Toxin genotypes, antibiotic resistance and their correlations in Clostridioides difficile isolated from hospitals in Xi'an, China.

BACKGROUND: Clostridioides difficile is the main pathogen of antimicrobial-associated diarrhoea and health care facility-associated infectious diarrhoea. This study aimed to investigate the prevalence, toxin genotypes, and antibiotic resistance of C. difficile among hospitalized patients in Xi'an, China. RESULTS: We isolated and cultured 156 strains of C. difficile, representing 12.67% of the 1231 inpatient stool samples collected. Among the isolates, tcdA + B + strains were predominant, accounting for 78.2% (122/156), followed by 27 tcdA-B + strains (27/156, 17.3%) and 6 binary toxin gene-positive strains. The positive rates of three regulatory genes, tcdC, tcdR, and tcdE, were 89.1% (139/156), 96.8% (151/156), and 100%, respectively. All isolates were sensitive to metronidazole, and the resistance rates to clindamycin and cephalosporins were also high. Six strains were found to be resistant to vancomycin. CONCLUSION: Currently, the prevalence rate of C. difficile infection (CDI) in Xi'an is 12.67% (156/1231), with the major toxin genotype of the isolates being tcdA + tcdB + cdtA-/B-. Metronidazole and vancomycin were still effective drugs for the treatment of CDI, but we should pay attention to antibiotic management and epidemiological surveillance of CDI.

The role of type VI secretion system genes in antibiotic resistance and virulence in Acinetobacter baumannii clinical isolates.

Introduction: The type VI secretion system (T6SS) is a crucial virulence factor in the nosocomial pathogen Acinetobacter baumannii. However, its association with drug resistance is less well known. Notably, the roles that different T6SS components play in the process of antimicrobial resistance, as well as in virulence, have not been systematically revealed. Methods: The importance of three representative T6SS core genes involved in the drug resistance and virulence of A. baumannii, namely, tssB, tssD (hcp), and tssM was elucidated. Results: A higher ratio of the three core genes was detected in drug-resistant strains than in susceptible strains among our 114 A. baumannii clinical isolates. Upon deletion of tssB in AB795639, increased antimicrobial resistance to cefuroxime and ceftriaxone was observed, alongside reduced resistance to gentamicin. The ΔtssD mutant showed decreased resistance to ciprofloxacin, norfloxacin, ofloxacin, tetracycline, and doxycycline, but increased resistance to tobramycin and streptomycin. The tssM-lacking mutant showed an increased sensitivity to ofloxacin, polymyxin B, and furazolidone. In addition, a significant reduction in biofilm formation was observed only with the ΔtssM mutant. Moreover, the ΔtssM strain, followed by the ΔtssD mutant, showed decreased survival in human serum, with attenuated competition with Escherichia coli and impaired lethality in Galleria mellonella. Discussion: The above results suggest that T6SS plays an important role, participating in the antibiotic resistance of A. baumannii, especially in terms of intrinsic resistance. Meanwhile, tssM and tssD contribute to bacterial virulence to a greater degree, with tssM being associated with greater importance.

The optimized carbapenem inactivation method for objective and accurate detection of carbapenemase-producing Acinetobacter baumannii.

The modified carbapenem inactivation method (mCIM) recommended by the Clinical and Laboratory Standards Institute is not applicable for detecting carbapenemases in Acinetobacter baumannii. Four currently reported phenotypic detection methods, namely, the modified Hodge test, the mCIM, the adjusted mCIM, and the simplified carbapenem inactivation method (sCIM), did not perform well in our 90 clinical A. baumannii isolates. Thus, the minimal inhibitory concentrations (MICs) of carbapenems and the existence and expression of carbapenemase-encoding genes were detected to explain the results. According to the E-test, which was more accurate than the VITEK 2 system, 80.0 and 41.1% were resistant to imipenem (IPM) and meropenem (MEM), respectively, and 14.4 and 53.3% exhibited intermediate resistance, respectively. Five ß-lactamase genes were found, of which blaOXA-51-like, blaTEM, and blaOXA-23-like were detected more frequently in 85 non-susceptible strains. The expression of blaOXA-23-like was positively correlated with the MIC values of IPM and MEM. Therefore, an improved approach based on the mCIM, designated the optimized CIM (oCIM), was developed in this study to detect carbapenemases more accurately and reproducibly. The condition was improved by evaluating the factors of A. baumannii inoculum, incubation broth volume, and MEM disk incubation time. Obvious high sensitivity (92.94%) and specificity (100.00%) were obtained using the oCIM, which was cost-effective and reproducible in routine laboratory work.

