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.

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.

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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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.

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.

Photodynamic inactivation of antibiotic-resistant bacteria and biofilms by hematoporphyrin monomethyl ether.

The worldwide increase in bacterial antibiotic resistance has led to a search for alternative antibacterial therapies. A promising approach to killing antibiotic-resistant bacteria is photodynamic antimicrobial chemotherapy, which uses light in combination with a photosensitizer to induce a phototoxic reaction. We evaluated the photodynamic inactivation (PDI) efficiency of hematoporphyrin monomethyl ether (HMME) on antibiotic-resistant bacteria and biofilms. HMME exhibited no significant dark toxicity and provided dose-dependent inactivation of antibiotic-resistant bacteria and biofilms. After incubation with 100-µM HMME and irradiation with 72-J cm(-2) white light, 4.19-7.59 log10 reductions in survival were achieved in planktonic suspension. Antibiotic-resistant strains were as susceptible to PDI in biofilms as in planktonic suspensions, but the inactivation of bacterial cells in biofilms was attenuated. In addition, gram-positive bacterial strains and biofilms were more susceptible than gram-negative strains and biofilms to the PDI effect of HMME. Thus, HMME is a promising photosensitizer for the treatment of infectious diseases caused by antibiotic-resistant bacteria, especially gram-positive bacteria.

Photodynamic inactivation of Klebsiella pneumoniae biofilms and planktonic cells by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

The treatment of Klebsiella pneumoniae, particularly extended-spectrum ß-lactamase (ESBL)-producing K. pneumoniae, is currently a great challenge. Photodynamic antimicrobial chemotherapy is a promising approach for killing antibiotic-resistant bacteria. The aim of this study was to evaluate the capacity of 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL) in the presence of white light to cause photodynamic inactivation (PDI) of K. pneumoniae planktonic and biofilm cells. In the presence of white light, 5-ALA and MAL inactivated planktonic cells in a concentration-dependent manner. Biofilms were also sensitive to 5-ALA and MAL-mediated PDI. The mechanisms by which 5-ALA and MAL caused PDI of ESBL-producing K. pneumonia were also investigated. Exposure of K. pneumonia to light in the presence of either 5-ALA or MAL induced cleavage of genomic DNA and the rapid release of intracellular biopolymers. Intensely denatured cytoplasmic contents and aggregated ribosomes were also detected by transmission electron microscopy. Scanning electron microscopy showed that PDI of biofilms caused aggregated bacteria to detach and that the bacterial cell envelope was damaged. This study provides insights into 5-ALA and MAL-mediated PDI of ESBL-producing K. pneumoniae.

Factors on trough teicoplanin levels, associations between levels, efficacy and safety in patients with gram-positive infections.

OBJECTIVE: The objective of this study was to identify the factors influencing trough teicoplanin concentrations (C(min)), to investigate the relationship between teicoplanin C(min) with efficacy and safety, and to determine a target therapeutic concentration. METHODS: An analysis was performed on 95 serum concentrations from 50 patients with gram-positive infections who received teicoplanin treatment. Teicoplanin serum concentrations were measured by high-performance liquid chromatography. Univariate and multivariable analysis were performed to investigate the effect of independent variables on teicoplanin C(min). A logistic regression analysis was used to determine the relationship between teicoplanin C(min) and efficacy and safety. RESULTS: Teicoplanin therapy was effective in 74.0% (37/50) of patients, and 10.0% (5/50) of patients exhibited signs of adverse events. Using multivariable linear regression, two covariates were found to be a significant effect on teicoplanin C(min): dosage (mg/kg), and creatinine clearance rate (CL(cr). There was no covariate that has a significant impact on the safety of teicoplanin and only teicoplanin C(min) has a significant impact on the efficacy of treatment in the logistics regression. The logistics regression analysis showed that teicoplanin C(min) of 10 mg/L was associated with a 79.4% probability of success response. CONCLUSIONS: This study highlighted that teicoplanin C(min) was strongly influenced by the values of dosage (mg/kg) and CL(cr) and the teicoplanin C(min) range of 10 -€“ 20 mg/L was identified as the therapeutic target with optimum clinical efficacy and safety.

Mechanistic aspects of the photodynamic inactivation of vancomycin-resistant Enterococci mediated by 5-aminolevulinic acid and 5-aminolevulinic acid methyl ester.

