Patterns of Human Milk Oligosaccharides in Mature Milk Are Associated with Certain Gut Microbiota in Infants.

Human milk oligosaccharides (HMOs) are complexes that play a crucial role in shaping the early-life gut microbiota. This study intends to explore whether HMO patterns are associated with the gut microbiota of infants. We included 96 Chinese breastfeeding mother-infant dyads. Breast milk and infant faecal samples were collected and tested. With milk 2'-fucosyllactose, difucosyllactose, and lacto-N-fucopentaose-I as biomarkers, we divided the mothers into secretor and non-secretor groups. HMO patterns were extracted using principal component analysis. The majority (70.7%) of mothers were categorised as secretor and five different HMO patterns were identified. After adjustment, the infants of secretor mothers exhibited a lower relative abundance of Bifidobacterium bifidum (ß = -0.245, 95%CI: -0.465~-0.025). An HMO pattern characterised by high levels of 3-fucosyllactose, lacto-N-fucopentaose-III, and lacto-N-neodifucohexaose-II was positively associated with the relative abundance of Bifidobacterium breve (p = 0.014), while the pattern characterised by lacto-N-neotetraose, 6'-sialyllactose, and sialyllacto-N-tetraose-b was negatively associated with Bifidobacterium breve (p = 0.027). The pattern characterised by high levels of monofucosyl-lacto-N-hexaose-III and monofucosyl-lacto-N-neohexaose was positively associated with Bifidobacterium dentium (p = 0.025) and Bifidobacterium bifidum (p < 0.001), respectively. This study suggests that HMO patterns from mature breast milk were associated with certain gut microbiota of breastfed infants.

Sialic acid-based probiotic intervention in lactating mothers improves the neonatal gut microbiota and immune responses by regulating sialylated milk oligosaccharide synthesis via the gut-breast axis.

Human milk oligosaccharides (HMOs) are vital milk carbohydrates that help promote the microbiota-dependent growth and immunity of infants. Sialic acid (SA) is a crucial component of sialylated milk oligosaccharides (S-MOs); however, the effects of SA supplementation in lactating mothers on S-MO biosynthesis and their breastfed infants are unknown. Probiotic intervention during pregnancy or lactation demonstrates promise for modulating the milk glycobiome. Here, we evaluated whether SA and a probiotic (Pro) mixture could increase S-MO synthesis in lactating mothers and promote the microbiota development of their breastfed neonates. The results showed that SA+Pro intervention modulated the gut microbiota and 6'-SL contents in milk of maternal rats more than the SA intervention, which promoted Lactobacillus reuteri colonization in neonates and immune development. Deficient 6'-SL in the maternal rat milk of St6gal1 knockouts (St6gal1-/-) disturbed intestinal microbial structures in their offspring, thereby impeding immune tolerance development. SA+Pro intervention in lactating St6gal1± rats compromised the allergic responses of neonates by promoting 6'-SL synthesis and the neonatal gut microbiota. Our findings from human mammary epithelial cells (MCF-10A) indicated that the GPR41-PI3K-Akt-PPAR pathway helped regulate 6'-SL synthesis in mammary glands after SA+Pro intervention through the gut - breast axis. We further validated our findings using a human-cohort study, confirming that providing SA+Pro to lactating Chinese mothers increased S-MO contents in their breast milk and promoted gut Bifidobacterium spp. and Lactobacillus spp. colonization in infants, which may help enhance immune responses. Collectively, our findings may help alter the routine supplementation practices of lactating mothers to modulate milk HMOs and promote the development of early-life gut microbiota and immunity.

Isolation of Human Milk Difucosyl Nona- and Decasaccharides by Ultrahigh-Temperature Preparative PGC-HPLC and Identification of Novel Difucosylated Heptaose and Octaose Backbones by Negative-Ion ESI-MSn.