Programmed Cell Death Protein 1/Programmed Cell Death Ligand 1 Signaling Pathway Mediated Interleukin-10 and Bacterial Biofilm Formation to Drug Resistance Mechanism of Pneumoniae Meningitis.

It aimed to explore the resistance and biofilm formation characteristics of pneumococcal meningitis (PM) and the mechanism of programmed cell death protein 1/programmed cell death ligand 1 (PD-1/PD-L1) signaling pathway (SPW). Firstly, the drug susceptibility test of 32 Streptococcus pneumoniae strains isolated from patients with PM and the biofilm semi-quantitative determination was performed. Then, the PM mouse model was constructed. The differences in brain morphology, blood-brain barrier (BBB) permeability, water content, cytokines such as interferon-γ (IFN-γ), interleukin-10 (IL-10), and chemokine C-X-C ligand 10 (CXCL10), and levels of PD-1 and PD-L1 in the normal control (NC), sham operation, PM, and PD-1 antibody (PM + PD-1 Ab)groups were compared and analyzed. The results showed that streptococcus pneumoniae had multidrug resistance, and the thickness of biofilm decreased with the increase of penicillin minimum inhibitory concentration (MIC). Compared with the NC and Sham groups, BBB permeability, water content, levels of IFN-γ and IL-10, and PD-1 and PD-L1 were signally increased in the PM and PM + PD-1 Ab groups, while CXCL10 level was decreased, exhibiting differences withP<0.05. Based on the PM group, BBB permeability, water content, levels of IFN-γ and CXCL10, and PD-1 and PD-L1 were remarkably decreased in the PM + PD-1 Ab group, while the IL-10 level was observably increased (P<0.05). Therefore, high-MIC penicillin could inhibit the thickness of Streptococcus pneumoniae biofilm, while blocking the PD-1/PD-L1 pathway exerted an improving effect on the PM symptoms.

Hemorrhagic Fever with Renal Syndrome Complicated with Rhino Mucormycosis: A Case Report.

Mucormycosis (MCR) is a rare but aggressive fungal disease. Rhino-orbito-cerebral mucormycosis is the most common clinical form of MCR infection, and sinonasal inoculation is the primary site of infection. The morbidity and mortality rates associated with MCR remain high. In this case report, we describe the successful use of amphotericin B in a 40-year-old male with hemorrhagic fever with renal syndrome (HFRS) complicated by rhinomucormycosis. This case report provides evidence for the successful treatment of HFRS.

Molecular epidemiology and carbapenem resistance characteristics of Acinetobacter baumannii causing bloodstream infection from 2009 to 2018 in northwest China.

Bloodstream infection (BSI) caused by Acinetobacter baumannii poses a serious threat to health and is correlated with high mortality in patients with hospital-acquired infections, so the molecular epidemiology and antimicrobial resistance characteristics of this pathogen urgently need to be explored. A. baumannii isolates from BSI patients were collected in three tertiary hospitals in northwest China from 2009 to 2018. Antimicrobial susceptibility testing was used to determine the MICs of the A. baumannii isolates. Whole-genome sequencing based on the Illumina platform was performed for molecular epidemiological analyses and acquired resistance gene screening. The efflux pump phenotype was detected by examining the influence of an efflux pump inhibitor. The expression of efflux pump genes was evaluated by RT-PCR. In total, 47 A. baumannii isolates causing BSI were collected and they presented multidrug resistance, including resistance to carbapenems. Clone complex (CC) 92 was the most prevalent with 30 isolates, among which a cluster was observed in the phylogenetic tree based on the core genome multi-locus sequence type, indicating the dissemination of a dominant clone. BSI-related A. baumannii isolates normally harbour multiple resistance determinants, of which oxacillinase genes are most common. Except for the intrinsic bla OXA-51 family, there are some carbapenem-resistant determinants in these A. baumannii isolates, including bla OXA-23, which is encoded within the Tn2006, Tn2008 or Tn2009 transposon structures and bla OXA-72. The transfer of bla OXA-72 was suggested by XerC/D site-specific recombination. The AdeABC efflux pump system contributed to carbapenem resistance in A. baumannii isolates, as evidenced by the high expression of some of its encoding genes. Both the clone dissemination and carbapenem resistance mediated by oxacillinase or efflux pumps suggest an effective strategy for hospital infection control.

