Antibiotic resistance and virulence genes in Helicobacter pylori strains isolated from children in Shanghai, China (2019-2022).

BACKGROUND: The increasing prevalence of antibiotic-resistant Helicobacter pylori strains poses a significant threat to children's health. This study investigated antibiotic resistance rates in Helicobacter pylori strains isolated from children in Shanghai and analyzed the presence of virulence genes in these strains. METHODS: We obtained 201 Helicobacter pylori strains from pediatric patients with upper gastrointestinal symptoms who underwent gastrointestinal endoscopy between 2019 and 2022. Subsequently, we performed antibiotic susceptibility tests and virulence gene PCR assays on these strains. RESULTS: Helicobacter pylori resistance rates of 45.8%, 15.4%, 1.0%, and 2.5% were detected for metronidazole, clarithromycin, amoxicillin, and levofloxacin, respectively. Among all isolates, 64.7% exhibited resistance to at least one antibiotic. Resistance to metronidazole and clarithromycin increased from 2019 to 2022. The predominant vacA gene subtype was vacA s1a/m2. The prevalence of vacA m2 and dupA exhibited an upward trend, while oipA presented a decreasing trend from 2019 to 2022. The prevalence of dupA was significantly higher in gastritis than peptic ulcer disease, and in non-treatment compared to treatment groups. CONCLUSIONS: Helicobacter pylori antibiotic resistance remains high in children and has risen in recent years. Therefore, the increasing use of metronidazole and clarithromycin requires increased monitoring in children. No association was observed between antibiotic resistance and virulence gene phenotypes.

[Prognostic analysis of childhood T-lymphoblastic lymphoma treated with leukemia regimen]. / 儿童Tæ·‹å·´æ¯ç»†èƒžæ·‹å·´ç˜¤é‡‡ç”¨ç™½è¡€ç— æ–¹æ¡ˆæ²»ç–—çš„é¢„åŽåˆ†æž.

OBJECTIVES: To investigate the prognosis of childhood T-lymphoblastic lymphoma (T-LBL) treated with acute lymphoblastic leukemia (ALL) regimen and related influencing factors. METHODS: A retrospective analysis was performed for the prognostic characteristics of 29 children with T-LBL who were treated with ALL regimen (ALL-2009 or CCCG-ALL-2015 regimen) from May 2010 to May 2022. RESULTS: The 29 children with T-LBL had a 5-year overall survival (OS) rate of 84%±7% and an event-free survival (EFS) rate of 81%±8%. The children with B systemic symptoms (unexplained fever >38°C for more than 3 days; night sweats; weight loss >10% within 6 months) at initial diagnosis had a lower 5-year EFS rate compared to the children without B symptoms (P<0.05). The children with platelet count >400×109/L and involvement of both mediastinum and lymph nodes at initial diagnosis had lower 5-year OS rates (P<0.05). There were no significant differences in 5-year OS and EFS rates between the children treated with CCCG-ALL-2015 regimen and those treated with ALL-2009 regimen (P>0.05). Compared with the ALL-2009 regimen, the CCCG-ALL-2015 regimen reduced the frequency of high-dose methotrexate chemotherapy and the incidence rate of severe infections (P<0.05). CONCLUSIONS: The ALL regimen is safe and effective in children with T-LBL. Children with B systemic symptoms, platelet count >400×109/L, and involvement of both mediastinum and lymph nodes at initial diagnosis tend to have a poor prognosis. Reduction in the frequency of high-dose methotrexate chemotherapy can reduce the incidence rate of severe infections, but it does not affect prognosis.

Protectin Conjugates in Tissue Regeneration 1 Inhibits Macrophage Pyroptosis by Restricting NLRP3 Inflammasome Assembly to Mitigate Sepsis via the cAMP-PKA Pathway.

Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel anti-inflammatory and proresolving lipid mediator biosynthesized from docosahexaenoic acid. Excessive activation of NLR family pyrin domain containing 3 (NLRP3) inflammasome and consequent pyroptosis are involved in diverse inflammatory diseases. However, how PCTR1 affects NLRP3 inflammasome activation and pyroptosis are still unclear. Here, we demonstrated that PCTR1 inhibited NLRP3 inflammasome activation and pyroptosis. These results show that PCTR1 dose-dependently inhibited gasdermin D cleavage in lipopolysaccharide (LPS)-primed murine primary macrophages upon nigericin stimulation. Additionally, PCTR1 treatment after LPS priming inhibited caspase-1 activation and subsequent mature interleukin-1ß release independent of the nuclear factor-kappa B pathway. PCTR1 exerted its inhibitory effects by blocking NLRP3-apoptosis-associated speck-like protein containing a CARD (ASC) interaction and ASC oligomerization, thereby restricting NLRP3 inflammasome assembly. However, the inhibitory effect of PCTR1 could be reversed by KH7 and H89, which are the inhibitors of the cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) signaling pathway. Moreover, PCTR1 treatment alleviated lung tissue damage and improved mouse survival in LPS-induced sepsis. Our study unveils the molecular mechanism of negative regulation of NLRP3 inflammasome activation and pyroptosis by a novel lipid mediator and suggests that PCTR1 may serve as a potential treatment option for NLRP3-inflammasome driven diseases.

Molecular Evolution and Increasing Macrolide Resistance of Bordetella pertussis, Shanghai, China, 2016-2022.

Resurgence and spread of macrolide-resistant Bordetella pertussis (MRBP) threaten global public health. We collected 283 B. pertussis isolates during 2016-2022 in Shanghai, China, and conducted 23S rRNA gene A2047G mutation detection, multilocus variable-number tandem-repeat analysis, and virulence genotyping analysis. We performed whole-genome sequencing on representative strains. We detected pertussis primarily in infants (0-1 years of age) before 2020 and older children (>5-10 years of age) after 2020. The major genotypes were ptxP1/prn1/fhaB3/ptxA1/ptxC1/fim2-1/fim3-1 (48.7%) and ptxP3/prn2/fhaB1/ptxA1/ptxC2/fim2-1/fim3-1 (47.7%). MRBP increased remarkably from 2016 (36.4%) to 2022 (97.2%). All MRBPs before 2020 harbored ptxP1, and 51.4% belonged to multilocus variable-number tandem-repeat analysis type (MT) 195, whereas ptxP3-MRBP increased from 0% before 2020 to 66.7% after 2020, and all belonged to MT28. MT28 ptxP3-MRBP emerged only after 2020 and replaced the resident MT195 ptxP1-MRBP, revealing that 2020 was a watershed in the transformation of MRBP.

Clinical and molecular characteristics of methicillin-resistant Staphylococcus aureus in bone and joint infection among children.