Despite their many important physiological functions, past work on the diverse sequences of human milk oligosaccharides (HMOs) has been focused mainly on the highly abundant HMOs with a relatively low degree of polymerization (DP) due to the lack of efficient methods for separation/purification and high-sensitivity sequencing of large-sized HMOs with DP ≥ 10. Here we established an ultrahigh-temperature preparative HPLC based on a porous graphitized carbon column at up to 145 °C to overcome the anomeric α/ß splitting problem and developed further the negative-ion ESI-CID-MS/MS into multistage MSn using a combined product-ion scanning of singly charged molecular ion and doubly charged fragment ion of the branching Gal and adjacent GlcNAc residues. The separation and sequencing method allows efficient separation of a neutral fraction with DP ≥ 10 into 70 components, among which 17 isomeric difucosylated nona- and decasaccharides were further purified and sequenced. As a result, novel branched difucosyl heptaose and octaose backbones were unambiguously identified in addition to the conventional linear and branched octaose backbones. The novel structures of difucosylated DF-novo-heptaose, DF-novo-LNO I, and DF-novo-LNnO I were corroborated by NMR. The various fucose-containing Lewis epitopes identified on different backbones were confirmed by oligosaccharide microarray analysis.

Carbon dots as a novel photosensitizer for photodynamic therapy of cancer and bacterial infectious diseases: recent advances.

Carbon dots (CDs) are novel carbon-based nanomaterials that have been used as photosensitizer-mediated photodynamic therapy (PDT) in recent years due to their good photosensitizing activity. Photosensitizers (PSs) are main components of PDT that can produce large amounts of reactive oxygen species (ROS) when stimulated by light source, which have the advantages of low drug resistance and high therapeutic efficiency. CDs can generate ROS efficiently under irradiation and therefore have been extensively studied in disease local phototherapy. In tumor therapy, CDs can be used as PSs or PS carriers to participate in PDT and play an extremely important role. In bacterial infectious diseases, CDs exhibit high bactericidal activity as CDs are effective in disrupting bacterial cell membranes leading to bacterial death upon photoactivation. We focus on recent advances in the therapy of cancer and bacteria with CDs, and also briefly summarize the mechanisms and requirements for PSs in PDT of cancer, bacteria and other diseases. We also discuss the role CDs play in combination therapy and the potential for future applications against other pathogens.

Protocol for automated N-glycan sequencing using mass spectrometry and computer-assisted intelligent fragmentation.

Biological functions of glycans are intimately linked to fine details in branches and linkages, which make structural identification extremely challenging. Here, we present a protocol for automated N-glycan sequencing using multi-stage mass spectrometry (MSn). We describe steps for release/purification and derivation of glycans and procedures for MSn scanning. We then detail "glycan intelligent precursor selection" to computationally guide MSn experiments. The protocol can be used for both discrete individual glycans and isomeric glycan mixtures. For complete details on the use and execution of this protocol, please refer to Sun et al.,1 Huang et al.,2 and Huang et al.3.

Multifaceted Materials for Enhanced Osteogenesis and Antimicrobial Properties on Bioplastic Polyetheretherketone Surfaces: A Review.

Implant-associated infections and the increasing number of bone implants loosening and falling off after implantation have become urgent global challenges, hence the need for intelligent alternative solutions to combat implant loosening and falling off. The application of polyetheretherketone (PEEK) in biomedical and medical therapy has aroused great interest, especially because its elastic modulus close to bone provides an effective alternative to titanium implants, thereby preventing the possibility of bone implants loosening and falling off due to the mismatch of elastic modulus. In this Review, we provide a comprehensive overview of recent advances in surface modifications to prevent bone binding deficiency and bacterial infection after implantation of bone implants, starting with inorganics for surface modification, followed by organics that can effectively promote bone integration and antimicrobial action. In addition, surface modifications derived from cells and related products of biological activity have been proposed, and there is increasing evidence of clinical potential. Finally, the advantages and future challenges of surface strategies against medical associated poor osseointegration and infection are discussed, with promising prospects for developing novel osseointegration and antimicrobial PEEK materials.

The application of small intestinal submucosa in tissue regeneration.

The distinctive three-dimensional architecture, biological functionality, minimal immunogenicity, and inherent biodegradability of small intestinal submucosa extracellular matrix materials have attracted considerable interest and found wide-ranging applications in the domain of tissue regeneration engineering. This article presents a comprehensive examination of the structure and role of small intestinal submucosa, delving into diverse preparation techniques and classifications. Additionally, it proposes approaches for evaluating and modifying SIS scaffolds. Moreover, the advancements of SIS in the regeneration of skin, bone, heart valves, blood vessels, bladder, uterus, and urethra are thoroughly explored, accompanied by their respective future prospects. Consequently, this review enhances our understanding of the applications of SIS in tissue and organ repair and keeps researchers up-to-date with the latest research advancements in this area.

