Genetic and virulence characteristics of hybrid Shiga toxin-producing and atypical enteropathogenic Escherichia coli strains isolated in South Korea.

Introduction: The predominant hybrid pathogenic E. coli, enterohemorrhagic E. coli (EHEC), combines characteristics of Shiga toxin-producing E. coli (STEC) and enteropathogenic E. coli (EPEC), contributing to global outbreaks with severe symptoms including fatal consequences. Since EHEC infection was designated as a notifiable disease in 2000 in South Korea, around 2000 cases have been reported, averaging approximately 90 cases annually. Aim: In this work, genome-based characteristic analysis and cell-based assay of hybrid STEC/aEPEC strains isolated from livestock feces, animal source foods, and water in South Korea was performed. Methods: To identify the virulence and antimicrobial resistance genes, determining the phylogenetic position of hybrid STEC/aEPEC strains isolated in South Korea, a combination of real-time PCR and whole-genome sequencing (WGS) was used. Additionally, to assess the virulence of the hybrid strains and compare them with genomic characterization, we performed a cell cytotoxicity and invasion assays. Results: The hybrid STEC/aEPEC strains harbored stx and eae genes, encoding Shiga toxins and E. coli attachment/effacement related protein of STEC and EPEC, respectively. Furthermore, all hybrid strains harbored plasmid-carried enterohemolysin(ehxCABD), a key virulence factor in prevalent pathogenic E. coli infections, such as diarrheal disease and hemolytic-uremic syndrome (HUS). Genome-wide phylogenetic analysis revealed a close association between all hybrid strains and specific EPEC strains, suggesting the potential acquisition of Stx phages during STEC/aEPEC hybrid formation. Some hybrid strains showed cytotoxic activity against HeLa cells and invasive properties against epithelial cells. Notably, all STEC/aEPEC hybrids with sequence type (ST) 1,034 (n = 11) exhibited higher invasiveness than those with E2348/69. This highlights the importance of investigating potential correlations between STs and virulence characteristics of E. coli hybrid strains. Conclusion: Through genome-based characterization, we confirmed that the hybrid STEC/aEPEC strains are likely EPEC strains that have acquired STEC virulence genes via phage. Furthermore, our results emphasize the potential increased danger to humans posed by hybrid STEC/aEPEC strains isolated in South Korea, containing both stx and eaeA, compared to STEC or EPEC alone.

Comparative analysis of LC-MS/MS and real-time PCR assays for efficient detection of potential allergenic silkworm.

The silkworm (Bombyx mori) has long been valued food and feed in East Asia for its abundant nutritional and medicinal attributes, conversely, it can elicit allergic responses in susceptible individuals. Therefore, the development of silkworm detection method is required to avert allergenic incidents. In this study, two methodologies, tandem mass spectrometry (LC-MS/MS) and real-time PCR, were developed to achieve effective silkworm detection. These methods exhibited exceptional sensitivity in identifying silkworm presence in processed foods. Furthermore, model cookies spiked with silkworm were used to validate the sensitivities of LC-MS/MS (0.0005%) and real-time PCR (0.001%). Overall, these techniques were useful for trace silkworm detection in food products; therefore, they may help prevent allergic reactions. To the best of our knowledge, this study represents the first comparison of LC-MS/MS and real-time PCR methods for silkworm detection, marking an important contribution to the field. Data are available from ProteomeXchange under identifier PXD042494.

Rapid and Simultaneous Authentication of Six Laver Species Using Capillary Electrophoresis-Based Multiplex PCR.

Lavers are typically consumed in dried or seasoned forms. However, commercially processed lavers can lead to seafood fraud because it is impossible to authenticate the original species based on morphological characteristics alone. In this study, we developed a capillary electrophoresis-based multiplex polymerase chain reaction (PCR) to authenticate six different laver species. The species-specific primer sets to target the chloroplast rbcL or rbcS genes were newly designed. We successfully established both singleplex and multiplex conditions, which resulted in specific amplicons for each species (N. dentata, 274 bp; N. yezoensis, 211 bp; N. seriata, 195 bp; N. tenera, 169 bp; N. haitanensis, 127 bp; P. suborbiculata, 117 bp). Moreover, the assays were sensitive enough to detect DNA ranging from 10 to 0.1 pg of DNA. The optimized capillary electrophoresis-based multiplex PCR was successfully applied to 40 commercial laver products. In addition to detecting the laver species as stated on the commercial label, the assay discovered cases where less expensive species were mixed in. With its advantageous properties, such as short amplicon size, high specificity, and superior sensitivity, this assay could be used for the authentication of the six laver species.