OBJECTIVE: To investigate the characteristics of Methicillin-Resistant Staphylococcus aureus (MRSA) in bone and joint infection (BJI) among children. METHODS: A total of 338 patients diagnosed with BJI from 2013 to 2022 in Children's Hospital of Fudan University were enrolled. Demographic information, microbiology culture results and laboratory findings, including white blood counts (WBC), C-reactive protein (CRP), procalcitonin (PCT), interleukin-6 (IL-6), and erythrocyte sedimentation rate (ESR) were collected and analyzed. MRSA was confirmed by antimicrobial susceptibility testing. Other MRSA-caused infections were randomly selected for comparison. Twenty-three virulence and antimicrobial resistance (AMR) genes were screened for MRSA strains. Multilocus sequence typing (MLST) and Staphylococcal protein A (spa) typing were performed using PCR amplification and sequencing. RESULTS: Of the identified pathogens in BJI, MRSA accounted for 21.0% (47/224). Patients with BJI had high levels of initial CRP, white blood cell count (WBC) and IL-6. ST59 (43.9%) and t437 (37.6%) were the main MRSA subtypes isolated from the children. The major genotypes in BJI were ST59-t437 (29.8%) and ST22-t309 (14.9%), with high carriage of hemolysins including hla (94.4-100%), hlb (66.2-93.3%), and hld (100%). Notably, Panton-Valentine leukocidin (pvl) had a high prevalence (53.3%) in ST22-t309-MRSA. Other virulence genes including tst, seg and sei were more commonly detected in ST22-t309-MRSA (40.0-46.7%) than in ST59-t437-MRSA (4.2-9.9%). High-carriage AMR genes in MRSA included aph(3')/III (66.7-80%), ermB (57.5-73.3%) and ermC (66.7-78.9%). MRSA presented high-resistance to erythromycin (52.0-100%) and clindamycin (48.0-92.5%), different genotypes displayed variation in their susceptibilities to antibiotics. CONCLUSIONS: The major MRSA genotype in BJI was ST59-t437, followed by ST22-t309, with a higher prevalence of the pvl gene. Continuous surveillance of pvl-positive ST22-t309-MRSA in pediatric BJI infections is thus required.

An Emerging Fluorescent Carbon Nanobead Label Probe for Lateral Flow Assays and Highly Sensitive Screening of Foodborne Toxins and Pathogenic Bacteria.

By virtue of the fascinating merits of low cost, rapid screening, and on-site detection, fluorescence lateral flow assays (FLFAs) have attracted considerable attention. Their detection limits are closely associated with the label probes used. The development of high-performance and robust phosphors remains a great challenge. Herein, we presented a new label probe, i.e., fluorescent carbon nanobeads (FCNBs), for FLFAs. Monodispersive, water-soluble, and highly emissive FCNBs were facilely prepared via a hydrothermal carbonization manner. Their abundant amino groups were beneficial for versatile surface functionalization. After being modified by biomolecules, the fabricated FCNB reporter probes were adopted for the construction of lateral flow test strips toward representative foodborne toxins, i.e., aflatoxin B1 (AFB1), and pathogenic bacteria, i.e., Staphylococcus aureus (S. aureus), respectively. The detection limits (0.01 ng/mL for AFB1 and 102 cfu/mL for S. aureus) were about 1 or 2 orders of magnitude lower than most reported methods. Furthermore, the proposed test strips were successfully applied for the quantitative, accurate, and rapid screening of AFB1 and S. aureus in food samples. This work provided a promising label probe for FLFAs and would open the opportunity to exploit a sensing platform by modifying different ligands onto the FCNBs.

DNA Nanoribbon for Efficient Anti-miRNA Peptide Nucleic Acid Delivery and Synergistic Enhancement of Cancer Cell Apoptosis.

Antisense peptide nucleic acid (asPNA), an effective antisense drug, has been employed as a gene therapy agent and a useful tool in molecular biology. Gaining control over the delivery of asPNA to target tissues has been a major hindrance to its wide application in clinical practice. A simple and efficient DNA nanoribbon (DNR)-based drug delivery process has been designed in this study that releases the asPNA agent to inhibit oncogenic microRNAs (miRNAs). Furthermore, we demonstrated how the AS1411 aptamer that binds nucleolin on the cell membranes works as a control mechanism capable of identifying target cancer cells and enhancing the enrichment capacity of DNR. With the biodegradability of DNR, we can efficiently initiate the release of asPNA into the cytoplasm, particularly targeting the intended miR-21 and synergistically increasing programmed cell death 4 (PDCD4) expression to enhance cell apoptosis. We assume that this well-defined delivery mechanism will aid in designing antisense site-specific treatments for various diseases, including cancer.

Peroxidase-Mimetic Copper-Doped Carbon-Dots for Oxidative Stress-Mediated Broad-Spectrum and Efficient Antibacterial Activity.

