AcrAB-TolC efflux pump overexpression and tet(A) gene mutation increase tigecycline resistance in Klebsiella pneumoniae.

Tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) is increasing and has emerged as a global public health issue. However, the mechanism of tigecycline resistance remains unclear. The objective of this study was to investigate the potential role of efflux pump system in tigecycline resistance. 29 tigecycline-non-susceptible Klebsiella pneumoniae (TNSKP) strains were collected and their minimum inhibitory concentrations (MIC) were determined by the broth microdilution method. The ramR, acrR, rpsJ, tet(A), and tet(X) were amplified by polymerase chain reaction (PCR). The mRNA expression of different efflux pump genes and regulator genes were analyzed by real-time PCR. Additionally, KP14 was selected for genome sequencing. KP14 genes without acrB, oqxB, and TetA were modified using suicide plasmids and MIC of tigecycline of KP14 with target genes knocked out was investigated. It was found that MIC of tigecycline of 20 out of the 29 TNSKP strains decreased by over four folds once combined with phenyl-arginine-ß-naphthylamide dihydrochloride (PaßN). Most strains exhibited upregulation of AcrAB and oqxAB efflux pumps. The strains with acrB, oqxB, and tetA genes knocked out were constructed, wherein the MIC of tigecycline of KP14∆acrB and KP14∆tetA was observed to be 2 µg/mL (decreased by 16 folds), the MIC of tigecycline of KP14ΔacrBΔTetA was 0.25 µg/mL (decreased by 128 folds), but the MIC of tigecycline of KP14∆oqxB remained unchanged at 32 µg/mL. The majority of TNSKP strains demonstrated increased expression of AcrAB-TolC and oqxAB, while certain strains showed mutations in other genes associated with tigecycline resistance. In KP14, both overexpression of AcrAB-TolC and tet(A) gene mutation contributed to the mechanism of tigecycline resistance.

A Method for Detecting Five Carbapenemases in Bacteria Based on CRISPR-Cas12a Multiple RPA Rapid Detection Technology.

Introduction: As the last line of defense for clinical treatment, Carbapenem antibiotics are increasingly challenged by multi-drug resistant bacteria containing carbapenemases. The rapid spread of these multidrug-resistant bacteria is the greatest threat to severe global health problems. Methods: To solve the problem of rapid transmission of this multidrug-resistant bacteria, we have developed a rapid detection technology using CRPSPR-Cas12a gene editing based on multiple Recombinase polymerase amplification. This technical method can directly isolate the genes of carbapenemase-containing bacteria from samples, with a relatively short detection time of 30 minutes. The instrument used for the detection is relatively inexpensive. Only a water bath can complete the entire experiment of Recombinase polymerase amplification and trans cleavage. This reaction requires no lid during the entire process while reducing a large amount of aerosol pollution. Results: The detection sensitivity of this method is 1.5 CFU/mL, and the specificity is 100%. Discussion: This multi-scene detection method is suitable for screening populations in wild low-resource environments and large-scale indoor crowds. It can be widely used in hospital infection control and prevention and to provide theoretical insights for clinical diagnosis and treatment.

Three-dimensional finite element analysis of the biomechanical properties of different material implants for replacing missing teeth.

The purpose of this study was to analyze the biomechanical properties of implants made of different materials to replace missing teeth by using three-dimensional finite element analysis and provide a theoretic basis for clinical application. CBCT data was imported into the Mimics and 3-Matic to construct the three-dimensional finite element model of a missing tooth restored by an implant. Then, the model was imported into the Marc Mentat. Based on the variations of the implant materials (titanium, titanium-zirconia, zirconia and poly (ether-ether-ketone) (PEEK)) and bone densities (high and low), a total of eight models were created. An axial load of 150 N was applied to the crown of the implant to simulate the actual occlusal situation. Both the maximum values of stresses in the cortical bone and implant were observed in the Zr-low model. The maximum displacements of the implants were also within the normal range except for the PEEK models. The cancellous bone strains were mainly distributed in the apical area of the implant, and the maximum value (3225 µstrain) was found in PEEK-low model. Under the premise of the same implant material, the relevant data from various indices in low-density bone models were larger than that in high-density bone models. From the biomechanical point of view, zirconia, titanium and titanium-zirconia were all acceptable implant materials for replacing missing teeth and possessed excellent mechanical properties, while the application of PEEK material needs to be further optimized and modified.

