OBJECTIVES: The prevalence of carbapenem-resistant Enterobacterales (CRE) presents a significant challenge in clinical anti-infective treatment. This study aims to investigate drug resistance and the molecular epidemiological characteristics of CRE in our area. Additionally, we seek to evaluate practicality of utilizing carbapenemase inhibitor enhancement test in clinical laboratory. METHODS: Non-repeated CREs isolated from clinical specimens at Xiangya Hospital, Central South University, were collected. Minimum inhibitory concentration (MIC) combined with Kirby-Bauer (KB) assay was used to detect the drug susceptibility of the strains, and 13 carbapenemase-producing genes were detected by PCR. The phenotype of 126 strains of carbapenemase-producing Enterobacterales identified by PCR was detected by the carbapenemase inhibitor enhancement test to understand the agreement between the method and the gold standard PCR results. RESULTS: Among 704 CRE strains examined, we observed significant drug resistance in 501 strains dentified as carbapenemase-producing Enterobacterales (CPE). Klebsiella pneumoniae was the predominant CPE strain, followed by Enterobacter cloacae and Escherichia coli. A total of 9 carbapenemase types were detected, including Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), Verona integron- encoded metallo-β-lactamases (VIM), imipenemase (IMP), oxacillinase-48 (OXA-48), and rare imipenem-hydrolyzing β-lactamase (IMI), adelaide imipenemase (AIM), Bicêtre carbapenemase (BIC), and guiana extended-spectrum β-lactamase (GES). The detection rate of KPC serine carbapenemase was 61.7% (309/501). The carbapenemase inhibitor enhancement test exhibited a 100% consistency rate for the strains producing Class A serine carbapenemase and/or Class B metallo-β-lactamases. CONCLUSIONS CRE strains in Changsha, Hunan, China, are wide distribution and exhibit carbapenemase production. The main mechanism of carbapenem resistance in these bacterias is predominatly attributed to the production of KPC serine carbapenemase. The presence of GES and IMI genes carried by Enterobacterales has been detected for the first time in this region. The carbapenemase inhibitor enhancement test has been proven to be an accurate method for detecting CRE producing Class A serine carbapenemase and/or Class B metallo-β-lactamases. This method offers simpicity of operation and ease of results interpretation, making it weel-suited meeting the clinical microbiology laboratory's reguirements for the detection of serine carbapenemase and metallo-β-lactamases.
Humans ; Carbapenems/pharmacology* ; Molecular Epidemiology ; Bacterial Proteins/analysis* ; beta-Lactamases/analysis* ; Klebsiella pneumoniae/genetics* ; Escherichia coli ; Microbial Sensitivity Tests ; Serine ; Anti-Bacterial Agents/pharmacology*

Objective: To study the polymorphism in P66 and its human B-cell epitopes of Methods: Polymerase chain reaction (PCR) and sequencing were used to obtain the P66 sequences of 59 Chinese Results: Results showed that genetic and amino acid diversity presented in the 66 kD protein of all 59 Chinese strains, especially in Conclusion In P66 of 59 Chinese strains, polymorphisms were widely distributed. More importantly, the P66 amino acid sequences of
Bacterial Proteins/genetics* ; Borrelia burgdorferi/genetics* ; China ; Cluster Analysis ; Epitopes, B-Lymphocyte/genetics* ; Genetic Markers ; Genotype ; Humans ; Mutation ; Polymerase Chain Reaction ; Polymorphism, Genetic ; Porins/genetics*

Objective: To evaluate multidrug resistant loop-mediated isothermal amplification (MDR-LAMP) assay for the early diagnosis of multidrug-resistant tuberculosis and to compare the mutation patterns associated with the Methods: MDR-LAMP assay was evaluated using 100 Results: The sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of MDR-LAMP were 85.5%, 93.6%, 96.7%, and 74.4% for the detection of resistance to isoniazid and rifampicin, respectively, and 80.5%, 92.3%, 98.6%, and 41.4% for the detection of Conclusion MDR-LAMP is a rapid and accessible assay for the laboratory identification of rifampicin and isoniazid resistance of
Antitubercular Agents ; Bacterial Proteins/genetics* ; Catalase/genetics* ; DNA, Bacterial/analysis* ; DNA-Directed RNA Polymerases/genetics* ; Drug Resistance, Multiple, Bacterial/genetics* ; Isoniazid ; Molecular Diagnostic Techniques/methods* ; Mutation ; Mycobacterium tuberculosis/isolation & purification* ; Nucleic Acid Amplification Techniques/methods* ; Oxidoreductases/genetics* ; Phenotype ; Rifampin ; Whole Genome Sequencing

OBJECTIVETo investigate the prokaryotic expression of proteins pneumococcal endopeptidase O (PepO) and pneumococcal surface adhesin A (PsaA) in Streptococcus pneumoniae and their immunoprotective effect as vaccine candidate proteins.