Application of mNGS in the Etiological Diagnosis of Thoracic and Abdominal Infection in Patients With End-Stage Liver Disease.

Background: Despite the obvious advantages of metagenomic next-generation sequencing (mNGS) in etiological diagnosis of various infectious diseases, there are few reports on etiological diagnosis of suspected thoracic and abdominal infections in patients with end-stage liver disease (ESLD). Methods: Seventy-three ESLD patients were enrolled from January 2019 to May 2021 due to suspected complicated thoracic and abdominal infections with poor response to empirical anti-infective treatment. Pleural effusion and ascites samples of these patients were collected for mNGS detection and conventional pathogen culture. The application value of mNGS in etiological diagnosis of thoracic and abdominal infections in ESLD patients was finally evaluated. Results: A total of 96 pathogens were detected using mNGS method, including 47 bacteria, 32 viruses, 14 fungi, 2 Mycobacterium tuberculosis, and 1 parasite. The positive rate of mNGS reached 42.5%, which was significantly higher than that of conventional culture method (21.9%) (p = 0.008). Considering neutrophil counts, the overall positive rate of bacteria detection of both methods in Polymorphonuclear Neutrophils (PMN) ≥250/mm3 group was 64.3% and in PMN <250/mm3 group was 23.7%. Compared with the final clinical diagnosis, the agreement rate of mNGS in patients with positive bacteria detection and with suspected positive bacteria detection was 78.6% (11/14) and 44.4% (8/18), respectively. In addition, the agreement rate of mNGS was 66.7% (4/6, respectively) in patients with positive and suspected fungal detection. Interestingly, of the 11 patients with fungal detection, 5 had alcoholic liver disease, accounting for 45.5% of all patients with alcoholic liver disease. We also detected 32 strains of viruses using mNGS, mainly cytomegalovirus (62.5%). Conclusions: The mNGS method is a useful supplement to conventional culture methods, which performs a higher positive rate, higher sensitivity, and broader pathogen spectrum, especially for rare pathogens and those difficult to culture. For ESLD patients, mNGS has great prospects in early etiological diagnosis of thoracic and abdominal infections. In addition, the cutoff values for the diagnosis of bacterial infection (PMN ≥250/mm3) in the thoracic and abdominal cavities may need to be redefined.

Microbial Predominance and Antimicrobial Resistance in a Tertiary Hospital in Northwest China: A Six-Year Retrospective Study of Outpatients and Patients Visiting the Emergency Department.

