transmission operates within spatially structured reservoir populations and heterogeneous environments, while additional environmental and bacterial persistence mechanisms remain plausible but require further experimental validation.

Rapid and reliable molecular detection of Salmonella is important for laboratory investigation and infectious disease surveillance. This study compared six candidate molecular targets (hilA, invA, phoP, fimA, rpoS, and spvA) and developed a hilA-targeting TaqMan real-time PCR assay for Salmonella enterica detection. During conventional PCR screening, the hilA-M1-F/R primer pair demonstrated the most consistent and reproducible amplification across the tested isolate panel under the experimental conditions used and was therefore selected for subsequent assay development. The developed assay showed an amplification efficiency of 93.4% and an R² of 0.9988 over a linear genomic DNA range from 1 ng/μL to 10 fg/μL. Experimentally determined detection limits under the conditions tested were 10² CFU/mL for bacterial suspensions and 10 fg/μL for genomic DNA. All 24 whole-genome sequencing-confirmed S. enterica isolates representing multiple serovars and sequence types were reproducibly detected, whereas no specific amplification was observed within the expanded panel of 15 non-target bacterial strains or isolates. The assay also detected S. enterica DNA in artificially contaminated milk and carrot matrices in preliminary proof-of-concept experiments. These findings demonstrate promising analytical performance of the hilA-targeting TaqMan assay within the evaluated experimental framework. However, broader validation using geographically diverse S. enterica isolate collections, additional non-target organisms, internal amplification controls, naturally contaminated food products, primary clinical, veterinary, and environmental specimens, and independent laboratories is required before routine diagnostic, food-testing, or epidemiological surveillance applications can be established.

Reliable detection of Salmonella is essential for public health surveillance and food safety, particularly given the diversity of circulating serovars and the potential limitations of individual molecular targets. This study comparatively evaluated six candidate targets (hilA, invA, phoP, fimA, rpoS, and spvA) and developed a hilA-targeting TaqMan real-time PCR assay for Salmonella detection. To the best of our knowledge, this is the first study in Kazakhstan to apply a comparative target-selection approach to the development of a hilA-based real-time PCR assay. Among the targets evaluated, hilA demonstrated the broadest amplification coverage across the tested isolate panel, including less common serovars, whereas invA was not amplified in a subset of iso-lates. The developed assay demonstrated limits of detection of 10² CFU/mL for bacterial suspensions and 10 fg/μL for genomic DNA, with an amplification efficiency of 93.4% and an R² of 0.9988 over the linear range from 1 ng/μL to 10 fg/μL. No cross-reactivity was observed within the tested non-target bacterial panel. All 24 whole-genome sequencing-confirmed Salmonella isolates yielded positive amplification within the predefined Ct threshold (≤37), including S. enterica serovar Dublin strain 25S, which showed the highest mean Ct value (36.66 ± 0.14). The Proteus mirabilis comparator, initially identified as Salmonella by conventional bacteriology, showed no specific amplification. Salmonella DNA was also detected in both artificially contaminated food matrices tested. These findings support hilA as a promising molecular target and demonstrate the potential of the developed assay for laboratory detection, food safety investigations, and epidemiological surveillance. Further validation using broader isolate collections, naturally contaminated samples, and independent laboratories is warranted.

Plague remains a globally significant zoonotic infection maintained in natural foci, with ongoing epizootic activity and periodic human cases reported in different regions of the world. Continuous monitoring of antimicrobial susceptibility of Yersinia pestis is essential due to the potential emergence and spread of resistant strains. A total of 75 Yersinia pestis isolates, including clinical and epizootic strains obtained from plague outbreaks in Kazakhstan, were analyzed. Antimicrobial susceptibility was evaluated using standard phenotypic methods, and molecular screening for resistance determinants was performed by real-time PCR. Genome-level analysis based on whole-genome sequencing (WGS) data from the NCBI BioProject PRJNA1249055 was conducted to assess the presence of acquired antimicrobial resistance genes and chromosomal mutations associated with resistance. All isolates demonstrated high susceptibility to clinically relevant antibiotics. No resistance genes were detected by molecular screening. Genome-based analysis confirmed the absence of acquired antimicrobial resistance determinants, resistance-associated mutations in key loci (rpsL, gyrA, parC), and plasmid-mediated resistance mechanisms. Minor lineage-associated variation in phoP was identified in a limited number of isolates and was not associated with antimicrobial resistance. These findings indicate a stable antimicrobial susceptibility profile of Yersinia pestis in Kazakhstan and confirm the absence of emerging resistance despite long-term circulation in natural plague foci. The results highlight the importance of integrated surveillance and support the continued effectiveness of current therapeutic strategies for plague.