Rifampicin-Loaded Polyelectrolyte Complex Eliminates Intracellular Bacteria through Thiol-Mediated Cellular Uptake and Oxidative Stress Enhancement.

The poor accumulation of antibiotics in the cytoplasm leads to the poor eradication of intracellular bacteria. Herein, a polyelectrolyte complex (PECs@Rif) allowing direct cytosolic delivery of rifampicin (Rif) was developed for the treatment of intracellular infections by complexation of poly(α-lipoic acid) (pLA) and oligosaccharide (COS) in water and loading Rif. Due to the thiol-mediated cellular uptake, PECs@Rif delivered 3.9 times higher Rif into the cytoplasm than that of the free Rif during 8 h of incubation. After entering cells, PECs@Rif released Rif by dissociating pLA into dihydrolipoic acid (DHLA) in the presence of intracellular thioredoxin reductase (TrxR). Notably, DHLA could reduce endogenous Fe(III) to Fe(II) and provide a catalyst for the Fenton reaction to produce a large amount of reactive oxygen species (ROS), which would assist Rif in eradicating intracellular bacteria. In vitro assay showed that PECs@Rif reduced almost 2.8 orders of magnitude of intracellular bacteria, much higher than 0.7 orders of magnitude of free Rif. The bacteremia-bearing mouse models showed that PECs@Rif reduced bacterial levels in the liver, spleen, and kidney by 2.2, 3.7, and 2.3 orders of magnitude, respectively, much higher than free Rif in corresponding tissues. The direct cytosolic delivery in a thiol-mediated manner and enhanced oxidative stress proposed a feasible strategy for treating intracellular bacteria infection.

A PCR-Reverse Dot Blot Hybridization Based Microfluidics Detection System for the Rapid Identification of 13 Fungal Pathogens Directly After Blood Cultures Over a Period of Time.

Introduction: It is time-consuming to identify fungal pathogens from positive blood cultures using the standard culture-based method. And delayed diagnosis of bloodstream infection leads to significantly increased mortality. Methods: We developed a PCR-reverse dot blot hybridization combined with microfluidic chip techniques to rapidly identify 13 fungal pathogens within 3-4 h using the sample of blood cultured over a period of time. Results: We performed clinical validation using 43 blood culture-positive samples with a sensitivity of 96.7%, a specificity of 100%, and a concordance rate of 97.7%. Samples with different culture durations were evaluated using our approach, showing a detection rate of 85.2% at 16 h and 96.3% at 24 h; the platform could reach a detection limit of 103cfu/mL for the Candida spp. and 103 copies/mL for Aspergillus spp. Discussion: The detection rate of the platform is much higher than the positive rates of concurrent blood cultures. This method bears substantial clinical application potential as it incorporates the microfluidic platform with low reagent consumption, automation, and low cost (about 10 dollars).

Comprehensive Study of Untargeted Metabolomics and 16S rRNA Reveals the Mechanism of Fecal Microbiota Transplantation in Improving a Mouse Model of T2D.