Carbon dots (CDs) have recently emerged as antibacterial agents and have attracted considerable attention owing to their fascinating merits of small size, facile fabrication, and surface functionalization. Most of them are involved in external light activation or hybridization with other functional nanomaterials. Herein, we present peroxidase-like Cu-doped CDs (Cu-CDs) for in vitro antibacterial applications. The unique peroxidase-mimicking property of the Cu-CDs was demonstrated by tetramethylbenzidine chromogenic assay, electron paramagnetic resonance spectra, and hydroxy radical probe. Escherichia coli and Staphylococcus aureus were chosen as representative gram-negative/positive models against which Cu-CDs exhibited superior antimicrobial activity even at a dosage down to 5 µg/mL. A possible mechanism of action was that the Cu-CDs triggered a catalytic redox reaction of endogenous H2 O2 and glutathione depletion in the bacteria cells, with subsequent oxidative stress and membrane disruption. This work provides a new strategy for the design of microenvironment-responsive antimicrobial nano-agents.

High responsivity graphene-InGaAs near-infrared photodetector realized by hole trapping and its response saturation mechanism.

Graphene is an ideal material for wide spectrum detector owing to its special band structure, but its low light absorption and fast composite of photogenerated carriers lead to a weak response performance. In this paper, we designed a unique photoconductive graphene-InGaAs photodetector. The built-in electric field was formed between graphene and InGaAs, which can prolong the lifetime of photogenerated carriers and improve the response of devices by confining the holes. Compared with graphene-Si structure, a higher built-in electric field and reach to 0.54 eV is formed. It enables the device to achieve a responsivity of 60 AW-1 and a photoconductive gain of 79.4 at 792 nm. In the 1550 nm communication band, the responsivity of the device is also greater than 10 AW-1 and response speed is less than 2 ms. Meanwhile, the saturation phenomenon of light response was also found in this photoconductive graphene heterojunction detector during the experiment, we have explained the phenomenon by the capacitance theory of the built-in electric field, and the maximum optical responsivity of the detector is calculated theoretically, which is in good agreement with the measurement result.

Protectin conjugates in tissue regeneration 1 restores lipopolysaccharide-induced pulmonary endothelial glycocalyx loss via ALX/SIRT1/NF-kappa B axis.

BACKGROUND: Endothelial glycocalyx loss is integral to increased pulmonary vascular permeability in sepsis-related acute lung injury. Protectin conjugates in tissue regeneration 1 (PCTR1) is a novel macrophage-derived lipid mediator exhibiting potential anti-inflammatory and pro-resolving benefits. METHODS: PCTR1 was administrated intraperitoneally with 100 ng/mouse after lipopolysaccharide (LPS) challenged. Survival rate and lung function were used to evaluate the protective effects of PCTR1. Lung inflammation response was observed by morphology and inflammatory cytokines level. Endothelial glycocalyx and its related key enzymes were measured by immunofluorescence, ELISA, and Western blot. Afterward, related-pathways inhibitors were used to identify the mechanism of endothelial glycocalyx response to PCTR1 in mice and human umbilical vein endothelial cells (HUVECs) after LPS administration. RESULTS: In vivo, we show that PCTR1 protects mice against lipopolysaccharide (LPS)-induced sepsis, as shown by enhanced the survival and pulmonary function, decreased the inflammatory response in lungs and peripheral levels of inflammatory cytokines such as tumor necrosis factor-α, interleukin-6, and interleukin-1ß. Moreover, PCTR1 restored lung vascular glycocalyx and reduced serum heparin sulphate (HS), syndecan-1 (SDC-1), and hyaluronic acid (HA) levels. Furthermore, we found that PCTR1 downregulated heparanase (HPA) expression to inhibit glycocalyx degradation and upregulated exostosin-1 (EXT-1) protein expression to promote glycocalyx reconstitution. Besides, we observed that BAY11-7082 blocked glycocalyx loss induced by LPS in vivo and in vitro, and BOC-2 (ALX antagonist) or EX527 (SIRT1 inhibitor) abolished the restoration of HS in response to PCTR1. CONCLUSION: PCTR1 protects endothelial glycocalyx via ALX receptor by regulating SIRT1/NF-κB pathway, suggesting PCTR1 may be a significant therapeutic target for sepsis-related acute lung injury.