Biosafety and chemical solubility studies of multiscale crystal-reinforced lithium disilicate glass-ceramics.

Lithium disilicate (Li2 Si2 O5 ) glass-ceramics are currently a more widely used all-ceramic restorative material due to their good mechanical properties and excellent aesthetic properties. However, they have a series of problems such as high brittleness and low fracture toughness, which has become the main bottleneck restricting its development. Therefore, in order to compensate for these shortcomings, we propose to prepare a reinforced glass-ceramics with better mechanical properties and to test the biosafety and chemical solubility of the material. Li2 Si2 O5 whiskers were synthesized by a one-step hydrothermal method, and multi-scale crystal-enhanced Li2 Si2 O5 glass-ceramics were prepared by reaction sintering. The biosafety of multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics was investigated by in vitro cytotoxicity test, rabbit pyrogen test, mice bone marrow micronucleus test, skin sensitization test, sub-chronic systemic toxicity test, and chronic systemic toxicity test. Additionally, the chemical solubility of multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics was investigated. The test results showed that the material was non-cytotoxic, non-thermogenic, non-mutagenic, non-sensitizing, and non-systemic. The chemical solubility, determined to be 377 ± 245 µg/cm2 , complied with the ISO 6872 standard for the maximum solubility of ceramic materials. Multi-scale crystal-reinforced Li2 Si2 O5 glass-ceramics' biosafety and chemical solubility met current normative criteria, and they can move on to mechanical property measurements (such as flexural strength test, fatigue life test, friction and wear property study, etc.) and bonding property optimization, which shows promise for future clinical applications.

Recent advances in glass-ceramics: Performance and toughening mechanisms in restorative dentistry.

The use of glass-ceramics in the medical field has grown significantly since the 1980s. With excellent aesthetic properties, semi-translucency, outstanding mechanical properties, corrosion resistance, wear resistance and great biocompatibility and workability glass-ceramics is one of the most commonly used materials in restorative dentistry and is widely used in veneers, inlays, onlays, all-ceramic crowns, and implant abutments. This review provides an overview of the research progress of glass-ceramics in restorative dentistry, focusing on the classification, performance requirements, toughening mechanisms and their association with clinical performance, as well as the manufacturing and fabrication of glass-ceramics in restorative dentistry. Finally, the developments and prospects of glass-ceramics in restorative dentistry are summarized and discussed.

Recent advances in composite hydrogels: synthesis, classification, and application in the treatment of bone defects.

Bone defects are often difficult to treat due to their complexity and specificity, and therefore pose a serious threat to human life and health. Currently, the clinical treatment of bone defects is mainly surgical. However, this treatment is often more harmful to patients and there is a potential risk of rejection and infection. Hydrogels have a unique three-dimensional structure that can accommodate a variety of materials, including particles, polymers and small molecules, making them ideal for treating bone defects. Therefore, emerging composite hydrogels are considered one of the most promising candidates for the treatment of bone defects. This review describes the use of different types of composite hydrogel in the treatment of bone defects. We present the basic concepts of hydrogels, different preparation techniques (including chemical and physical crosslinking), and the clinical requirements for hydrogels used to treat bone defects. In addition, a review of numerous promising designs of different types of hydrogel doped with different materials (e.g., nanoparticles, polymers, carbon materials, drugs, and active factors) is also highlighted. Finally, the current challenges and prospects of composite hydrogels for the treatment of bone defects are presented. This review will stimulate research efforts in this field and promote the application of new methods and innovative ideas in the clinical field of composite hydrogels.

Alterations of milk oligosaccharides in mothers with gestational diabetes mellitus impede colonization of beneficial bacteria and development of RORγt+ Treg cell-mediated immune tolerance in neonates.