METHODSSpecific primers of target gene fragments were designed, and then PCR amplification was performed to establish recombinant plasmids pET28a(+)-pepO and pET28a(+)-psaA, which were transformed into host cells, Escherichia coli BL21 and DE3, respectively, to induce expression. Highly purified target proteins PepO and PsaA were obtained after purification. Mucosal immunization was performed for BALB/c mice and specific antiserum was prepared. ELISA was used to measure the antibody titer, and Western blot was used to analyze the specificity of the antiserum of target proteins. The mice were randomly divided into negative control group, PepO group, PsaA group, and PepO+PsaA combined immunization group, with 18 mice in each group. The models of different serotypes of Streptococcus pneumoniae infection were established to evaluate the immunoprotective effect of target proteins used alone or in combination.

RESULTSThe target proteins PepO and PsaA were successfully obtained and Western blot demonstrated that the antiserum of these proteins had good specificity. There was no significant difference in the titers of IgA in saliva and IgG in serum between the PepO group and the combined immunization group (P>0.05); however, these two groups had significantly higher antibody titers than the PsaA group (P<0.05). The PepO, PsaA, and combined immunization groups had significantly higher protection rates for mice infected with Streptococcus pneumoniae D39 and CMCC31436 in the nasal cavity than the negative control group (P<0.05). The PepO and combined immunization groups had a significantly higher protection rate for mice infected with Streptococcus pneumoniae D39 than the PsaA group (P<0.05). The results of colonization experiment showed that compared with the control group, the PepO, PsaA, and combined immunization groups showed a significant reduction in the colonization of Streptococcus pneumoniae (CMCC31693 and CMCC31207) in the nasopharynx and lung (P<0.05). The combined immunization group showed a better effect on reducing the colonization of CMCC31207 in the lung than the PepO and PsaA alone groups.

CONCLUSIONSCombined PepO/PsaA vaccines may produce a better protective effect by mucosal immunization compared with the vaccine used alone in mice. The combined vaccines can effectively reduce the colonization of Streptococcus pneumoniae in the nasopharynx and lung. Therefore, such protein vaccines may have a great potential for research and development.

Adhesins, Bacterial ; immunology ; Animals ; Antibodies, Bacterial ; analysis ; Bacterial Proteins ; immunology ; Female ; Immunization ; Lipoproteins ; immunology ; Lung ; microbiology ; Metalloendopeptidases ; immunology ; Mice ; Mice, Inbred BALB C ; Pneumococcal Infections ; prevention & control ; Pneumococcal Vaccines ; immunology ; Saliva ; immunology

Valienone is a significant natural carbasugar member of the C7-cyclitol family as a valuable precursor for glycosidase inhibitor drugs. It is an intermediate of validamycin A biosynthesis pathway and exhibits minimal accumulation in the fermentation broth of the natural Streptomyces producer. A quantitative analytical method is crucial for the development of a breakthrough microbial process overcoming the consumption of the natural metabolic flux. The present study was designed to develop a pre-column derivatization high-performance liquid chromatography method for quantification of valienone and to help establish a straightforward fermentation process for valienone production by metabolically engineered Streptomyces hygroscopicus 5008. Valienone was derivatized by 2, 4-dinitrophenylhydrazine (DNPH) in 10 mmol·L HPO at 37 °C for 45 min and the derivatives were separated on Eclipse XDB-C (5 μm, 4.6 mm × 150 mm) column at 30 °C eluted with 50% acetonitrile for 18 min. The derivatives were detected by diode array detector at 380 nm and the configurations of the derivatives were determined by computational studies. The method was shown to be effective, sensitive, and reliable. Good linearity was found in the range of 5-2 000 μg·mL. The intra- and inter-day precisions were 1.1%-2.7% and 1.7%-2.2%, respectively. The absolute recovery of the spiked samples was 97.2%-102.6%. To date, this is the first reversed-phase high-performance liquid chromatography detection method for valienone in microbial culture medium. This method successfully helped evaluate the valienone production capability of the engineered Streptomyces hygroscopicus 5008 and could be promising for C7-cyclitol profiling of different engineered mutants combined with the metabonomics methods.
Bacterial Proteins ; genetics ; metabolism ; Biosynthetic Pathways ; Chromatography, Reverse-Phase ; methods ; Cyclohexenes ; analysis ; Hexosamines ; analysis ; biosynthesis ; Metabolic Engineering ; Streptomyces ; genetics ; metabolism