Background: Fecal microbiota transplantation (FMT) has emerged as a new therapy targeting gastrointestinal microbiota for the treatment of a growing number of diseases in recent years. Previous studies have suggested that FMT may be a potential therapy for type 2 diabetes (T2D), but the underlying mechanism remains unclear. Therefore, in the present study, we aimed to investigate the role of FMT in T2D and its underlying mechanisms. Methods: To induce T2D, mice were fed a high-fat diet and injected with low-dose streptozotocin (STZ) for four weeks. The mice were then randomly divided into four groups: control group (n = 7), T2D group (n = 7), metformin (MET)-treated group (n = 7), and FMT group (n = 7). The MET group was orally administered 0.2 g/kg MET, the FMT group was orally administered 0.3 mL of bacterial solution, and the other two groups were orally administered the same volume of saline for four weeks. Serum and fecal samples were collected for non-targeted metabolomics, biochemical indicators, and 16S rRNA sequencing, respectively. Results: Our results demonstrated that FMT had a curative effect on T2D by ameliorating hyperlipidemia and hyperglycemia. Using 16S rRNA sequencing and serum untargeted metabolomic analysis, we found that FMT could restore the disorders of gastrointestinal microbiota in T2D mice. Moreover, corticosterone, progesterone, L-urobilin, and other molecules were identified as biomarkers after FMT treatment. Our bioinformatics analysis suggested that steroid hormone biosynthesis, arginine, proline metabolism, and unsaturated fatty acid biosynthesis could be potential regulatory mechanisms of FMT. Conclusion: In summary, our study provides comprehensive evidence for the role of FMT in the treatment of T2D. FMT has the potential to become a promising strategy for the treatment of metabolic disorders, T2D, and diabetes-related complications.

Rapid visualization of Clostridioides difficile toxins A and B by multiplex RPA combined with CRISPR-Cas12a.

Purpose: Clostridioides difficile (C. difficile) infection is the most common cause of nosocomial infection, which is a severe challenge in modern medical care. Currently, many laboratory diagnostic methods for C. difficile are available, such as PCR, culture-based tests, and antigen-based tests. However, these methods are not suitable for rapid point-of-care testing (POCT). Therefore, it is of great significance to develop a rapid, sensitive, and cost-effective method to detect C. difficile toxin genes. Methods: Recently, the development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has emerged as a promising tool for rapid POCT. In this study, we developed a rapid and specific detection platform for dual C. difficile toxins by combining recombinase polymerase amplification (RPA) and CRISPR/Cas12a. Results: The platform includes multiplex RPA-cas12a-fluorescence assay and multiplex RPA-cas12a-LFS (Lateral flow strip) assay, through which the detection limit for tcdA and tcdB was 10 copies/µL and 1 copy/µL, respectively. The results can be more clearly distinguished using a violet flashlight, which realized a portable visual readout. The platform can be tested within 50 min. Furthermore, our method did not cross-react with other pathogens that cause intestinal diarrhea. The results of 10 clinical samples using our method was 100% consistent with those from real-time PCR detection. Conclusion: In conclusion, the CRISPR-based double toxin gene detection platform for C. difficile is an effective, specific, and sensitive detection method, which can be used as a powerful on-site detection tool for POCT in the future.

The role of HBV cccDNA in occult hepatitis B virus infection.

Occult hepatitis B virus (HBV) infection (OBI) refers to the presence of replication-competent HBV DNA in the liver, with or without HBV DNA in the blood, in individuals who tested negative for HBV surface antigen (HBsAg). In this peculiar phase of HBV infection, the covalently closed circular DNA (cccDNA) is in a low state of replication. Several advances have been made toward clarifying the mechanisms involved in such a suppression of viral activity, which seems to be mainly related to the host's immune control and epigenetic factors. Although the underlying mechanisms describing the genesis of OBI are not completely known, the presence of viral cccDNA, which remains in a low state of replication due to the host's strong immune suppression of HBV replication and gene expression, appears to be the causative factor. Through this review, we have provided an updated account on the role of HBV cccDNA in regulating OBI. We have comprehensively described the HBV cell cycle, cccDNA kinetics, current regulatory mechanisms, and the therapeutic methods of cccDNA in OBI-related diseases.

Establishment and Methodological Evaluation of a Method for Rapid Detection of Helicobacter pylori and Virulence Genes Based on CRISPR-Cas12a.