Gestational diabetes mellitus (GDM) is an increasing public health concern that significantly increases the risk of early childhood allergic diseases. Altered maternal milk glycobiome may strongly affect gut microbiota and enteric-specific Treg cell-mediated development of immune tolerance in GDM infants. In this study, we found that, compared with healthy Chinese mothers, mothers with GDM had significantly lower levels of total and specific human milk oligosaccharides (HMOs) in their colostrum that subsequently increased with extension of lactation. This alteration in HMO profiles significantly delayed colonization of Lactobacillus and Bifidobacterium spp. in their breast-fed infants, resulting in a distinct gut microbial structure and metabolome. Further experiments in GDM mouse models indicated that decreased contents of milk oligosaccharides, mainly 3'-sialyllactose (3'-SL), in GDM maternal mice reduced colonization of bacteria, such as L. reuteri and L. johnsonii, in the neonatal gut, which impeded development of RORγt+ regulatory T (Treg) cell-mediated immune tolerance. Treatment of GDM neonates with 3'-SL, Lactobacillus reuteri (L. reuteri) and L. johnsonii promoted the proliferation of enteric Treg cells and expression of transcription factor RORγt, which may have contributed to compromising ovalbumin (OVA)-induced allergic responses. In vitro experiments showed that 3'-SL, metabolites of L. johnsonii, and lysates of L. reuteri stimulated differentiation of mouse RORγt+ Treg cells through multiple regulatory effects on Toll-like receptor, MAPK, p53, and NOD-like receptor signaling pathways. This study provides new ideas for the development of gut microbiota and immune tolerance in GDM newborns.

Application of chromatography in purification and structural analysis of natural polysaccharides: A review.

Polysaccharides are widely distributed in natural sources from monocytic microorganisms to higher animals, and are found in a variety of biological activities in recent decades. Natural polysaccharides have the characteristics of large molecular weight, diverse composition, and complex structure, so their purification and structural analysis are difficult issues in research. Chromatography as a powerful separation technique, plays an irreplaceable role in the separation and structural analysis of natural polysaccharides, especially in the purification of polysaccharides, the separation of hydrolysates, and the analysis of monosaccharide composition. The separation mechanisms and application of different chromatographic methods in the studies of polysaccharides were summarized in this review. Moreover, the advantages and drawbacks of various chromatography methods were discussed as well.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Human Milk Oligosaccharides Are Associated with Lactation Stage and Lewis Phenotype in a Chinese Population.

BACKGROUND: Human milk oligosaccharides (HMOs) are the third most abundant component of human milk. Various factors may affect the concentration of HMOs, such as the lactation period, Lewis blood type, and the maternal secretor gene status. OBJECTIVES: The purpose of this study is to investigate factors associated with HMO concentrations in Chinese populations. METHODS: A sub-sample of 481 was randomly selected from a large cross-sectional study in China (n = 6481) conducted in eight provinces (Beijing, Heilongjiang, Shanghai, Yunnan, Gansu, Guangdong, Zhejiang, and Shandong) between 2011 and 2013. HMO concentrations were determined by a high-throughput UPLC-MRM method. Various factors were collected through face-to-face interviews. Anthropometric measurement was conducted by trained staff. RESULTS: Median total HMO concentration was 13.6 g/L, 10.7 g/L, and 6.0 g/L for colostrum, transitional milk, and mature milk, respectively. HMO concentration decreased significantly as the lactation period increased (p < 0.0001). There were significant differences of average total HMO concentration between secretor mothers and non-secretor mothers (secretor 11.3 g/L vs. non-secretor 5.8 g/L, p < 0.0001). There were significant differences of average total HMO concentrations among three Lewis blood types (p = 0.003). Comparing with the concentration of total oligosaccharides of Le+ (a-b+), average of total oligosaccharides concentrations increased by 3.9 (Le+ (a+b-), p = 0.004) and 1.1 g/L (Le- (a-b-), p = 0.049). The volume of breast milk expressed and the province the mother came from affected the concentration of total oligosaccharides (all p < 0.0001). Maternal BMI (p = 0.151), age (p = 0.630), prematurity (p = 0.850), mode of delivery (p = 0.486), infants' gender (p = 0.685), maternal education level (p = 0.989), maternal occupation (p = 0.568), maternal allergic history (p = 0.370), maternal anemia (p = 0.625), pregnancy-induced hypertension (p = 0.739), gestational diabetes (p = 0.514), and parity (p = 0.098) were not significantly correlated with the concentration of milk oligosaccharides. The concentrations of 2'-fucosyllactose (2'-FL), lacto-N-neotetraose (LNnT), sialyllacto-N-tetraose c (LSTc), lacto-N-fucopentaose I (LNFP-I), disialylated lacto-N-tetraose (DSLNT), difucosyl-para-lacto-N-neohexaose (DFpLNnH), difucosyl-lacto-N-hexaose (DFLNH[a]), and 3-sialyllactose (3'-SL) showed a gradual downward trend, while the concentration of 3-fucosyllactose (3-FL) showed a gradual upward trend among three lactation stages (p < 0.05). CONCLUSIONS: The concentration of HMOs changes throughout lactation, and it varies between different HMOs. HMO concentrations differed between lactation stage, maternal secretor gene status, Lewis blood type, volume of breast milk expressed, and the province the mother came from. Prematurity, mode of delivery, parity, infants' gender, and maternal characteristics did not affect the HMO concentration. Geographical region may be not associated with HMOs concentration in human milk. There may be a mechanism for co-regulation of the secretion of some of the oligosaccharides such as 2'FL vs. 3FL, 2'FL vs. LNnT, and lacto-N-tetraose (LNT).