BACKGROUND: With the wide use of antibiotics, antimicrobial resistance becomes a serious issue. Timely understanding of microbial pathogen profiles and the change of antimicrobial resistance provide an important guidance for effective and optimized use of antibiotics in local healthcare systems. The aim was to investigate the characteristics of microbial species and their antimicrobial resistances in a tertiary hospital with an Emergency Department and outpatient clinics for a period of six years. Methodology. A retrospective study was conducted using the HIS database of a tertiary hospital between 2013 and 2018. Antimicrobial susceptibility was tested by automated systems and/or the Kirby-Bauer disc diffusion method. The data were analyzed using the WHONET 5.6 software. The Cochran-Armitage test was used to study the trends over the period of research. RESULTS: In a total of 19,028 specimens submitted for microbial tests during the period from 49 units of the hospital, only the samples from the Emergency Department and Kidney Transplantation Clinic showed an annually significant increase (P < 0.001). More than 200 species with 46.4% gram-positive cocci and 45.3% gram-negative bacilli were identified in the 3,849 nonrepetitive isolates. The methicillin-resistant S. aureus and S. epidermidis rates were 25.1% and 74.6%, respectively. 60.9% E. coli and 33.5% K. pneumonia samples carried extended-spectrum-ß-lactamase. All Staphylococci and Enterococci samples were not resistant to linezolid, vancomycin, and tigecycline. In addition, only 0.01% E. coli, 1.1% K. pneumonia, and 18.7% P. aeruginosa isolates showed resistance to carbapenems. CONCLUSIONS: Vancomycin, linezolid and tigecycline were the most effective antibiotics for outpatients with gram-positive infection. Carbapenems were the most effective antibiotics for gram-negative infection. There was no significant annual increase of common multidrug resistances.

Clinical Efficacy and Cost-Effectiveness of ß-Lactam/ß-Lactamase Inhibitor Combinations and Carbapenems in Liver Cirrhosis Patients with Gram-Negative Bacteria Bloodstream Infection.

BACKGROUND: Gram-negative bacteria bloodstream infection (GNB-BSI) results in considerable mortality and hospitality costs in cirrhotic patients. ß-lactam/ß-lactamase inhibitor combinations (BLBLIs) and carbapenems (CARs) are widely recommended for treating GNB-BSI in cirrhotic patients, while the efficacy and cost-effectiveness of two strategies have never been evaluated. Therefore, we conducted a retrospective cohort study to evaluate the efficacy and the cost-effectiveness of BLBLIs and CARs. PATIENTS AND METHODS: Cirrhotic patients with GNB-BSI treated by BLBLIs or CARs were included. A propensity score-matching analysis was performed to compare the efficacy between BLBLIs and CARs. A decision tree was used to estimate the clinical outcomes and direct costs of treating BSI using two strategies from the patients' perspective. RESULTS: No statistically significant difference was found between the BLBLIs (n = 41) group and the CARs (n = 43) group regarding the time to defervescence (2.4 ± 0.2 vs 2.5 ± 0.3, P = 0.94). Thirty-seven patients from each group were matched in propensity-score-matched cohort, and there was no significant difference between two groups in terms of the time to defervescence (2.4 ± 0.3 vs 2.4 ± 0.3, P = 0.75) and success rate (86.5% vs 78.4%; OR = 0.57; P = 0.36). Based on the drug and hospital costs in China, cefoperazone/sulbactam was cost-effective in the present analysis under the willingness-to-pay threshold (¥64,644). CONCLUSION: The efficacy of BLBLIs is similar to CARs. Cefoperazone/sulbactam could be a cost-effective therapy in cirrhotic patients with GNB-BSI. Carbapenems-sparing regimens should be encouraged in regions with a low prevalence of MDR bacteria.

Clinical and Molecular Epidemiology of Invasive Group B Streptococcus Disease among Infants, China.

Invasive group B Streptococcus (GBS) remains a leading cause of illness and death among infants globally. We conducted prospective and retrospective laboratory-based surveillance of GBS-positive cultures from infants <3 months of age in 18 hospitals across China during January 1, 2015-December 31, 2017. The overall incidence of GBS was 0.31 (95% CI 0.27-0.36) cases/1,000 live births; incidence was 0-0.76 cases/1,000 live births across participating hospitals. The case-fatality rate was 2.3%. We estimated 13,604 cases of GBS and 1,142 GBS-associated deaths in infants <90 days of age annually in China. GBS isolates were most commonly serotype III (61.5%) and clonal complex 17 (40.6%). Enhanced active surveillance and implementation of preventive strategies, such as maternal GBS vaccination, warrants further investigation in China to help prevent these infections.

The assemblage of covalent and metal binding dual functional scaffold for cross-class metallo-ß-lactamases inhibition.