BACKGROUND: We evaluated the performance of four commercial nucleic acid amplification tests (NAATs: Xpert C. difficile, BD MAX Cdiff, IMDx C. difficile for Abbott m2000, and Illumigene C. difficile) for direct and rapid detection of Clostridium difficile toxin genes. METHODS: We compared four NAATs on the same set of 339 stool specimens (303 prospective and 36 retrospective specimens) with toxigenic culture (TC). RESULTS: Concordance rate among four NAATs was 90.3% (306/339). Based on TC results, the sensitivity and specificity were 90.0% and 92.9% for Xpert; 86.3% and 89.3% for Max; 84.3% and 94.4% for IMDx; and 82.4% and 93.7% for Illumigene, respectively. For 306 concordant cases, there were 11 TC-negative/NAATs co-positive cases and 6 TC-positive/NAATs co-negative cases. Among 33 discordant cases, 18 were only single positive in each NAAT (Xpert, 1; Max, 12; IMDx, 1; Illumigene, 4). Positivity rates of the four NAATs were associated with those of semi-quantitative cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). CONCLUSIONS: Commercial NAATs may be rapid and reliable methods for direct detection of tcdA and/or tcdB in stool specimens compared with TC. Some differences in the sensitivity of the NAATs may partly depend on the number of toxigenic C. difficile in stool specimens.
Bacterial Proteins/genetics ; Bacterial Toxins/genetics ; Clostridium Infections/*diagnosis/microbiology ; Clostridium difficile/*genetics/isolation & purification ; DNA, Bacterial/*analysis/metabolism ; Enterotoxins/genetics ; Feces/*microbiology ; Humans ; *Multiplex Polymerase Chain Reaction ; Reagent Kits, Diagnostic ; Sensitivity and Specificity

PURPOSE: In the gastric mucosa of Helicobacter pylori (H. pylori)-infected patients with gastritis or adenocarcinoma, proliferation of gastric epithelial cells is increased. Hyperproliferation is related to induction of oncogenes, such as β-catenin and c-myc. Even though transcription factors NF-κB and AP-1 are activated in H. pylori-infected cells, whether NF-κB or AP-1 regulates the expression of β-catenein or c-myc in H. pylori-infected cells has not been clarified. The present study was undertaken to investigate whether H. pylori-induced activation of NF-κB and AP-1 mediates the expression of oncogenes and hyperproliferation of gastric epithelial cells. MATERIALS AND METHODS: Gastric epithelial AGS cells were transiently transfected with mutant genes for IκBα (MAD3) and c-Jun (TAM67) or treated with a specific NF-κB inhibitor caffeic acid phenethyl ester (CAPE) or a selective AP-1 inhibitor SR-11302 to suppress activation of NF-κB or AP-1, respecively. As reference cells, the control vector pcDNA was transfected to the cells. Wild-type cells or transfected cells were cultured with or without H. pylori. RESULTS: H. pylori induced activation of NF-κB and AP-1, cell proliferation, and expression of oncogenes (β-catenein, c-myc) in AGS cells, which was inhibited by transfection of MAD3 and TAM67. Wild-type cells and the cells transfected with pcDNA showed similar activities of NF-κB and AP-1, proliferation, and oncogene expression regardless of treatment with H. pylori. Both CAPE and SR-11302 inhibited cell proliferation and expression of oncogenes in H. pylori-infected cells. CONCLUSION: H. pylori-induced activation of NF-κB and AP-1 regulates transcription of oncogenes and mediates hyperproliferation in gastric epithelial cells.
Blotting, Western ; Caffeic Acids ; Cell Line, Tumor ; Cell Proliferation ; DNA, Bacterial/analysis/genetics ; DNA-Binding Proteins/*metabolism ; Epithelial Cells/*metabolism ; Gastric Mucosa/*metabolism/pathology ; Gastritis/pathology ; Gene Expression Regulation, Bacterial ; Helicobacter Infections/metabolism/pathology/physiopathology ; Helicobacter pylori/pathogenicity/physiology ; Humans ; NF-kappa B/antagonists & inhibitors/*biosynthesis/metabolism ; Peptide Fragments ; Phenylethyl Alcohol/analogs & derivatives ; Proto-Oncogene Proteins c-jun ; Repressor Proteins ; Transcription Factor AP-1/*biosynthesis ; Transcription Factors/*metabolism ; beta Catenin/*metabolism

