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.

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.

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.

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.

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.

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.

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.

Novel approaches for the identification of microbial communities in kimchi: MALDI-TOF MS analysis and high-throughput sequencing.

Complex interactions occur within microbial communities during the fermentation process of kimchi. Identification of these microorganisms provides the essential information required to improve food quality and to understand their role in this process. This was the first study to compare two methods for accuracy in the identification of microbial community changes during the fermentation of kimchi by comparing a culture-dependent (MALDI-TOF MS analysis) and a culture-independent method (high-throughput sequencing) of 16S rRNA gene fragment). Members of the Lactobacillus-related genera, Leuconostoc, and Weissella were identified as the predominant microorganisms by both methods. The culture-independent method was able to additionally identify non-lactic acid bacteria and yeasts, such as Kazachstania in kimchi. However, high-throughput sequencing failed to accurately recognize Latilactobacillus sakei, Latilactobacillus curvatus, Lactiplantibacillus plantarum, and W. cibaria, which played an important role in kimchi fermentation, as this method only allowed for identification at the genus level. Conversely, MALDI-TOF MS analysis could identify the isolates at the species level. Also, culture-dependent method could identify predominant species in viable cell communities. The culture-dependent method and culture-independent method provided complementary information by producing a more comprehensive view of the microbial ecology in fermented kimchi.

Prevalence and Characteristics of Phenicol-Oxazolidinone Resistance Genes in Enterococcus Faecalis and Enterococcus Faecium Isolated from Food-Producing Animals and Meat in Korea.

The use of phenicol antibiotics in animals has increased. In recent years, it has been reported that the transferable gene mediates phenicol-oxazolidinone resistance. This study analyzed the prevalence and characteristics of phenicol-oxazolidinone resistance genes in Enterococcus faecalis and Enterococcus faecium isolated from food-producing animals and meat in Korea in 2018. Furthermore, for the first time, we reported the genome sequence of E. faecalis strain, which possesses the phenicol-oxazolidinone resistance gene on both the chromosome and plasmid. Among the 327 isolates, optrA, poxtA, and fexA genes were found in 15 (4.6%), 8 (2.5%), and 17 isolates (5.2%), respectively. Twenty E. faecalis strains carrying resistance genes belonged to eight sequence types (STs), and transferability was found in 17 isolates. The genome sequences revealed that resistant genes were present in the chromosome or plasmid, or both. In strains EFS17 and EFS108, optrA was located downstream of the ermA and ant(9)-1 genes. The strains EFS36 and EFS108 harboring poxtA-encoding plasmid cocarried fexA and cfr(D). These islands also contained IS1216E or the transposon Tn554, enabling the horizontal transfer of the phenicol-oxazolidinone resistance with other antimicrobial-resistant genes. Our results suggest that it is necessary to promote the prudent use of antibiotics through continuous monitoring and reevaluation.

Design of PCR assays to specifically detect and identify 37 Lactobacillus species in a single 96 well plate.

BACKGROUND: Lactobacillus species are used as probiotics and play an important role in fermented food production. However, use of 16S rRNA gene sequences as standard markers for the differentiation of Lactobacillus species offers a very limited scope, as several species of Lactobacillus share similar 16S rRNA gene sequences. In this study, we developed a rapid and accurate method based on comparative genomic analysis for the identification of 37 Lactobacillus species that are commonly used in probiotics and fermented foods. RESULTS: To select species-specific sequences or genes, a total of 180 Lactobacillus genome sequences were compared using Python scripts. In 14 out of 37 species, species-specific sequences could not be found due to the similarity of the 16S-23S rRNA gene. Selected unique genes were obtained using comparative genomic analysis and all genes were confirmed to be specific for 52,478,804 genomes via in silico analysis; they were found not to be strain-specific, but to exist in all strains of the same species. Species-specific primer pairs were designed from the selected 16S-23S rRNA gene sequences or unique genes of species. The specificity of the species-specific primer pairs was confirmed using reference strains, and the accuracy and efficiency of the polymerase chain reaction (PCR) with the standard curve were confirmed. The PCR method developed in this study is able to accurately differentiate species that were not distinguishable using the 16S rRNA gene alone. This PCR assays were designed to detect and identify 37 Lactobacillus species. The developed method was then applied in the monitoring of 19 probiotics and 12 dairy products. The applied tests confirmed that the species detected in 17 products matched those indicated on their labels, whereas the remaining products contained species other than those appearing on the label. CONCLUSIONS: The method developed in this study is able to rapidly and accurately distinguish different species of Lactobacillus, and can be used to monitor specific Lactobacillus species in foods such as probiotics and dairy products.

Novel real-time PCR assay for Lactobacillus casei group species using comparative genomics.