Aim: The discovery and development of novel broad-spectrum MßLs inhibitors are urgent to overcome antibiotic resistance mediated by MßLs. Methods & results: Herein, the synthesized 21 compounds exhibited potent inhibition to the clinically important MßLs (NDM-1, IMP-1 and ImiS) and effectively restored the antibacterial efficacy of cefazolin and imipenem against Escherichia coli harboring MßLs. 5b was first identified to be dual functional broad-spectrum MßLs inhibitor through assemblage of covalent and metal binding scaffold, which irreversibly inhibited B1, B2 MßLs via forming a Se-S covalent bond, and competitively inhibited B3 MßLs by coordinating the metals at active site. Conclusion: The designed compounds can serve as potent broad-spectrum MßLs inhibitors and combat MßLs-producing 'superbug' in combination with ß-lactams.

Antibiotic consumption versus the prevalence of carbapenem-resistant Gram-negative bacteria at a tertiary hospital in China from 2011 to 2017.

PURPOSE: China launched a 3-year rectification scheme on the clinical use of antibiotics in 2011, and a specific scheme on carbapenem use in February 2017. This study investigated the trends in and correlations between antibiotic consumption and the prevalence of carbapenem-resistant Gram-negative bacteria (CRGN) at a tertiary hospital during these years, particularly in carbapenem consumption. METHODS: The data were collected calculated per quarter from 2011 to 2017. The trends in antibiotic consumption and resistance were analyzed by regression analysis, while Spearman correlation analysis was used to assess the correlations. RESULT: The total consumption of antibiotics halved during the 7-year study period, from 770.15 DDDs/1000 PDs in quarter 1 of 2011 to 395.07 DDDs/1000 PDs in quarter 4 of 2017. Meantime, carbapenem consumption showed the significant increase, from 28.71 DDDs/1000 PDs to 49.2 DDDs/1000 PDs. The detection rates of CRGN (carbapenem-resistant Klebsiella pneumonia, Acinetobacter baumannii, and Pseudomonas aeruginosa) remained stable (P>0.05). The positive correlation was only discovered between the resistance rate of carbapenem-resistant K. pneumonia and the usage of carbapenems, which included meropenem and imipenem, with coefficients of 0.543, 0.537, and 0.497 (P<0.05), respectively. There was no more significant correlation in this study. CONCLUSION: The total consumption of antibiotics reduced significantly in the analysed hospital, which could be related to the antimicrobial stewardship programme. However, the carbapenem consumption was increased. The specific index should be established to limit the application of carbapenems. This study identified the positive correlation between the detection rate of carbapenem-resistant K. pneumonia and carbapenem consumption. More research is needed to confirm the impact of restricting and appropriated use of carbapenems on the prevalence of CRGN.

Label-Free Visualization of Carbapenemase Activity in Living Bacteria.

Evaluating enzyme activity intracellularly on natural substrates is a significant experimental challenge in biomedical research. We report a label-free method for real-time monitoring of the catalytic behavior of class A, B, and D carbapenemases in live bacteria based on measurement of heat changes. By this means, novel biphasic kinetics for class D OXA-48 with imipenem as substrate is revealed, providing a new approach to detect OXA-48-like producers. This in-cell calorimetry approach offers major advantages in the rapid screening (10 min) of carbapenemase-producing Enterobacteriaceae from 142 clinical bacterial isolates, with superior sensitivity (97 %) and excellent specificity (100 %) compared to conventional methods. As a general, label-free method for the study of living cells, this protocol has potential for application to a wider range and variety of cellular components and physiological processes.

Real-Time Monitoring of NDM-1 Activity in Live Bacterial Cells by Isothermal Titration Calorimetry: A New Approach To Measure Inhibition of Antibiotic-Resistant Bacteria.