Genomic characteristics of cfr and fexA carrying Staphylococcus aureus isolated from pig carcasses in Korea.

The emergence of transferable linezolid resistance genes poses significant challenges to public health, as it does not only confer linezolid resistance but also reduces susceptibility to florfenicol, which is widely used in the veterinary field. This study evaluated the genetic characteristics of linezolid-resistant Staphylococcus aureus strains isolated from pig carcasses and further clarified potential resistance and virulence mechanisms in a newly identified sequence type. Of more than 2500 strains isolated in a prior study, 15 isolated from pig carcasses exhibited linezolid resistance (minimum inhibitory concentration ≥ 8 mg/L). The strains were characterized in detail by genomic analysis. Linezolid-resistant S. aureus strains exhibited a high degree of genetic lineage diversity, with one strain (LNZ_R_SAU_64) belonging to ST8004, which has not been reported previously. The 15 strains carried a total of 21 antibiotic resistance genes, and five carried mecA associated with methicillin resistance. All strains harbored cfr and fexA, which mediate resistance to linezolid, phenicol, and other antibiotics. Moreover, the strains carried enterotoxin gene clusters, including the hemolysin, leukotoxin, and protease genes, which are associated with humans or livestock. Some genes were predicted to be carried in plasmids or flanked by ISSau9 and the transposon Tn554, thus being transmittable between staphylococci. Strains carrying the plasmid replicon repUS5 displayed high sequence similarity (99%) to the previously reported strain pSA737 in human clinical samples in the United States. The results illustrate the need for continuous monitoring of the prevalence and transmission of linezolid-resistant S. aureus isolated from animals and their products.

Molecular and Genomic Analysis of the Virulence Factors and Potential Transmission of Hybrid Enteropathogenic and Enterotoxigenic Escherichia coli (EPEC/ETEC) Strains Isolated in South Korea.

Hybrid strains Escherichia coli acquires genetic characteristics from multiple pathotypes and is speculated to be more virulent; however, understanding their pathogenicity is elusive. Here, we performed genome-based characterization of the hybrid of enteropathogenic (EPEC) and enterotoxigenic E. coli (ETEC), the strains that cause diarrhea and mortality in children. The virulence genes in the strains isolated from different sources in the South Korea were identified, and their phylogenetic positions were analyzed. The EPEC/ETEC hybrid strains harbored eae and est encoding E. coli attaching and effacing lesions and heat-stable enterotoxins of EPEC and ETEC, respectively. Genome-wide phylogeny revealed that all hybrids (n = 6) were closely related to EPEC strains, implying the potential acquisition of ETEC virulence genes during ETEC/EPEC hybrid emergence. The hybrids represented diverse serotypes (O153:H19 (n = 3), O49:H10 (n = 2), and O71:H19 (n = 1)) and sequence types (ST546, n = 4; ST785, n = 2). Furthermore, heat-stable toxin-encoding plasmids possessing estA and various other virulence genes and transporters, including nleH2, hlyA, hlyB, hlyC, hlyD, espC, espP, phage endopeptidase Rz, and phage holin, were identified. These findings provide insights into understanding the pathogenicity of EPEC/ETEC hybrid strains and may aid in comparative studies, virulence characterization, and understanding evolutionary biology.

Comprehensive metagenomic analysis of stress-resistant and -sensitive Listeria monocytogenes.

Listeria monocytogenes is a pathogenic bacterium which can live in adverse environments (low pH, high salinity, and low temperature). Even though there are various whole genome sequencing (WGS) data on L. monocytogenes, investigations on genetic differences between stress-resistant and -sensitive L. monocytogenes grown under stress environments have been not fully examined. This study aims to investigate and compare genetic characteristics between stress-resistant and -sensitive L. monocytogenes using whole genome sequencing (WGS). A total of 47 L. monocytogenes strains (43 stress-resistant and 4 stress-sensitive) were selected based on the stress-resistance tests under pH 3, 5% salt concentration, and 1 °C. The sequencing library for WGS was prepared and sequenced using an Illumina MiSeq. Genetic characteristics of two different L. monocytogenes groups were examined to analyze the pangenome, functionality, virulence, antibiotic resistance, core, and unique genes. The functionality of unique genes in the stress-resistant L. monocytogenes was distinct compared to the stress-sensitive L. monocytogenes, such as carbohydrate and nucleotide transport and metabolism. The lisR virulence gene was detected more in the stress-resistant L. monocytogenes than in the stress-sensitive group. Five stress-resistant L. monocytogenes strains possessed tet(M) antibiotic resistance gene. This is the first study suggesting that deep genomic characteristics of L. monocytogenes may have different resistance level under stress conditions. This new insight will aid in understanding the genetic relationship between stress-resistant and -sensitive L. monocytogenes strains isolated from diverse resources. KEY POINTS: • Whole genomes of L. monocytogenes isolated from three different sources were analyzed. • Differences in two L. monocytogenes groups were identified in functionality, virulence, and antibiotic resistance genes. • This study first examines the association between resistances and whole genomes of stress-resistant and -sensitive L. monocytogenes.