Dual cascade isothermal amplification reaction based glucometer sensors for point-of-care diagnostics of cancer-related microRNAs.

The practical use of a point-of-care (POC) device is of particular interest in performing liquid biopsies related to cancer. Herein, taking advantage of the practical convenience of a commercially available personal glucose meter (PGM), we report a convenient, low-cost and sensitive detection strategy for circulating microRNA-155 (miRNA155) in human serum. First, miRNA155 in serum triggers the catalyzed hairpin assembly (CHA) reaction, and then the CHA product is specifically captured by the peptide nucleic acid (PNA) probes attached to the surface of a 96-well plate, which in turn triggers the hybridization chain reaction (HCR), resulting in the local enrichment of invertase. Next, introduction of a substrate (sucrose) for the invertase results in the generation of glucose, which can be detected by a PGM. In this sensor, neutrally charged PNA (12 nt) is more likely to hybridize with the CHA products than with the negatively charged DNA in kinetics, which improves the detection sensitivity and specificity. Due to the synergistic isothermal amplification reaction between CHA and HCR, the sensor is able to achieve a broad dynamic range (from 1 fM to 10 nM) with a detection limit down to 0.36 fM (3 orders of magnitude lower than that without HCR) and is capable of distinguishing single-base mismatched sequences. Thus the convenient, sensitive, robust and low-cost PGM sensor makes on-site nucleic acids detection possible, suggesting its great application prospect as a promising POC device in cancer diagnostics.

Isolation and complete genome sequencing of the virulent phage vB_EcoS_XY3 infecting multidrug-resistant Escherichia coli.

A virulent phage, named vB_EcoS_XY3, was isolated from hospital wastewater in Xiangyang, China. Its morphological characteristics, growth parameters, adsorption rate, and pH and temperature stability were determined. Phage vB_EcoS_XY3 was found to be able to infect Escherichia coli laboratory strains and also some multidrug-resistant E. coli strains. Its complete genome consists of 51,345 base pairs of double-stranded DNA with an average GC content of 55.24% and 85 putative protein-coding genes. Forty-four genes were annotated with known functions. These results will not only provide further insights into E. coli phages but also have implications for the development of potential biocontrol agents.

A PNA-DNA2 Triple-Helix Molecular Switch-Based Colorimetric Sensor for Sensitive and Specific Detection of microRNAs from Cancer Cells.

Peptide nucleic acids (PNAs), the synthetic DNA mimics that can bind to oligonucleotides to form duplexes, triplexes, and quadruplexes, could be advantageous as probes for nucleic acid sequences owing to their unique physicochemical and biochemical properties. We have found that a homopurine PNA strand could bind to two homopyrimidine DNA strands to form a PNA-DNA2 triplex. Moreover, the cyanine dye DiSC2 (5) could bind with high affinity to this triplex and cause a noticeable color change. On the basis of this phenomenon, we have designed a label-free colorimetric sensing platform for miRNAs from cancer cells by using a PNA-DNA2 triple-helix molecular switch (THMS) and DiSC2 (5). This sensing platform can detect miRNA-21 specifically with a detection limit of 0.18 nM, which is comparable to that of the THMS-mediated fluorescence sensing platform. Moreover, this colorimetric platform does not involve any chemical modification or enzymatic signal amplification, which boosts its applicability and availability at the point of care in resource-limited settings. The universality of this approach can be simply achieved by altering the sequences of the probe DNA for specific targets.

Absence of the type I-E CRISPR-Cas system in Klebsiella pneumoniae clonal complex 258 is associated with dissemination of IncF epidemic resistance plasmids in this clonal complex.