The "superbug" infection caused by New Delhi metallo-ß-lactamase (NDM-1) has become an emerging threat. Monitoring NDM-1 has proven challenging due to its shuttling between pathogenic bacteria. Here, we report an isothermal titration calorimetry (ITC) method that can monitor activity and inhibition of NDM-1 in live bacterial cells in real time. This method has been exemplified by monitoring of the activity and inhibition of the target enzyme and evaluating the breakdown of antibiotics by pathogenic bacteria expressing ß-lactamases. Cell-based studies demonstrate that the NDM-1 expressed in bacterial cells was inhibited by four known inhibitors ethylene diamine tetraacetic acid (EDTA), d-captopril, ebselen and azolylthioacetamide with fifty percent inhibitory concentration (IC50) values of 3.8, 48, 0.55, and 17.5 µM, respectively, which are in good agreement with the data from inhibition kinetics using UV-vis and NMR spectroscopy in vivo. This approach could be applied to screen and evaluate small molecule inhibitors of metallo-ß-lactamases (MßLs) in whole cells or to identify drug resistant bacteria.

A strategy for designing voriconazole dosage regimens to prevent invasive pulmonary aspergillosis based on a cellular pharmacokinetics/pharmacodynamics model.

BACKGROUND: Invasive pulmonary aspergillosis (IPA) is a life-threatening disease in immunosuppressed patients. Voriconazole is commonly used to prevent and treat IPA in the clinic, but the optimal prophylactic antifungal regimen is unknown. The objective of this study was to clarify the mechanism underlying how voriconazole prevents IPA based on a target cellular pharmacokinetics/pharmacodynamics model, with the aim of identifying a way to design an optimal prophylactic antifungal regimen. METHODS: A nystatin assay was used to establish a target-cells model for A. fumigatus infection. An inhibitory effect sigmoid Emax model was developed to explore the cellular PK/PD breakpoint, and Monte Carlo simulation was used to design the prophylactic antifungal regimen. RESULTS: The intracellular activity of voriconazole in the target cells varied with its concentration, with the minimum inhibitory concentration (MIC) being an important determinant. For A. fumigatus strains AF293 and AF26, voriconazole decreased the intracellular inoculum by 0.79 and 0.84 lg cfu, respectively. The inhibitory effect sigmoid Emax model showed that 84.01% of the intracellular inoculum was suppressed by voriconazole within 24 h, and that a PK/PD value of 35.53 for the extracellular voriconazole concentration divided by MIC was associated with a 50% suppression of intracellular A. fumigatus. The Monte Carlo simulation results showed that the oral administration of at least 200 mg of voriconazole twice daily was yielded estimated the cumulative fraction of response value of 91.48%. Concentration of voriconazole in the pulmonary epithelial lining fluid and the plasma of > 17.77 and > 1.55 mg/L, respectively, would ensure the PK/PD > 35.53 for voriconazole against most isolates of A. fumigatus and may will be benefit to prevent IPA in clinical applications. CONCLUSIONS: This study used a target cellular pharmacokinetics/pharmacodynamics model to reveal a potential mechanism underlying how voriconazole prevents IPA and has provided a method for designing voriconazole prophylactic antifungal regimen in immunosuppressed patients.

Voriconazole therapeutic drug monitoring: Factors associated with supratherapeutic and subtherapeutic voriconazole concentrations.

OBJECTIVE: The voriconazole trough concentration (Cmin) varies widely, and Cmin outside the therapeutic range (COTR) is associated with response failure and toxicity. The objective of this study was to identify potential factors associated with COTR in patients, and specifically the population at a high risk of COTR. MATERIALS AND METHODS: We performed a retrospective study of patients who received voriconazole from 2009 to 2016. Voriconazole Cmin values were analyzed with high-performance liquid chromatography, and values of < 1 mg/L and > 4 mg/L were defined as COTR. Logistic regression and the classification and regression tree (CART) were used to explore the potential factors associated with COTR. RESULTS: In total, 134 voriconazole Cmin values were measured in 64 patients who met the eligibility criteria: 55 (41.0%) were subtherapeutic, and 79 (59.0%) were supertherapeutic. Logistic regression revealed that voriconazole COTR was significantly associated with age, CYP2C19 genetic status, and liver function after voriconazole treatment. CART identified the high-risk population of COTR: (1) patients' age < 47 years and with underlying liver disease, (2) patients' age > 47 years and with acute liver dysfunction after voriconazole treatment, (3) non-poor metabolizers, aged from 46 to 65 years and with normal liver function after voriconazole treatment, and (4) old (age > 65 years) patients with normal liver function and body weight < 66 kg. CONCLUSION: Our findings suggest that age, CYP2C19 genetic status, and liver function status are strongest predictors of voriconazole COTR. Clinically, these results can be used to estimate the probability of voriconazole COTR in individual patients.
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Disease burden and antimicrobial resistance of invasive group B streptococcus among infants in China: a protocol for a national prospective observational study.

