Bioisosteric investigation of ebselen: Synthesis and in vitro characterization of 1,2-benzisothiazol-3(2H)-one derivatives as potent New Delhi metallo-ß-lactamase inhibitors.

Carbapenem-resistant Enterobacteriaceae (CRE) producing New Delhi metallo-ß-lactamase (NDM-1) cause untreatable bacterial infections, posing a significant threat to human health. In the present study, by employing the concept of bioisosteric replacement of the selenium moiety of ebselen, we have designed, synthesized and characterized a small compound library of 2-substituted 1,2-benzisothiazol-3(2H)-one derivatives and related compounds for evaluating their cytotoxicity and synergistic activity in combination with meropenem against the E. coli Tg1 (NDM-1) strain. The most promising compound 3a demonstrated potent synergistic activity against a panel of clinically isolated NDM-1 positive CRE strains with FICI as low as 0.09. Moreover, its IC50 value and inhibition mechanism were also confirmed by using the enzyme inhibition assay and the ESI-MS analysis respectively. Importantly, compound 3a has acceptable toxicity and is not a PAINS. Because of its structural simplicity and potent synergistic activity in combination with meropenem, we propose that compound 3a may be a promising meropenem adjuvant and a new series of such compounds may worth further investigations.

Boosting the efficacy of anti-MRSA ß-lactam antibiotics via an easily accessible, non-cytotoxic and orally bioavailable FtsZ inhibitor.

The rapid emergence of methicillin-resistant Staphylococcus aureus (MRSA) strains has undermined the therapeutic efficacy of existing ß-lactam antibiotics (BLAs), prompting an urgent need to discover novel BLAs adjuvants that can potentiate their anti-MRSA activities. In this study, cytotoxicity and antibacterial screening of a focused compound library enabled us to identify a compound, namely 28, which exhibited low cytotoxicity against normal cells and robust in vitro bactericidal synergy with different classes of BLAs against a panel of multidrug-resistant clinical MRSA isolates. A series of biochemical assays and microscopic studies have revealed that compound 28 is likely to interact with the S. aureus FtsZ protein at the T7-loop binding pocket and inhibit polymerization of FtsZ protein without interfering with its GTPase activity, resulting in extensive delocalization of Z-ring and morphological changes characterized by significant enlargement of the bacterial cell. Animal studies demonstrated that compound 28 had a favorable pharmacokinetic profile and exhibited potent synergistic efficacy with cefuroxime antibiotic in a murine systemic infection model of MRSA. Overall, compound 28 represents a promising lead of FtsZ inhibitor for further development of efficacious BLAs adjuvants to treat the staphylococcal infection.

Investigation of synergistic antimicrobial effects of the drug combinations of meropenem and 1,2-benzisoselenazol-3(2H)-one derivatives on carbapenem-resistant Enterobacteriaceae producing NDM-1.

The worldwide prevalence of NDM-1-producing bacteria has drastically undermined the clinical efficacy of the last line antibiotic of carbapenems, prompting a need to devise effective strategy to preserve their clinical value. Our previous studies have shown that ebselen can restore the efficacy of meropenem against a laboratory strain that produces NDM-1. Here we report the construction of a focused compound library of 1,2-benzisoselenazol-3(2H)-one derivatives which comprise a total of forty-six candidate compounds. The structure-activity relationship of these compounds and their potential to serve as an adjuvant to enhance the antimicrobial efficacy of meropenem against a collection of clinical NDM-1-producing carbapenem-resistant Enterobacteriaceae isolates was examined. Drug combination assays indicated that these derivatives exhibited synergistic antimicrobial activity when used along with meropenem, effectively restoring the activity of carbapenems against the resistant strains tested in a Galleria mellonella larvae in vivo infection model. The mode of inhibition of one compound, namely 11_a38, which was depicted when tested on the purified NDM-1 enzyme, indicated that it could covalently bind to the enzyme and displaced one zinc ion from the active site. Overall, this study provides a novel 1,2-benzisoselenazol-3(2H)-one scaffold that exhibits strong synergistic antimicrobial activity with carbapenems, and low cytotoxicity. The prospect of application of such compounds as carbapenem adjuvants warrants further evaluation.