BACKGROUND: The pandemics caused by MDR Klebsiella pneumoniae are mostly due to the global dissemination of high-risk clonal complex 258 (CC258) and related IncF epidemic plasmids. However, the factors leading to the epidemiological advantages of CC258-IncF linkage remain obscure. The Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas) systems, providing adaptive immunity against invading DNA, play an important role in the interactions between plasmids and hosts. OBJECTIVES: To investigate the relationship between CRISPR-Cas systems and the high-risk linkage CC258-IncF. METHODS: CRISPR-Cas loci were detected among 381 collected K. pneumoniae clinical isolates and 207 K. pneumoniae complete genomes available in GenBank. MLST was used to determine the genetic relatedness of these isolates. Nucleotide BLAST was used to search for protospacers on K. pneumoniae plasmids. RESULTS: We observed an epidemic correlation between CRISPR-Cas loci, CC258 and IncF plasmids. Interestingly, most type I-E CRISPR-Cas systems identified carried spacers matching the backbone regions of IncF plasmids. CONCLUSIONS: Our results suggest that the absence of type I-E CRISPR-Cas systems in K. pneumoniae CC258 is strongly associated with the dissemination of IncF epidemic plasmids, contributing to the global success of the international high-risk linkage CC258-IncF. Our findings provide new information regarding the dissemination and evolution of the high-risk linkage of K. pneumoniae CC258-IncF and pave the way for new strategies to address the problem of antibiotic resistance.

Novel nonsense mutation p. Gln264Ter in the ANK1 confirms causative role for hereditary spherocytosis: a case report.

BACKGROUND: Hereditary spherocytosis (HS) is the most common haemolytic anaemia caused by congenital membrane defects of red blood cells. The name derives from the presence of spherical red blood cells in the peripheral blood. Clinical manifestations of HS are anaemia, haemolytic jaundice, and large spleen, and infection can worsen the condition, often with cholelithiasis. HS is mainly caused by abnormal functions of the products of six genes. Splenectomy is the main treatment for HS. CASE PRESENTATION: Half a day after birth, the proband exhibited HS-related symptoms, with progressive aggravation. Routine examination in the outpatient department showed an increase in white blood cells and a decrease in red blood cells. His mother had HS and a partial splenectomy. We suspected that the infant might also have HS. Genomic DNA samples were extracted from the three members of the HS trio pedigree, and genomic whole-exome sequencing (WES) was performed. The three DNA samples were amplified by polymerase chain reaction (PCR), followed by Sanger sequencing to identify mutation sites. A novel nonsense heterozygous mutation, c.790C > T (p. Gln264Ter), in the ANK1 gene, which causes premature termination of translation, was found in this Chinese family with autosomal dominant HS. CONCLUSIONS: This de novo nonsense mutation can cause the onset of HS in early childhood, with severe symptoms. Expanding the ANK1 genotype mutation spectrum will lay a foundation for the further application of mutation screening in genetic counselling.

A peptide nucleic acid-regulated fluorescence resonance energy transfer DNA assay based on the use of carbon dots and gold nanoparticles.

A convenient fluorometric method was developed for specific determination of DNA based on peptide nuclei acid (PNA)-regulated fluorescence resonance energy transfer (FRET) between carbon dots (CDs) and gold nanoparticles (AuNPs). In this system, CDs that display lake blue fluorescence with excitation/emission maxima at 345/445 nm were used as fluorometric reporter, while AuNPs were used as fluorescence nanoquencher. A neutral PNA probe, which is designed to recognize the target DNA, was used as a coagulant to control the dispersion and aggregation of AuNPs. Without DNA, PNA can induce immediate AuNP aggregation, thus leading to the recovery of the FRET-quenched fluorescence emission of CDs. However, the addition of the complementary target DNA can protect AuNPs from being aggregated due to the formation of DNA/PNA complexes, which subsequently produces a high fluorescence quenching efficiency of CDs by dispersed AuNPs. Under optimized conditions, quantitative evaluation of DNA was achieved in a linear range of 5-100 nM with a detection limit of 0.21 nM. This method exhibited an excellent specificity towards fully matched DNA. In addition, the application of this assay for sensitive determination of DNA in cell lysate demonstrates its potential for bioanalysis and biodetection. Graphical abstract A simple fluorometric biosensor for specific detection of DNA was developed based on peptide nuclei acid (PNA)-regulated fluorescence resonance energy transfer (FRET) between carbon dots (CDs) and gold nanoparticles (AuNPs).