BACKGROUND: Group B Streptococcus (GBS) is a cause of neonatal sepsis, pneumonia, and meningitis that can lead to neurological sequelae in infants less than 3 months of age. The GBS disease burden is not known in China, therefore it cannot receive major attention. The main objectives of this study are the evaluation of the incidence of neonatal GBS infection, GBS case-fatality ratio, its serotypes and genotypes, bacterial resistance, clinical treatment and outcomes in China. METHODS: We are conducting a nation-wide, population-based, multi-center, prospective, observational cohort study in China from May 2016 to December 2017. Eighteen large urban tertiary care hospitals from 16 provinces were selected that cover the eastern, southern, western, northern and central regions of China. Meanwhile, we retrospectively collected data and GBS strains from January 2015 to April 2016 from selected hospitals. The incidence rate per 1000 live births will be defined as the total number of confirmed GBS cases born in the selected hospitals divided by the number of live births in the hospitals during the study period. All GBS cases detected in selected hospitals will be used to calculate the case-fatality ratio and for the typing analysis. GBS isolates will be serotyped using the Strep-B-Latex® rapid latex agglutination test for serotyping of Group B streptococci. Multi-locus sequence typing (MLST) will be performed by sequencing the internal fragments of seven house-keeping genes. Antimicrobial susceptibility will be tested per interpretive standards established by the Clinical and Laboratory Standards Institute. The presence of the common resistance genes ermA, ermB, mefA, tetI, tetO and tetM will be tested by PCR. DISCUSSION: We are conducting the first national study to estimate the invasive GBS disease burden and antimicrobial resistance of GBS among infants in China. Study findings will provide important evidence for improving clinical practice to ensure timely diagnosis of GBS disease and decisions for preventive measures. Surveillance of antimicrobial resistance will promote the rational use of antimicrobials. TRIAL REGISTRATION: The study was retrospectively registered at http://clinicaltrials.gov on June 13, 2016. It was granted a registration number of "NCT02812576".

Characterization of ß-lactamase activity using isothermal titration calorimetry.

BACKGROUND: Hydrolysis of ß-lactam antibiotic by ß-lactamase is the most common mechanism of ß-lactam resistance in clinical isolates. Timely detection and characterization of ß-lactamases are therefore of utmost biomedical importance. Conventional spectrophotometric method is time-consuming and cannot provide thermodynamic information on ß-lactamases. METHODS: A new assay was developed for the study of ß-lactamase activity in protein solutions (Metallo-ß-lactamase L1) and in clinical bacterial cells, based on heat-flow changes derived from enzymatic hydrolysis of ß-lactams using isothermal titration calorimetry. RESULTS: (1) The thermokinetic parameters of three antibiotics (penicillin G, cefazolin and imipenem) and the inhibition constant of an azolylthioacetamide inhibitor were determined using the calorimetric assay. The results from the calorimetric assays were consistent with the data from the spectrophotometric assay. (2) The values of heat change in the calorimetric assay using two clinical Escherichia coli strains correlated well with their antibiotic susceptibility results from the broth dilution experiment. The subtypes of ß-lactamase were also determined in the calorimetric assay. CONCLUSIONS: The ITC assay is a reliable and fast method to study ß-lactamase enzyme kinetics and inhibition. It can also provide thermodynamic information on antibiotic hydrolysis, which has been taken advantage of in this work to study ß-lactamase activity in two clinical Escherichia coli isolates. GENERAL SIGNIFICANCE: As the first calorimetric study of ß-lactamase activity, it may provide a new assay to assist biomedical validation of new ß-lactamase inhibitors, and also has potential applications on rapid antibiotic susceptibility testing and screening ß-lactamase producing bacteria.

blaOXA-23-like and blaTEM rather than blaOXA-51-like contributed to a high level of carbapenem resistance in Acinetobacter baumannii strains from a teaching hospital in Xi'an, China.