Differentiation between Weissella cibaria and Weissella confusa Using Machine-Learning-Combined MALDI-TOF MS.

Although Weissella cibaria and W. confusa are essential food-fermenting bacteria, they are also opportunistic pathogens. Despite these species being commercially crucial, their taxonomy is still based on inaccurate identification methods. In this study, we present a novel approach for identifying two important Weissella species, W. cibaria and W. confusa, by combining matrix-assisted laser desorption/ionization and time-of-flight mass spectrometer (MALDI-TOF MS) data using machine-learning techniques. After on- and off-plate protein extraction, we observed that the BioTyper database misidentified or could not differentiate Weissella species. Although Weissella species exhibited very similar protein profiles, these species can be differentiated on the basis of the results of a statistical analysis. To classify W. cibaria, W. confusa, and non-target Weissella species, machine learning was used for 167 spectra, which led to the listing of potential species-specific mass-to-charge (m/z) loci. Machine-learning techniques including artificial neural networks, principal component analysis combined with the K-nearest neighbor, support vector machine (SVM), and random forest were used. The model that applied the Radial Basis Function kernel algorithm in SVM achieved classification accuracy of 1.0 for training and test sets. The combination of MALDI-TOF MS and machine learning can efficiently classify closely-related species, enabling accurate microbial identification.

Genome-Based Characterization of Hybrid Shiga Toxin-Producing and Enterotoxigenic Escherichia coli (STEC/ETEC) Strains Isolated in South Korea, 2016-2020.

The global emergence of hybrid diarrheagenic E. coli strains incorporating genetic markers from different pathotypes is a public health concern. Hybrids of Shiga toxin-producing and enterotoxigenic E. coli (STEC/ETEC) are associated with diarrhea and hemolytic uremic syndrome (HUS) in humans. In this study, we identified and characterized STEC/ETEC hybrid strains isolated from livestock feces (cattle and pigs) and animal food sources (beef, pork, and meat patties) in South Korea between 2016 and 2020. The strains were positive for genes from STEC and ETEC, such as stx (encodes Shiga toxins, Stxs) and est (encodes heat-stable enterotoxins, ST), respectively. The strains belong to diverse serogroups (O100, O168, O8, O155, O2, O141, O148, and O174) and sequence types (ST446, ST1021, ST21, ST74, ST785, ST670, ST1780, ST1782, ST10, and ST726). Genome-wide phylogenetic analysis revealed that these hybrids were closely related to certain ETEC and STEC strains, implying the potential acquisition of Stx-phage and/or ETEC virulence genes during the emergence of STEC/ETEC hybrids. Particularly, STEC/ETEC strains isolated from livestock feces and animal source foods mostly exhibited close relatedness with ETEC strains. These findings allow further exploration of the pathogenicity and virulence of STEC/ETEC hybrid strains and may serve as a data source for future comparative studies in evolutionary biology.

Development of real-time PCR method for rapid and accurate detection of Centipedes (Scolopendra mutilans) in food.

Centipedes contain pharmacologically active compounds used as important medicinal material. However, the poisons produced by centipedes can cause human diseases; therefore, its use as a food ingredient is prohibited. This is the first report to develop a real-time PCR method for detection of centipedes. The primer and probe targeting the mitochondrial cytochrome c oxidase subunit 1 (COI) gene were newly designed. The specificity was verified using ten species and was confirmed to amplify only the centipede species. The real-time PCR method exhibited good linearity with a high-determination coefficient (R 2 = 0.999) and a detection limit was 0.001 ng. The performance of our method was also verified using five real-time PCR platforms under Universal and Fast PCR conditions. Finally, its applicability to processed food was evaluated using binary insect mixtures, and at least 0.1% of centipedes was detected. Therefore, our method can specifically and sensitively detect centipedes in food, contributing to food safety.