Dual Quantification of MicroRNAs and Telomerase in Living Cells.

The development of a unique and universal strategy for the simultaneous quantification of different types of biomolecules (i.e., nucleic acids and proteins) in living cells is extremely challenging. Herein, a two-signal platform, based upon surface-enhanced Raman scattering and upconversion, for the ultrasensitive and quantitative in situ detection of microRNA (miR)-21 and telomerase in living cells is reported. In the presence of miR-21 and telomerase, the hybridization of miR-21 with a molecular beacon leads to the separation of 3,3'-diethylthiocarbamyl cyanine iodide-modified Au NR dimers, resulting in a decrease in Raman signal. Also, the target telomerase triggers elongation of the telomerase primer strands, followed by substitutional hybridization and release of upconversion nanoparticles, leading to an increase in luminescence. A linear relationship between the Raman intensities and logarithmic concentration of intracellular miR-21 between 0.021 and 22.36 amol/ngRNA is observed, and the limit of detection (LOD) was determined to be 0.011 amol/ngRNA. The luminescence data show a linear response between 0.6 × 10-12 and 31 × 10-12 IU for logarithmic concentration of intracellular telomerase with a LOD of 3.2 × 10-13 IU. These results are in good agreement with Raman and confocal imaging. Importantly, the ultrasensitive detection of miR-21 was possible due to strong plasmonic "hot spots". This innovative two-signal approach can be utilized for the quantitative and precise detection of many types of signaling molecules in living cells and to understand the chemistry within cellular systems and its application in the diagnosis of disease.

The LspC3-41I restriction-modification system is the major determinant for genetic manipulations of Lysinibacillus sphaericus C3-41.

BACKGROUND: Lysinibacillus sphaericus has been widely used in integrated mosquito control program and it is one of the minority bacterial species unable to metabolize carbohydrates. In consideration of the high genetic conservation at genomic level and difficulty of genetic horizontal transfer, it is hypothesized that effective restriction-modification (R-M) systems existed in mosquitocidal L. sphaericus. RESULTS: In this study, six type II R-M systems including LspC3-41I were predicted in L. sphaericus C3-41 genome. It was found that the cell free extracts (CFE) from this strain shown similar restriction and methylation activity on exogenous Bacillus/Escherichia coli shuttle vector pBU4 as the HaeIII, which is an isoschizomer of BspRI. The Bsph_0498 (encoding the predicted LspC3-41IR) knockout mutant Δ0498 and the complement strain RC0498 were constructed. It was found that the unmethylated pBU4 can be digested by the CFE of C3-41 and RC0498, but not by that of Δ0498. Furthermore, the exogenous plasmid pBU4 can be transformed at very high efficacy into Δ0498, low efficacy into RC0498, but no transformation into C3-41, indicating that LspC3-41I might be a major determinant for the genetic restriction barrier of strain C3-41. Besides, lspC3-41IR and lspC3-41IM genes are detected in other two strains besides C3-41 of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and MLST sequence type (ST) 1. Furthermore, the three strains are not horizontal transferred, and this restriction could be overcome by in vitro methylation either by the host CFE or by commercial methytransferase M. HaeIII. The results provide an insight to further study the genetic restriction, modification and evolution of mosquitocidal L. sphaericus, also a theoretical basis and a method for the genetic manipulations of L. sphaericus. CONCLUSIONS: LspC3-41I is identified as the major determinant for the restriction barrier of L. sphaericus C3-41. Only three strains of the tested 16 L. sphaericus strains, which all belonging to serotype H5 and ST1 by MLST scheme, contain LspC3-41I system. Two different methods can be used to overcome the restriction barrier of the three isolates to get transformants efficiently: 1) to methylate plasmid DNA prior to the electroporation; and 2) to delete the major restriction endonuclease encoding gene lspC3-41IR.