GIPS-Mix for Accurate Identification of Isomeric Components in Glycan Mixtures Using Intelligent Group-Opting Strategy.

Accurate identification of glycan structures is highly desirable as they are intimately linked to their different functions. However, glycan samples generally exist as mixtures with multiple isomeric structures, making assignment of individual glycan components very challenging, even with the aid of multistage mass spectrometry (MSn). Here, we present an approach, GIPS-mix, for assignment of isomeric glycans within a mixture using an intelligent group-opting strategy. Our approach enumerates all possible combinations (groupings) of candidate glycans and opts in the best-matched glycan group(s) based on the similarity between the simulated spectra of each glycan group and the acquired experimental spectra of the mixture. In the case that a single group could not be elected, a tie break is performed by additional MSn scanning using intelligently selected precursors. With 11 standard mixtures and 6 human milk oligosaccharide fractions, we demonstrate the application of GIPS-mix in assignment of individual glycans in mixtures with high accuracy and efficiency.

Effect of Breastmilk Microbiota and Sialylated Oligosaccharides on the Colonization of Infant Gut Microbial Community and Fecal Metabolome.

The complex microbiota and sialylated oligosaccharides in breastmilk are important bioactive components that affect the gut microbiota. However, the effect of breastmilk microbiota and sialylated oligosaccharides on the gut microbiota during the neonatal period has been largely overlooked. Here, 16S rRNA gene sequencing and metabolomics analysis were applied to the breastmilk and feces of 69 newborns to clarify the link between breastmilk components and the newborn gut. Results showed that Staphylococcus, Enterococcus, and Bacteroides were commonly shared and positively correlated between breastmilk and the neonatal intestine and they were the main bacteria of breastmilk that interacted with the newborn fecal metabolome. Breastmilk Staphylococcus mainly interacted with amino acids, whereas Bacteroides was involved in the tryptophan, nucleotide, and vitamin metabolism. Breastmilk sialylated oligosaccharides were related to Bacteroides and amino acids of the newborn fecal metabolites. Moreover, Bacteroides was related to the interaction between breastmilk 3'-sialyllactose and newborn fecal metabolites in the mediation effect models. Finally, we pointed out that breastmilk Bacteroides was important in the milk-gut interaction, and it was negatively associated with waist circumference in infants aged 1 year. Our study provides a scientific basis for understanding the role of breastmilk in the development of newborn gut microbiota and metabolome.

Development of high-throughput UPLC-MS/MS using multiple reaction monitoring for quantitation of complex human milk oligosaccharides and application to large population survey of secretor status and Lewis blood group.