Development of a Novel Quantum Dots and Graphene Oxide Based FRET Assay for Rapid Detection of invA Gene of Salmonella.

A novel, rapid and simple fluorescence resonance energy transfer (FRET) based Salmonella specific gene, invA, detection system was developed, in which quantum dots (QDs) and graphene oxide (GO) worked as fluorescent donor and quencher, respectively. By measuring the fluorescence intensity signal, the Salmonella specific invA gene could be sensitively and specifically detected with a limit of detection (LOD) of ∼4 nM of the invA gene in 20 min. The developed system has the potential to be used for Salmonella detection in food and environmental samples and further developed into a platform for detection of other bacterial pathogens.

Efficient Synthesis of Amine-Linked 2,4,6-Trisubstituted Pyrimidines as a New Class of Bacterial FtsZ Inhibitors.

We have recently identified a new class of filamenting temperature-sensitive mutant Z (FtsZ)-interacting compounds that possess a 2,4,6-trisubstituted pyrimidine-quinuclidine scaffold with moderate antibacterial activity. Employing this scaffold as a molecular template, a compound library of amine-linked 2,4,6-trisubstituted pyrimidines with 99 candidates was successfully established by employing an efficient convergent synthesis designed to explore their structure-activity relationship. The results of minimum inhibitory concentration (MIC) assay against Staphylococcus aureus strains and cytotoxicity assay against the mouse L929 cell line identified those compounds with potent antistaphylococcal properties (MIC ranges from 3 to 8 µg/mL) and some extent of cytotoxicity against normal cells (IC50 ranges from 6 to 27 µM). Importantly, three compounds also exhibited potent antibacterial activities against nine clinically isolated methicillin-resistant S. aureus (MRSA) strains. One of the compounds, 14av_amine16, exhibited low spontaneous frequency of resistance, low toxicity against Galleria mellonella larvae, and the ability to rescue G. mellonella larvae (20% survival rate at a dosage of 100 mg/kg) infected with a lethal dose of MRSA ATCC 43300 strain. Biological characterization of compound 14av_amine16 by saturation transfer difference NMR, light scattering assay, and guanosine triphosphatase hydrolysis assay with purified S. aureus FtsZ protein verified that it interacted with the FtsZ protein. Such a property of FtsZ inhibitors was further confirmed by observing iconic filamentous cell phenotype and mislocalization of the Z-ring formation of Bacillus subtilis. Taken together, these 2,4,6-trisubstituted pyrimidine derivatives represent a novel scaffold of S. aureus FtsZ inhibitors.

Rapid and simultaneous detection of Mycobacterium tuberculosis complex and Beijing/W genotype in sputum by an optimized DNA extraction protocol and a novel multiplex real-time PCR.

Rapid diagnosis and genotyping of Mycobacterium tuberculosis by molecular methods are often limited by the amount and purity of DNA extracted from body fluids. In this study, we evaluated 12 DNA extraction methods and developed a highly sensitive protocol for mycobacterial DNA extraction directly from sputa using surface-coated magnetic particles. We have also developed a novel multiplex real-time PCR for simultaneous identification of M. tuberculosis complex and the Beijing/W genotype (a hypervirulent sublineage of M. tuberculosis) by using multiple fluorogenic probes targeting both the M. tuberculosis IS6110 and the Rv0927c-pstS3 intergenic region. With reference strains and clinical isolates, our real-time PCR accurately identified 20 non-Beijing/W and 20 Beijing/W M. tuberculosis strains from 17 different species of nontuberculosis Mycobacterium (NTM). Further assessment of our DNA extraction protocol and real-time PCR with 335 nonduplicate sputum specimens correctly identified all 74 M. tuberculosis culture-positive specimens. In addition, 15 culture-negative specimens from patients with confirmed tuberculosis were also identified. No cross-reactivity was detected with NTM specimens (n = 31). The detection limit of the assay is 10 M. tuberculosis bacilli, as determined by endpoint dilution analysis. In conclusion, an optimized DNA extraction protocol coupled with a novel multiprobe multiplex real-time PCR for the direct detection of M. tuberculosis, including Beijing/W M. tuberculosis, was found to confer high sensitivity and specificity. The combined procedure has the potential to compensate for the drawbacks of conventional mycobacterial culture in routine clinical laboratory setting, such as the lengthy incubation period and the limitation to viable organisms.