Droplet digital PCR method for the absolute quantitative detection and monitoring of Lacticaseibacillus casei.

Droplet digital polymerase chain reaction (ddPCR) is an emerging molecular detection assay that provides an absolute quantification of targets. Despite its emerging applications in the detection of food microorganisms, there are limited reports of its use for the monitoring of microorganisms utilized as starters in the dairy industry. This study investigated the applicability of ddPCR as a detection platform for Lacticaseibacillus casei, a probiotic found in fermented foods and exerts beneficial effects on human health. In addition, this study compared the performance of ddPCR with that of real-time PCR. The ddPCR targeting the haloacid dehalogenase-like hydrolase (LBCZ_1793) exhibited high specificity against 102 nontarget bacteria, including Lacticaseibacillus species that is very closely related to L. casei. The ddPCR exhibited high linearity and efficiency within the quantitation range (105-100 CFU/ml), with the limit of detection being 100 CFU/ml. The ddPCR also demonstrated a higher sensitivity than real-time PCR in detecting low bacterial concentration in spiked milk samples. Furthermore, it provided an accurate absolute quantification of the concentration of L. casei, without the need for standard calibration curves. This study demonstrated that ddPCR is a useful method for monitoring starter cultures in dairy fermentations and detecting L. casei in foods.

Comprehensive genome analysis of Burkholderia contaminans SK875, a quorum-sensing strain isolated from the swine.

The Burkholderia cepacia complex (BCC) is a Gram-negative bacterial, including Burkholderia contaminans species. Although the plain Burkholderia is pervasive from taxonomic and genetic perspectives, a common characteristic is that they may use the quorum-sensing (QS) system. In our previous study, we generated the complete genome sequence of Burkholderia contaminans SK875 isolated from the respiratory tract. To our knowledge, this is the first study to report functional genomic features of B. contaminans SK875 for understanding the pathogenic characteristics. In addition, comparative genomic analysis for five B. contaminans genomes was performed to provide comprehensive information on the disease potential of B. contaminans species. Analysis of average nucleotide identity (ANI) showed that the genome has high similarity (> 96%) with other B. contaminans strains. Five B. contaminans genomes yielded a pangenome of 8832 coding genes, a core genome of 5452 genes, the accessory genome of 2128 genes, and a unique genome of 1252 genes. The 186 genes were specific to B. contaminans SK875, including toxin higB-2, oxygen-dependent choline dehydrogenase, and hypothetical proteins. Genotypic analysis of the antimicrobial resistance of B. contaminans SK875 verified resistance to tetracycline, fluoroquinolone, and aminoglycoside. Compared with the virulence factor database, we identified 79 promising virulence genes such as adhesion system, invasions, antiphagocytic, and secretion systems. Moreover, 45 genes of 57 QS-related genes that were identified in B. contaminans SK875 indicated high sequence homology with other B. contaminans strains. Our results will help to gain insight into virulence, antibiotic resistance, and quorum sensing for B. contaminans species.

Weissella and the two Janus faces of the genus.

The genus Weissella belongs to the lactic acid bacteria group. It occurs naturally in foods and is a component of the human microbiome. A few Weissella species are candidate probiotics due to their potential for survival under the harsh conditions present in the gastrointestinal tract of humans and animals. Various species have also shown potential for treating and preventing periodontal disease, skin pathologies, and atopic dermatitis; some are used as starters for the fermentation of foods due to their production of exopolysaccharides; and others are used as protective cultures due to their production of weissellicin, a bacteriocin. However, a few Weissella species are opportunistic pathogens, such as W. ceti, which is the etiological agent of weissellosis, a disease in rainbow trout. Additionally, most Weissella species are intrinsically vancomycin-resistant. Thus, the Weissella genus is important from both medical and industrial points of view, and the Janus faces of this genus should be considered in any expected biotechnological applications. In this review, we present an overview of the probiotic potential and pathogenic cases of the Weissella genus reported in the literature.

Seasonal Changes in the Microbial Communities on Lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea.