Human milk oligosaccharides (HMOs) have attracted increasing attention due to the emerging evidence of their positive roles for infant's health. A high-throughput method for absolute quantitation of the complex HMOs including multiple isomeric structures is important but very challenging, due to the highly divers nature and wide variation in content of HMOs from different individuals. Here we used UPLC-MS-MRM in the negative-ion mode for accurate quantitation of 23 complex HMOs in just 15 min. The selected oligosaccharides are in their native forms and include neutral and sialylated, fucosylated and non-fucosylated, linear and branched, and secretor and Lewis phenotype indicators. The well validated method with good sensitivity, recovery and reproducibility was then applied to a large population quantitative survey of 251 Chinese mothers from five different ethnic groups (Han, Zhuang, Hui, Mongolian and Tibetan) living in different geographical regions for their secretor's status and Lewis phenotypes.

TfOH-catalyzed regioselective N2-alkylation of indazoles with diazo compounds.

Selective alkylation of indazoles is still a highly challenging topic in chemistry and the synthesis of important molecules. Herein, a novel highly selective N2-alkylation of indazoles with diazo compounds is described in the presence of TfOH. Unlike the traditional metal- and base-catalysed version, this protocol highlights the regioselectivity of alkylation of indazoles and a metal-free catalysis system, affording N2-alkylated products in good to excellent yields with high regioselectivity (N2/N1 up to 100/0) and excellent functional group tolerance. Furthermore, a gram scale synthesis was conducted successfully to give rise to the corresponding products. Mechanistic studies through control experiments provide plausible mechanistic proposals.

Biosafety evaluation of Li2Si2O5whisker-reinforced glass-ceramics.

Lithium disilicate (Li2Si2O5) glass-ceramic is a commonly used dental ceramic material. In this study, Li2Si2O5whiskers were prepared by the hydrothermal method, mixed with Li2Si2O5glass powders, and Li2Si2O5whisker-reinforced glass-ceramics were prepared by reaction sintering. The biosafety of the new Li2Si2O5glass-ceramics were evaluated byin vitrocytotoxicity, hemolysis, oral mucosal irritation, acute systemic toxicity, and subacute systemic toxicity (oral route) tests according to ISO 7405/ISO 10993 standards. The cytotoxicity test results showed that the cell growth of the experimental group was good, and the cell number and morphology were not significantly different from those of the blank group (P> 0.05). The toxicity grading for both experimental and blank control groups were 0. The hemolysis rate of the material was 1.25%, which indicated that it did not cause hemolytic reaction. The material was non-irritating to the oral mucosa. In acute systemic toxicity test, animals in the experimental group showed increased body weight, moved freely, with no signs of poisoning. The food utilization rate and relative growth rate (change of the weight) of rats in the subacute systemic toxicity test were not statistically different from those of the control group (P> 0.05). Preliminary evaluation of the biosafety of the Li2Si2O5whisker-reinforced glass-ceramics showed that it met the existing regulatory standards, and further biosafety experiments can be conducted, following which the material may be expected to be applied in clinical practice.

The effectiveness of a three-step sterilization method for Goldmann tonometer prism: A cross-sectional study.

PURPOSE: To propose a three-step sterilization method for Goldmann tonometer prism (GTP) and to analyze the sterilization effects of each step. METHODS: 120 patients (240 eyes) who underwent Goldmann applanation tonometer (GAT) IOP measurement were enrolled in this study. GTPs were used individually for each patient and wiped by swabs soaked with 75% ethyl alcohol, ofloxacin eye drops, and 75% ethyl alcohol for at least 5 s. GTPs were directly pressed onto the surface of agar plates before (W0) and after three-step sterilization (W1, W2, and W3). All the agars were sent to the laboratory in 2 h and incubated at 37°C for 48 h. Subsequently, the growth of microbial species was assessed through visual inspection of the colonies at the inoculation points on the agar surface. RESULTS: Staphylococcus. epidermidis was the most frequently isolated bacterium and was observed in 23.33% of all prisms. Most of the bacteria were eliminated at W3 except Staphylococcus. epidermidis and Kocuria roseus in one case. The isolation rates of Staphylococcus genus and Staphylococcus. epidermidis were significantly decreased (both with P < 0.001). The number of bacteria types isolated from prisms at time point W2 and W3 had a statistically significant difference compared with W1 and W (both with P < 0.05), while W2 and W3 exhibited no significant difference. CONCLUSION: This three-step sterilization method for GTP proved to be effective and safe for repeated use. We recommend using ofloxacin to prevent the transmission of pathogens based on ethyl alcohol, which could also bring some economic benefits.