Acinetobacter baumannii is one of the major threats in clinical infections due to its antibiotic resistance ability. It shows increasing resistance to carbapenems, mainly due to ß-lactamase mediated mechanisms. The aim of this study was to investigate carbapenem resistance (CR) profiles and analyze ß-lactamases genes composition of clinical A. baumannii strains from a teaching hospital in Xi'an. The resistance patterns to imipenem and meropenem were checked for 51 clinical A. baumannii strains. The existence of 15 ß-lactamases genes was detected by polymerase chain reaction (PCR), and the positive genes were sequenced. The correlation between PCR-positive genes and CR phenotype was analyzed using Chi-square test and contingency coefficient. The expressions of PCR-positive genes were investigated. Forty-five out of 51 strains were resistant to imipenem and meropenem. blaTEM, blaOXA-23-like, and blaOXA-51-like were positive among 15 ß-lactamases genes, and TEM-1, OXA-23, and OXA-66/69 were their subtypes. TEM and OXA-23-like only showed up in CR isolates, with the occurrence rate of 91.1% and 97.8%, respectively, whereas OXA-51-like appeared in all strains. ISAba1 was present in the upstream of OXA-23-like, but absent from that of OXA-51-like in our strains. OXA-23-like had highest relationship with CR, followed by TEM, but OXA-51-like had no correlation. This was verified by RT-qPCR that the expression was positive for OXA-23 and TEM-1, but negative for OXA-66/-69. A high rate of CR A. baumannii was detected in this study. Coexistence of TEM, OXA-23-like, and OXA-51-like was the primary resistance profile. The expressions of OXA-23-like and TEM genes were closely related with CR, while OXA-51-like had no contribution to the CR phenotype.

Uptake and efflux kinetics, and intracellular activity of voriconazole against Aspergillus fumigatus in human pulmonary epithelial cells: a new application for the prophylaxis and early treatment of invasive pulmonary aspergillosis.

Invasive pulmonary aspergillosis (IPA), most caused by Aspergillus fumigatus, is a serious life-threatening infection in immunocompromised patients. Voriconazole is used to prevent and treat IPA. However, little is known about the pharmacological characteristics of voriconazole in pulmonary epithelial cells, which are the target site for the prophylaxis and early treatment of IPA. The aim of the study was to evaluate the kinetics and activity of voriconazole against A. fumigatus in A549 cells. High-performance liquid chromatography/tandem mass spectrometry and time-kill method were used to study the cellular pharmacokinetic and pharmacodynamics of voriconazole. Voriconazole exerted a concentration-dependent toxic effect on A549 cells and could penetrate into cells, reaching plateau concentrations of 1.14 ± 0.64, 3.72 ± 1.38 and 6.36 ± 0.95 ng/mg protein after A549 cells were exposed to voriconazole at extracellular concentrations of 2, 8 and 16 mg/L for 2 h, respectively. The efflux of voriconazole was rapid, with a half-life of 10.2 min. Voriconazole can decrease the A. fumigatus conidia invade cells, and the number of viable A. fumigatus conidia in cells can be decreased 2.1- to 20.6-fold when A549 cells were cultured in medium containing voriconazole. After 24-h incubation, 75.6% and 80.5% of intracellular A. fumigatus were killed when extracellular voriconazole concentration was 8 and 16 mg/L, respectively. This study illustrated a new application for the prophylaxis and early treatment of IPA from the cellular pharmacokinetics and pharmacodynamics and emphasized the importance of monitoring concentrations of voriconazole in epithelial lining fluid in immunocompromised patients receiving voriconazole therapy.