Lettuce is one of the most consumed vegetables worldwide. However, it has potential risks associated with pathogenic bacterial contamination because it is usually consumed raw. In this study, we investigated the changes in the bacterial community on lettuce (Lactuca sativa L.) in Chungcheong-do, South Korea, and the prevalence of foodborne pathogens on lettuce in different seasons using 16S rRNA gene-based sequencing. Our data revealed that the Shannon diversity index showed the same tendency in term of the number of OTUs, with the index being greatest for summer samples in comparison to other seasons. Moreover, the microbial communities were significantly different between the four seasons. The relative abundance of Actinobacteriota varied according to the season. Family Micrococcaceae was most dominant in all samples except summer, and Rhizobiaceae was predominant in the microbiome of the summer sample. At the genus level, the relative abundance of Bacillus was greatest in spring samples, whereas Pseudomonas was greatest in winter samples. Potential pathogens, such as Staphylococcus and Clostridium, were detected with low relative abundance in all lettuce samples. We also performed metagenome shotgun sequencing analysis on the selected summer and winter samples, which were expected to be contaminated with foodborne pathogens, to support 16S rRNA gene-based sequencing dataset. Moreover, we could detect seasonal biomarkers and microbial association networks of microbiota on lettuce samples. Our results suggest that seasonal characteristics of lettuce microbial communities, which include diverse potential pathogens, can be used as basic data for food safety management to predict and prevent future outbreaks.

Genomic characteristics and comparative genomics analysis of Salmonella enterica subsp. enterica serovar Thompson isolated from an outbreak in South Korea.

Salmonella infections represent an important public health problem. In 2018, a multistate outbreak of S. enterica subsp. enterica serovar Thompson infection associated with contaminated chocolate cakes in schools was reported in South Korea. In this study, we sequenced the 37 S. Thompson strains isolated from chocolate cakes, egg whites, preserves, and cookware associated with the outbreak. In addition, we analyze the genomic sequences of 61 S. Thompson strains (37 chocolate cake-related outbreak strains, 4 strains isolated from outbreaks in South Korea and 20 strains available in the National Center for Biotechnology Information) to assess the genomic characteristics of outbreak-related strains by comparative genomics and phylogenetic analysis. The results showed that identically classified clusters divided strains into two clusters, sub-clusters A & I (with strains from 2018 in South Korea) and sub-clusters B & II (with strains from 2014 to 2015 in South Korea). S. Thompson isolated from South Korea were accurately distinguished from publicly-available strains. Unlike other S. Thompson genomes, those of chocolate cake outbreak-related strains had three Salmonella phages (SEN8, vB SosS Oslo, and SI7) integrated into their chromosome. Comparative genomics revealed several genes responsible for the specific genomic features of chocolate cake outbreak-related strains and three bacteriophages that may contribute to the pathogenicity of other S. Thompson strains.

Identification of Leuconostoc species based on novel marker genes identified using real-time PCR via computational pangenome analysis.

Leuconostoc species are important microorganisms in food fermentation but also cause food spoilage. Although these species are commercially important, their taxonomy is still based on inaccurate identification methods. Here, we used computational pangenome analysis to develop a real-time PCR-based method for identifying and differentiating the 12 major Leuconostoc species found in food. Analysis of pan and core-genome phylogenies showed clustering of strains into 12 distinct groups according to the species. Pangenome analysis of 130 Leuconostoc genomes from these 12 species enabled the identification of each species-specific gene. In silico testing of the species-specific genes against 143 publicly available Leuconostoc and 100 other lactic acid bacterial genomes showed that all the assays had 100% inclusivity/exclusivity. We also verified the specificity for each primer pair targeting each specific gene using 23 target and 124 non-target strains and found high specificity (100%). The sensitivity of the real-time PCR method was 102 colony forming units (CFUs)/ml in pure culture and spiked food samples. All standard curves showed good linear correlations, with an R 2 value of ≥0.996, suggesting that screened targets have good specificity and strong anti-interference ability from food sample matrices and non-target strains. The real-time PCR method can be potentially used to determine the taxonomic status and identify the Leuconostoc species in foods.

Rapid On-Site Identification for Three Arcidae Species (Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii) Using Ultrafast PCR Combined with Direct DNA Extraction.