Determination of total urinary 2,5-hexanedione in the Chinese general population.

OBJECTIVE: Determination of the urinary levels of 2.5-hexanedione (2,5-HD) was performed in subjects belonging to the Chinese general population to define the reference value for this metabolite. METHODS: Urine samples were collected from 8235 individuals (4216 men and 4019 women) from the healthy general population who had not been occupationally exposed to n-hexane or methyl-n-butyl ketone. The determination was performed by a gas chromatography mass spectrometry method using an ion-trap mass spectrometer. RESULTS: The result showed that the urinary 2,5-HD median level was 0.159mg/L for the total samples. Males had statistically significant higher excretion of 2,5-HD in urine than females (median 0.171mg/L compared to 0.147mg/L, Z=-8.21, P<0.001). There was a statistically significant difference in urinary 2,5-HD levels among age groups. The excretion of 2,5-HD in urine was related to increasing age (r=-0.160, P<0.05). There was statistically significant difference in urinary 2,5-HD levels among people from difference provinces. The results showed that there was also a statistically significant effect in urinary 2,5-HD levels between current smokers and non-smokers. CONCLUSION: Finding a measurable amount of 2,5-HD in urine does not mean that the level of 2,5-HD causes an adverse health effect. Biomonitoring studies on levels of urinary 2,5-HD can provide physicians and public health officials with reference values so that they can determine whether people have been exposed to higher levels of 2,5-HD than are found in the Chinese general population. These data can also provide a foundation for scientists to make a plan for further study.

IncX3 plasmid-mediated spread of blaNDM gene in Enterobacteriaceae among children in China.

OBJECTIVES: The blaNDM gene was prevalent among children and became the predominant cause of severe infection in infants and children. This study aimed to investigate the epidemiology and molecular characteristics of blaNDM in Enterobacteriaceae among children in China. METHODS: Carbapenem-resistant Enterobacteriaceae (CRE) were collected in the Children's Hospital of Fudan University from January 2016 to December 2022. Five carbapenemase genes (blaKPC, blaNDM, blaVIM, blaIMP, blaOXA-48) were screened by PCR method. Multilocus sequence typing (MLST) was conducted for phylogenetic analyses. blaNDM-carrying plasmids were typed by PCR-based Incompatibility (Inc) typing method. Moreover, plasmid comparison was performed with 213 publicly available IncX3 plasmids. RESULTS: A total of 330 CRE strains were enrolled, 96.4% of which carried carbapenemase genes. blaNDM gene accounted for 64.8% (214 strains) and included four variants, including blaNDM-1 (59.8%), blaNDM-5 (39.3%), blaNDM-7 (0.5%), and blaNDM-9 (0.5%). There were no predominant MLST lineages of blaNDM carrying strains. IncX3 was the major plasmid carrying blaNDM-1 (68.0%) and blaNDM-5 (72.6%) and was dominant in blaNDM-Klebsiella penumoniae (79.8%), blaNDM-Escherichia coli (58.2%), and blaNDM-Enterobacter cloacae (61.0%), respectively. Most (79.0%) clinical IncX3 plasmids in the world carried blaNDM, and the prevalence of blaNDM in IncX3 plasmids was more common in China (95.8%) than other countries (58.1%, P <0.01). CONCLUSION: blaNDM is highly prevalent in CRE among children in China. The spread of blaNDM was mainly mediated by IncX3 plasmids. Surveillance and infection control on the spread of blaNDM among children are important.