Introduction: More than half of the world's people are infected or have been infected with Helicobacter pylori. This infection is related to many diseases, with its pathogenicity related to virulence factors. Therefore, the rapid diagnosis of H. pylori and genotyping of virulence genes play an extremely important role in the clinical treatment and control of transmission. Methods: To this end, we developed a molecular detection method based on RPA- CRISPR-Cas12a technology for the specific genes 16S rDNA gene, cytotoxin associated gene A(cagA), and vacuolating cytotoxin A (vacA) of H. pylori. Results: The results of which were displayed by lateral flow strips. Macroscopic observation takes only about 25 minutes and the sensitivity is 2ng/microliter. Discussion: The method is simple, convenient to operate and has low costs, and can therefore be applied widely to the detection and typing of H. pylori in various environments such as primary hospitals, community clinics, outdoors, and large medical institutions.

Durability of Antibody Response Against Hepatitis B Virus for a Decreased Crowd: A Retrospective Polycentric Cohort Study from a 10-Year Follow-Up Clinical Study.

Aim: Hepatitis B surface antibody (HBsAb) plays an important role in the prevention of hepatitis B virus (HBV) infection, especially in immunocompromised individuals and in those infected with HBV.HBsAb levels often fluctuate and decrease.This study aimed to determine the regularity of HBsAb persistence among different populations. Moreover, the risk factors and the optimal cutoff value were determined to predict a decreasing population in HBsAb level. Methods: The study involved 182 participants, including 76 patients with a 25% decrease in HBsAb levels and 106 patients with an HBsAb decrease rate of >50%. Both hepatitis B core antibody negative and positive patients were included.These patients were followed up for 10 years. The follow-up demographic and laboratory data were recorded and compared among the groups. Fluctuations in HBsAb data and HBsAb persistent immunity were evaluated. The independent factors and the optimal cutoff value were recorded. Results: The first HBsAb median of Group 4 was lower than that of the other groups, and its median was 50.8 mlU/mL. In addition, the persistent immunity of the case groups was shorter than that of the control groups (p < 0.05). Furthermore, previous HBV history, use of antiviral drugs, and low levels of first HBsAb were independent risk factors in people with obviously decreased antibody levels. Also, when the optimum cutoff value on the receiver operating characteristic curve of the HBsAb difference value was taken as 8.53 mIU/mL, its sensitivity and specificity were 94% and 70% between the control and case groups, respectively. Conclusion: To maintain optimal immunity against HBV infection, patients with a previous HBV history, those taking antiviral drugs, and/or those with low levels of HBsAb should be reimmunized with the hepatitis B vaccine in a timely manner.

In vitro and in vivo Antimicrobial Activities of Ceftazidime/Avibactam Alone or in Combination with Aztreonam Against Carbapenem-Resistant Enterobacterales.

Introduction: To examine the in vitro and in vivo antimicrobial activities of ceftazidime/avibactam (CZA) alone or in combination with aztreonam (ATM) against KPC-, NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains. Methods: A total of 67 clinical non-repetitive carbapenem-resistant Enterobacterales (CRE) strains were selected for the microdilution broth method that was performed to analyze the minimal inhibitory concentration (MIC) and the combination antimicrobial susceptibility test using checkerboard titration method. The fractional inhibitory concentration (FIC) was calculated to determine the antimicrobial effect. The time-kill assays and the mouse infection model were used to study the bactericidal effect and therapeutic effect of CZA alone or in combination with ATM. Results: The CZA minimal inhibitory concentration (MIC) values of CZA revealed that 29 KPC-producing strains and 1 OXA-producing strain were ≤4µg/mL. The CZA MIC values of 37 metal-ß-lactamase (MBLs)-producing strains such as NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains were ≥128µg/mL, after combining with ATM, the FIC values were all below 0.51. The time-kill assays revealed that CZA at various concentrations of 2, 4 and 8 MIC showed significant bactericidal efficiency to the KPC-producing strains. For NDM-, IMP-producing strains, no colony growth was detected after 8 hours of incubation with CZA in combination with ATM. Six percent of the mice in the treatment group and 58% of the mice in the infection group died within 3 days. Conclusion: Our in vitro results showed that CZA had a good antimicrobial effect on the KPC-producing and OXA-producing strains. CZA combined with ATM showed synergistic bacteriostatic or bactericidal activity against NDM-, IMP-, KPC+IMP-, KPC+NDM-producing strains. The combination of CZA and ATM reduced mortality and prolonged lifespan of mice infected with NDM-, IMP-, KPC+IMP-, and KPC+NDM-producing strains, which provides fundamental knowledge for improving treatment strategies and initializing clinical trials.