Vancomycin-resistant Enterococci (VRE) is a serious concern for public health. Serious infections with VRE have very limited effective antimicrobial therapy, and alternative treatment approaches are highly desirable. One promising approach might be the photodynamic antimicrobial chemotherapy. In the present study, we investigated the photodynamic inactivation (PDI) of two VRE strains mediated by 5-aminolevulinic acid (5-ALA) and its derivative 5-ALA methyl ester (MAL). The photodynamic damages to bacteria on the level of genomic DNA, the leakage of cell components, and the changes of membrane structure were investigated. After treated with 10 mM 5-ALA and irradiated by the 633 ± 10 nm LED for 60 min, 5.37 and 5.22 log10 reductions in bacterial survival were achieved for the clinical isolate of VRE and E. faecalis (ATCC 51299), respectively. After treated with 10 mM MAL and irradiated by the LED for 60 min, 5.02 and 4.91 log10 reductions in bacterial survival were observed for the two VRE strains, respectively. In addition, the photocleavage on genomic DNA and the rapid release of intracellular biopolymers were detected in PDI-treated bacteria. The intensely denatured cytoplasm and the aggregated ribosomes were also found in PDI-treated bacteria by transmission electron microscopy. Although 5-ALA and MAL-mediated PDI could induce the photocleavage on genomic DNA, the PDI of the two VRE strains might be predominantly attributed to the envelope injury, the intracellular biopolymers leakage, and the cytoplasm denature.

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.

Septicemia caused by Leifsonia aquatica in a healthy patient after retinal reattachment surgery.

Leifsonia aquatica is an aquatic bacterium that is typically found in environmental water habitats. Infections due to L. aquatica are rare and commonly catheter associated in immunocompromised patients. We report the first case of an acute septicemia caused by L. aquatica in a healthy immunocompetent host after cryopexy in the absence of a catheter.

An improved susceptibility test based on Amberlite reveals the potential antilisterial activity of fosfomycin in vitro.

Listeria monocytogenes is resistant to fosfomycin in vitro but is susceptible in vivo due to increased expression of positive regulator factor A (PrfA) and its dependent factor, hexose phosphate transporter (Hpt), upon infection of host cells. Amberlite, a polymeric adsorbent resin, could induce PrfA-dependent gene expression and thus, in theory, improve the sensitivity of L. monocytogenes to fosfomycin in vitro. In the current study, an improved susceptibility test based on Amberlite was developed using reference strains. Thirty-five clinical isolates were further examined to verify those preliminary results. Briefly, Amberlite increased in vitro fosfomycin sensitivity of all strains. Optimal Amberlite concentrations, as evaluated through the expression of phospholipase B (PlcB) and Hpt, were 10% and 15% (w/v) in agar media and 3% (w/v) in broth media. Mueller-Hinton (MH) medium, tryptone soya (TS) medium, and brain heart infusion (BHI) medium were used to verify the results in the control strains using agar dilution and broth micro- and macro-dilution methods. Better listerial growth was shown in TS and BHI than in MH. Both broth dilution methods yielded lower minimal inhibitory concentration (MIC) of fosfomycin than the agar dilution method. The MIC of fosfomycin for 35 clinical isolates was 2-32 µg/mL, suggesting improved susceptibility. In conclusion, in vitro sensitivity of L. monocytogenes to fosfomycin was substantially improved in the presence of 3% Amberlite-supplemented TSB or BHIB and the broth microdilution method. This improved method revealed the potential antilisterial activity of fosfomycin in vitro and could facilitate the therapy of listeriosis using fosfomycin.

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.

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.

[An antimicrobial resistance surveillance of gram-positive cocci isolated from 12 teaching hospitals in China in 2009].

OBJECTIVE: To investigate antimicrobial resistance among gram-positive cocci in China in 2009. METHODS: From June to December 2009, 1169 consecutive and non-repetitive gram-positive cocci were collected from 12 teaching hospitals at 9 cities. The minimal inhibitory concentration (MIC) of antibacterial agents was determined by agar dilution method. RESULTS: The prevalences of methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant coagulase-negative Staphylococci (MRCoNS) were 45.3% (211/466) and 89.5% (214/239), respectively. The isolation rate of MRSA was 33.3% - 68.1% from different samples. All Staphylococci isolates were susceptible to vancomycin, teicoplanin and linezolid. Five point five percent (7/128) E.faecium strains were resistant to vancomycin. All E. faecalis strains were susceptible to vancomycin. About 99.1% (108/109) of E.faecalis and E.faecium were susceptible to linezolid. The prevalence of penicillin-intermediate Streptococcus pneumoniae (PISP) was 21.6% (48/222). Only 1 (0.5%, 1/222) Streptococcus pneumoniae strain was resistant to penicillin. Teicoplanin, vancomycin, linezolid and tigecycline were the most active agents against Streptococcus pneumoniae (susceptible rate 100%). CONCLUSIONS: The high prevalence of methicillin-resistance is among Staphylococcus strains. Different samples show a different MRSA prevalence. Teicoplanin, vancomycin and linezolid show very high activity to Staphylococci, E. faecalis, E. faecium and Streptococcus pneumoniae.

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.

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.

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.

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.