Granular ark (Tegillarca granosa), broughton's ribbed ark (Anadara broughtonii), and half-crenate ark (Anadara kagoshimensis) are important fishery resources throughout Asia; granular ark exhibiting a higher economic value due to its rarity. However, due to the similar morphological characteristics of the three species, the less valuable species could be exploited for food fraud. In this study, we developed a rapid on-site identification method based on a microfluidic chip for the detection of the three ark shell species. We designed new species-specific primers, targeting the genes encoding mitochondrial cytochrome b or cytochrome c oxidase I, for the identification of the three ark shells and estimated their specificity against 17 species, which amplified only the target species. The sensitivity of each primer was 0.001 ng. In addition, this method was further improved to develop a direct ultrafast polymerase chain reaction (PCR) for on-site food monitoring, which would allow for completing the entire procedure (from sampling to obtaining the results) within 25 min without DNA extraction. Our direct, ultrafast PCR was successfully applied to differentiate the three species from 29 commercial products. Therefore, this assay could be used as a rapid and cost-effective approach for the on-site identification of ark shells in commercial food products.

Evaluation of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the discrimination of Lacticaseibacillus species.

The closely related species, Lacticaseibacillus casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, are difficult to accurately discriminate by conventional identification methods. In this study, the bioTyper and in-house database of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was evaluated to discriminate five Lacticaseibacillus species. From the mass spectra of 130 isolates aligned with databases, 118 strains were correctly identified. On the other hand, databases could not accurately differentiate 12 isolates such as L. casei, L. rhamnosus and L. chiayiensis because the same colony was identified as two species with similar score. To overcome the database's limitations, the mass spectra were analyzed to discover species-specific protein peaks. The peaks at 6731 ± 1, 6849 ± 1, 7008 ± 1, 7376 ± 1, and 2593 ± 1 m/z were specifically found in the reference strains of L. casei, L. paracasei, L. rhamnosus, L. chiayiensis, and L. zeae, respectively. These peaks confirmed that the five peaks were consistently present in each species using 130 strains isolated from food samples. Our results demonstrate the high-resolution of MALDI-TOF MS technique for rapid and accurate classification of five species when used with databases coupled to specific peaks.

Identification of novel molecular targets for Weissella species-specific real-time PCR based on pangenome analysis.

Some Weissella species are used in probiotic products because of their beneficial effects in humans, whereas some species are considered as opportunistic pathogens that cause infections in humans. Therefore, an accurate and rapid identification of Weissella species is essential to control pathogenic Weissella species or isolate new functional strains with probiotic effects from their habitat. The objective of our study was to extract novel molecular targets using pangenome analysis for the identification of major Weissella species present in food. With 50 genomes representing 11 Weissella species, novel molecular targets were mined based on their 100% presence in the respective strains of the target species and absence in the strains of non-target bacteria. Primers based on molecular targets showed positive results for the corresponding species, whereas 79 non-target strains showed negative results. Standard curves revealed good linearity in the range of 103-108 colony-forming units per reaction. Our method was successfully applied to 74 Weissella strains isolated from food samples to demonstrate that the molecular targets provided a viable alternative to the 16S rRNA sequence. Furthermore, it was possible to identify and quantify Weissella communities in fermented foods. These results demonstrate that our method can be used for effective and accurate screening for the presence of Weissella species in foods. KEY POINTS: • This is first study to mine novel targets for differentiating 11 Weissella species. • The novel targets showed higher resolution than the 16S rRNA gene sequence. • The PCR method effectively detected Weissella species with opposing properties.

Comparison of Real-Time PCR and Droplet Digital PCR for the Quantitative Detection of Lactiplantibacillus plantarum subsp. plantarum

Droplet digital polymerase chain reaction (ddPCR) is one of the newest and most promising tools providing absolute quantification of target DNA molecules. Despite its emerging applications in microorganisms, few studies reported its use for detecting lactic acid bacteria. This study evaluated the applicability of a ddPCR assay targeting molecular genes obtained from in silico analysis for detecting Lactiplantibacillus plantarum subsp. plantarum, a bacterium mainly used as a starter or responsible for fermentation in food. The performance characteristics of a ddPCR were compared to those of a quantitative real-time PCR (qPCR). To compare the linearity and sensitivity of a qPCR and ddPCR, the calibration curve for a qPCR and the regression curve for a ddPCR were obtained using genomic DNA [102−108 colony-forming units (CFU)/mL] extracted from a pure culture and spiked food sample. Both the qPCR and ddPCR assays exhibited good linearity with a high coefficient of determination in the pure culture and spiked food sample (R2 ≥ 0.996). The ddPCR showed a 10-fold lower limit of detection, suggesting that a ddPCR is more sensitive than a qPCR. However, a ddPCR has limitations in the absolute quantitation of high bacterial concentrations (>106 CFU/mL). In conclusion, a ddPCR can be a reliable method for detecting and quantifying lactic acid bacteria in food.