Rapid Identification and Drug Sensitivity Test to Urinary Tract Infection Pathogens by DOT-MGA.

Aim: To reduce the inspection time for urinary tract pathogens and provide a rapid and effective therapeutic plan for clinical anti-infection treatment, this study developed a rapid identification (ID) and antimicrobial sensitivity test (AST) method by DOT-MGA. Methods: We grouped midstream urine samples with single bacteria according to the number of bacteria (≤5/5-15/≥ 15) under per oil microscope after Gram staining. Then we adopted differential centrifugation to process the grouped samples to collect precipitate. MALDI-TOF MS was performed using precipitate directly or after short-term culture. If succeed, we resuspended the precipitate into droplets with or without antibiotics at a MALDI target. Four hours later, mass spectrometer (MS) was used to identify the culture on the target and to analyse AST. Results: Samples (count ≥ 15), which precipitate can be directly identified by MS; otherwise, the precipitate need a short-term cultured for 3-6 h before ID. The consistency of the ID results between conventional culture and the precipitate is 100%. Compared with broth microdilution method, DOT-MGA for predicting AST had a high consistency. EA and CA for IPM, LEV, CAZ, NIT, and FOT were 100%/100%, 98%/90%, 98%/92%, 100%/90%, 98%/94%, respectively. No VME was observed in all tests. Besides, MIC50 for the five antibiotics by DOT-MGA and broth microdilution method were ≤1/≤0.5,>2/2,≤4/≤2,≤32/≤16,≤64/≤32 and MIC90 were ≤1/≤0.5, >2/>4, 16/16, 128/128, 128/64. Conclusion: This study can shorten the ID time (minimum 0.5h) and AST (minimum 4h) of the main pathogens of urinary tract infection to 5-10 hours, which greatly reduce the inspection time and provide substantial help for the rapid diagnosis and treatment of patients with urinary tract infection.

A New Method Based on LAMP-CRISPR-Cas12a-Lateral Flow Immunochromatographic Strip for Detection.

INTRODUCTION: Carbapenemase-mediated antimicrobial resistance is currently a hot spot of global concern. Carbapenem-resistant organisms are highly prevalent in hospitals associated with difficult-to-treat infections, resulting in poor clinical outcome due to limited treatment options. It is urgently needed to have a rapid, efficient, and convenient molecular assay for identifying such resistant strains. METHODS: For this end, we developed a new laboratory assay targeting Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-ß-lactamase (NDM) based on loop-mediated isothermal amplification, CRISPR-Cas12a, and lateral flow immunochromatographic strip (CRISPR-Cas-LAMP-lateral flow strip). The method was designed to use a guide RNA (gRNA) to recognize the target DNA and guide Cas12a to cleave the target DNA, and simultaneously cleave any single-stranded DNA within the cleavage reaction system. RESULTS: The cleavage products are visible to the naked eye on the lateral flow strip. This method is highly sensitive in direct detection of bacteria in samples containing at least 3×105 CFU/mL without the need for bacterial culture. DISCUSSION: It provides shorter turnaround time and higher specificity than the conventional bacterial culture and susceptibility testing method. This new assay is applicable for extensive use in hospital infection control, as well as identification and treatment of resistant strains due to simple operation and inexpensive apparatuses.

Direct-on-Target Microdroplet Growth Assay for Detection of Bacterial Resistance in Positive Blood Cultures.