The Lactobacillus casei group, which includes the closely related species L. casei, L. paracasei, L. rhamnosus, and L. chiayiensis, has been under debate regarding its taxonomy because of the difficulty in distinguishing the species from each other. In the present study, we developed a novel real-time PCR assay for distinguishing the L. casei group species. The pan-genome, as determined by the genomes of 44 strains, comprised 6789 genes, comparative genomic analysis showed that L. casei group strains were classified by species. Based on these results, species-specific genes were identified, and primers were designed from those genes. Real-time PCR clearly distinguished each species of the L. casei group and specifically amplified only to the target species. The method was applied to 29 probiotic products, and the detected results and label claims were compared. Total 23 products were in accordance with the label claims, and the remaining products contained species different from those stated in the label claims. Our method can rapidly and accurately distinguish the L. casei group species in a single reaction. Hence, our assay can be applied to identify L. casei group species from food or environmental samples and to accurately determine the nomenclature of the species.

Hydraulic Transcrestal Sinus Lift: Different Patterns of Elevation in Pig Sinuses.

PURPOSE: The aim of this study was to evaluate different patterns of sinus membrane elevation in pig jaws. MATERIALS AND METHODS: A total of 30 pig jaws (60 sinuses) were used for the present investigation. The hydraulic Crestal Approach Sinus kit was used to elevate sinus membrane, and different elevation patterns were recorded. RESULTS: There were 4 different scenarios of membrane separation patterns: center dome-shaped elevation, off-center dome-shaped elevation, horizontally spreading membrane elevation, and perforation. The incidence of each different type was 35.0% (n = 21) in center dome-shaped separation, 51.7% (n = 31) in off-center dome-shaped separation, 10.0% (n = 6) in horizontally spreading separation, and 3.3% (n = 2) in membrane perforation. CONCLUSION: Different patterns of membrane elevations are observed in pig sinuses and introduced in this study. The off-center dome-shaped elevation was the most common pattern followed by the center dome-shaped elevation and horizontally spreading elevation, respectively.

H1 linker histone promotes epigenetic silencing by regulating both DNA methylation and histone H3 methylation.

Epigenetic silencing in mammals involves DNA methylation and posttranslational modifications of core histones. Here we show that the H1 linker histone plays a key role in regulating both DNA methylation and histone H3 methylation at the H19 and Gtl2 loci in mouse ES cells. Some, but not all, murine H1 subtypes interact with DNA methyltransferases DNMT1 and DNMT3B. The interactions are direct and require a portion of the H1 C-terminal domain. Expression of an H1 subtype that interacts with DNMT1 and DNMT3B in ES cells leads to their recruitment and DNA methylation of the H19 and Gtl2 imprinting control regions. H1 also interferes with binding of the SET7/9 histone methyltransferase to the imprinting control regions, inhibiting production of an activating methylation mark on histone H3 lysine 4. H1-dependent recruitment of DNMT1 and DNMT3B and interference with the binding of SET7/9 also were observed with chromatin reconstituted in vitro. The data support a model in which H1 plays an active role in helping direct two processes that lead to the formation of epigenetic silencing marks. The data also provide evidence for functional differences among the H1 subtypes expressed in somatic mammalian cells.

Analysis of the respirogram phase of Korean wrestling athletes compared with nonathletes for sports physiotherapy research.

[Purpose] Respiratory function is important for patients including athletes who require physical therapy for respiratory dysfunction. The purpose of the present study was to analyze the differences in the respirograms between Korean wrestling athletes and nonathletes according to phase for the study of sports physiotherapy. [Subjects and Methods] Respiratory function was measured using spirometry in both the athletes and nonathletes while they were in a sitting position. [Results] Spirometry parameters in the athletes were significantly higher than in the nonathletes. In respirogram phasic analysis, the expiratory area and total area of forced vital capacity were significantly increased in the athletes compared with the nonathletes. The slopes of the forced vital capacity for athletes at slopes 1, 2, and 3 of the A area were significantly increased. In correlative analysis, chest circumference was significantly correlated with slope 3 of the A area of the forced vital capacity. [Conclusion] The results suggest that the differences in changes in the phases of the respirogram between the Korean wrestling athletes and nonathletes may in part contribute to our understanding of respiratory function in sports physiotherapy research.

Differences in the body pressure-related sensory changes between the floor and mattress in a static supine position for physiotherapy research: a randomized controlled pilot trial.