No abstract available.
Adult ; Anti-Bacterial Agents/therapeutic use ; Bacterial Proteins/chemistry/genetics/metabolism ; Brucella melitensis/classification/genetics/*isolation & purification ; Brucellosis/*diagnosis/drug therapy/microbiology ; Doxycycline/therapeutic use ; Humans ; Magnetic Resonance Imaging ; Male ; Phylogeny ; Polymerase Chain Reaction ; Republic of Korea ; Rifampin/therapeutic use ; Sequence Analysis, DNA ; Spondylitis/diagnostic imaging

BACKGROUND: We investigated the whole genome sequence (WGS) of a carbapenem-resistant Acinetobacter baumannii isolate belonging to the global clone 2 (GC2) and predicted resistance islands using a software tool. METHODS: A. baumannii strain YU-R612 was isolated from the sputum of a 61-yr-old man with sepsis. The WGS of the YU-R612 strain was obtained by using the PacBio RS II Sequencing System (Pacific Biosciences Inc., USA). Antimicrobial resistance genes and resistance islands were analyzed by using ResFinder and Genomic Island Prediction software (GIPSy), respectively. RESULTS: The YU-R612 genome consisted of a circular chromosome (ca. 4,075 kb) and two plasmids (ca. 74 kb and 5 kb). Its sequence type (ST) under the Oxford scheme was ST191, consistent with assignment to GC2. ResFinder analysis showed that YU-R612 possessed the following resistance genes: four β-lactamase genes bla(ADC-30), bla(OXA-66), bla(OXA-23), and bla(TEM-1); armA, aadA1, and aacA4 as aminoglycoside resistance-encoding genes; aac(6')Ib-cr for fluoroquinolone resistance; msr(E) for macrolide, lincosamide, and streptogramin B resistance; catB8 for phenicol resistance; and sul1 for sulfonamide resistance. By GIPSy analysis, six putative resistant islands (PRIs) were determined on the YU-R612 chromosome. Among them, PRI1 possessed two copies of Tn2009 carrying bla(OXA-23), and PRI5 carried two copies of a class I integron carrying sul1 and armA genes. CONCLUSIONS: By prediction of resistance islands in the carbapenem-resistant A. baumannii YU-R612 GC2 strain isolated in Korea, PRIs were detected on the chromosome that possessed Tn2009 and class I integrons. The prediction of resistance islands using software tools was useful for analysis of the WGS.
Acinetobacter Infections/*drug therapy/microbiology ; Acinetobacter baumannii/drug effects/*genetics/isolation & purification ; Anti-Bacterial Agents/pharmacology/*therapeutic use ; Bacterial Proteins/genetics ; Carbapenems/*therapeutic use ; DNA, Bacterial/chemistry/*genetics/metabolism ; Drug Resistance, Bacterial ; Genomic Islands/genetics ; Humans ; Microbial Sensitivity Tests ; Multilocus Sequence Typing ; Plasmids/genetics/metabolism ; Polymerase Chain Reaction ; Sequence Analysis, DNA

No abstract available.
Anti-Bacterial Agents/pharmacology ; Bacterial Proteins/genetics ; Ceftriaxone/pharmacology/therapeutic use ; Ciprofloxacin/pharmacology/therapeutic use ; DNA, Bacterial/analysis/metabolism ; Disk Diffusion Antimicrobial Tests ; *Drug Resistance, Bacterial ; Female ; Humans ; Neisseria meningitidis/drug effects/genetics/*isolation & purification ; Polymerase Chain Reaction ; Sepsis/*diagnosis/drug therapy/microbiology ; Transcription Factors/genetics ; Young Adult