INTRODUCTION: The recently developed DOT-MGA (direct-on-target microdroplet growth assay) has shown the desirability of direct application of this approach in positive blood cultures and its good performance in detection. This study selected 44 Enterobacteriaceae strains and implemented a DOT-MGA assay on blood cultures to detect their resistance to seven antibiotics. The results of DOT-MGA were compared with the other two antimicrobial susceptibility testing (AST) methods to analyze the detection performance of DOT-MGA. METHODS: We adopted the differential centrifugation to process positive blood-culture (BC). Processed BC broth was directly used for rapid AST using DOT-MGA. Droplets of 6 µL with and without antibiotics at the EUCAST breakpoint concentration were spotted in triplicates onto the surface of a MALDI target. The plates were incubated in a wet box for 4 h before the broth was removed with filter paper. Bruker Biotyper software was used to analyze the test results compared with standard database, and the scores were used to quantify and determine the results. RESULTS: DOT-MGA results were compared with the direct-from-BC disk-diffusion method and results were reported by broth microdilution method, respectively. The comparison demonstrated a 100% growth efficiency in DOT-MGA, a 100% classification consistency for ampicillin, ceftriaxone, and gentamicin, and >93% classification consistency for tobramycin, aztreonam, trimethoprim-sulfamethoxazole (TMP-SMX), and ceftazidime. DISCUSSION: These study results have shown that DOT-MGA is suitable for directly identifying bacterial resistance to positive blood cultures in clinical microbiology laboratories. Furthermore, it is conducive for early diagnosis and treatment of patients with bloodstream infection due to its convenience, time efficiency, and good performance in identifying multiple antibiotic-insensitive bacteria.

[Effect and postoperative reactions of acupoint catgut embedding for prediabetes].

OBJECTIVE: To observe the effect and safety of acupoint catgut embedding for prediabetes. METHODS: Eighty-six patients with prediabetes were randomly assigned into an observation group and a control group, 43 cases in each one. Diabetes health education was applied in the two groups. Catgut embedding was used at bilateral Pishu (BL 20), Weiwanxiashu (EX-B 3), Ganshu (BL 18), Shenshu (BL 23), and Zusanli (ST 36) in the observation group, once every 15 days for 6 times. The type and number of postoperative reactions of the last time had been recorded since the second embedding, and the situation of the last postoperative reactions was observed 15 days after treatment. Total cholesterol (TC), triglyceride (TG), fasting plasma glucose (FPG), 2 hours postprandial blood glucose (2hPG), glycosylated hemoglobin (HbA1c), empty stomach insulin (Ins) and TCM syndrome score were tested before and after treatment as well as 6 months after treatment. The clinical efficacy was evaluated. RESULTS: After treatment, the levels of TC, TG, FPG, 2hPG, HbA1c of the observation group were lower than those before treatment and those of the control group (all P<0.05). The Ins changed little after treatment and was not significantly different from that of the control group (both P>0.05). All the above indices had no significant difference at follow-up compared with those before treatment (all P>0.05). After treatment, the TCM syndrome score in the observation group was lower than that before treatment and that in the control group (both P<0.05). The TCM syndrome score at follow-up was better than that before treatment in the observation group (P<0.05). The total effective rate of the observation group was 76.7% (33/43) after treatment, which was superior to 30.2% (13/43) of the control group (P<0.05). The rates at follow-up was 37.2% (16/43) in the observation group and was 25.6% (11/43) in the control group (P>0.05). 18 postoperative reactions occurred 168 times totally after embedding, which could be divided into 5 categories, including 75 times (44.6%) of dietary inhibition, 41 times (24.4%) of abnormal feeling, 23 times (13.7%) of local discomfort, 21 times (12.5%) of general malaise, and 8 times (4.8%) of other symptoms. The postoperative peak responses were after the second and third operations. CONCLUSIONS: 3-month catgut embedding can improve some of the indices of prediabetes, but long-term healthy living habits need to be kept. There exist various postoperative reactions after embedding without blocking the treatment.