[Purpose] This study was performed to investigate the difference in body pressure-related sensory changes between the floor and mattress in a static supine position for physiotherapy research. [Subjects and Methods] To analyze body pressure, the Body Pressure Measurement System was used. Body pressure sensors were attached to mattresses and the floor beneath the subjects. The level of pain was evaluated using pain score tools before the static supine position was adopted, at 1, 5, 10, and 15 min, and in total for specific body points. [Results] In analysis of digitized images, there was no significant difference observed between floor and mattress body pressure values at the start position. However, the head pressure intensity was significantly higher than that of the other body parts. In analysis of pain scores, all body part pain scores except those for both legs were significantly higher for the floor than for the mattress. Furthermore, the pain scores of the floor group were significantly increased at minute 1 compared with those of the mattress group. [Conclusion] These results suggest that properties that change in a time-dependent manner and postural changes need to be carefully considered when applying physical therapy.

Differences in respirogram phase between taekwondo poomsae athletes and nonathletes.

[Purpose] Respiratory physiotherapy is an effective approach to improving lung function in patient, including athletes with respiratory dysfunction caused by sports injury. The purpose of this study was to analyze the differences in the respirograms between taekwondo poomsae athletes and nonathletes according to the respirogram phase. [Subjects and Methods] Respiratory measurements for 13 elite taekwondo poomsae athletes were obtained. Respiratory function was measured using spirometry while the participant was seated. [Results] In respirogram phasic analysis, the inspiratory area of forced vital capacity were significantly increased in the athletes than in the nonathletes. The slopes of the forced vital capacity for athletes at slopes 1, 2, and 3 of the A area were significantly higher than those for the nonathletes. In correlation analysis, chest circumference was significantly correlated with slope 1 of the A area of the forced vital capacity. [Conclusion] Results indicate that differences in respirogram phasic changes between athletes and nonathletes may contribute to better understanding of respiratory function, which is important to sports physiotherapy research.

A comparative study of the effects of trunk exercise program in aquatic and land-based therapy on gait in hemiplegic stroke patients.

[Purpose] The purpose of this study was to compare the effects of aquatic and land-based trunk exercise program on gait in stroke patients. [Subjects and Methods] The subjects were 28 hemiplegic stroke patients (20 males, 8 females). The subjects performed a trunk exercise program for a total of four weeks. [Results] Walking speed and cycle, stance phase and stride length of the affected side, and the symmetry index of the stance phase significantly improved after the aquatic and land-based trunk exercise program. [Conclusion] These results suggest that the aquatic and land-based trunk exercise program may help improve gait performance ability after stroke.

Differences in strength-duration curves of electrical diagnosis by physiotherapists between DJ-1 homozygous knockout and wild-type mice: a randomized controlled pilot trial.

[Purpose] Strength-duration (SD) curves are used in electrical diagnosis by physiotherapists to confirm muscle degeneration. However, the usefulness of SD curves in comparing muscle degeneration in DJ-1 homozygous knockout (DJ-1(-/-)) and wild-type mice (DJ-1(+/+)) is not yet fully understood. The electrical properties of the gastrocnemius muscles of DJ-1(-/-) and DJ-1(+/+) mice were compared in the current study. [Subjects and Methods] The electrode of an electrical stimulator was applied to the gastrocnemius muscle to measure the rheobase until the response of contractive muscle to electrical stimulation became visible in mice. [Results] The rheobase of DJ-1(-/-) mice showed a significant increase in a time-dependent manner, compared to that of DJ-1(+/+) mice. [Conclusion] These results demonstrate that the DJ-1 protein may be implicated in the regulation of neuromuscular activity of gastrocnemius muscles of mice.

Surface properties correlated with the human gingival fibroblasts attachment on various materials for implant abutments: a multiple regression analysis.

OBJECTIVES: To reveal the suitable surface condition of an implant abutment for fibroblast attachment, the correlation between the surface characteristics of various materials and the human gingival fibroblast (HGF-1) attachment to the surfaces were analyzed. METHODS: Six kinds of surfaces comprised of machined titanium alloy (SM), machined Co-Cr-Mo alloy (CCM), titanium nitride coated titanium alloy (TiN), anodized titanium alloy (AO), composite resin coating on titanium alloy (R) and zirconia (Zr) were used. The measured surface parameters were Sa, Sq, Sz, Sdr, Sdq, Sal, Str and water contact angle (WCA). The HGF-1 cell attachment was investigated and the correlations were analyzed using a multiple regression analysis. RESULTS: The HGF-1 cell attachment was greater in the SM, TiN and Zr groups than the other groups and smallest in the CCM group (p = 0.0096). From the multiple regression analysis, the HGF-1 cell attachment was significantly correlated with Sdr, Sdq and WCA. When the R group was excluded, only WCA showed significant correlation with the fibroblast attachment. CONCLUSIONS: Within the limitations of this study, the cell attachment of human gingival fibroblasts was correlated with WCA, developed interfacial area ratio and surface slope. When the surfaces with Sa values of ∼ 0.2 µm or less were concerned, only WCA showed a correlation in a third order manner.