Delineation of a bacterial starvation stress response network which can mediate antibiotic tolerance development.

This study aimed at elucidating the physiological basis of bacterial antibiotic tolerance. By use of a combined phenotypic and gene knockout approach, exogenous nutrient composition was identified as a crucial environmental factor which could mediate progressive development of tolerance with markedly varied drug specificity and sustainability. Deprivation of amino acids was a prerequisite for tolerance formation, conferring condition-specific phenotypes against inhibitors of cell wall synthesis and DNA replication (ampicillin and ofloxacin, respectively), according to the relative abundances of ammonium salts, phosphate, and nucleobases. Upon further depletion of glucose, this variable phase consistently evolved into a sustainable mode, along with enhanced capacity to withstand the effect of the protein synthesis inhibitor gentamicin. Nevertheless, all phenotypes produced during spontaneous nutrient depletion lacked the sustainable, multidrug-tolerant features exhibited by the stationary-phase population and were attributed to complex interaction between starvation-mediated metabolic and stress protection responses on the basis of the following reasons: (i) the nutrition-dependent tolerance characteristics observed suggested that adaptive biosynthetic mechanisms could suppress but not fully avert tolerance under transient starvation conditions; (ii) formation of specific phenotypes could be inhibited by suppressing protein synthesis prior to nutrient depletion; (iii) bacteriostatic drugs produced only weak tolerance in the absence of starvation signals; and (iv) the attenuation of the stringent and SOS responses, as well as the functionality of other putative tolerance determinants, including rpoS, hipA, glpD, and phoU, could alter the induction requirement and drug specificity of the resultant phenotypes. These data reveal the common physiological grounds characteristic of starvation responses and the onset of antibiotic tolerance in bacteria.

Application of denaturing HPLC to rapidly identify rifampicin-resistant Mycobacterium tuberculosis in low- and high-prevalence areas.

OBJECTIVES: Since the emergence of multidrug-resistant and extensively drug-resistant tuberculosis, there has been a call for a rapid assay to detect rifampicin-resistant strains that can be implemented into a routine service to analyse all strains in a specific geographical location. Denaturing HPLC (dHPLC) is a rapid screening test that can detect mutations in PCR amplicons. The aim of this study was to evaluate the dHPLC analysis of rifampicin-resistant Mycobacterium tuberculosis isolates using an extensive strain collection from Hong Kong and the UK and a collection of 84 consecutive clinical isolates. METHODS: DNA from 51 rifampicin-resistant M. tuberculosis strains from the UK and Hong Kong identified from 1996 to 2005 was extracted and each mutation was defined by capillary electrophoresis. A 400 bp PCR product was amplified from each strain, heteroduplexed with a known susceptible control (H37Rv) and analysed by dHPLC at 67.0 degrees C. RESULTS: Forty-five out of 51 (88.2%) rifampicin-resistant strains with known DNA mutations were detected by dHPLC. Two out of 84 clinical isolates were phenotypically rifampicin-resistant and dHPLC detected a mutation in the rpoB amplicon for both these isolates. dHPLC detected a mutation in 1 out of 82 phenotypically rifampicin-susceptible isolates (M482T, a non-cluster I/II mutation). In a combined analysis of all strains and isolates, mutation detection by dHPLC analysis exhibited 88.2% sensitivity and 98.8% specificity. CONCLUSIONS: This study shows that dHPLC analysis is sensitive and specific and could be implemented in a routine clinical service.

Rapid multiplex nested PCR for detection of respiratory viruses.

Respiratory tract infections can be caused by a heterogeneous group of viruses and bacteria that produce similar clinical presentations. Specific diagnosis therefore relies on laboratory investigation. This study developed and evaluated five groups of multiplex nested PCR assays that could simultaneously detect 21 different respiratory pathogens: influenza A virus (H1N1, H3N2, and H5N1); influenza B virus; parainfluenza virus types 1, 2, 3, 4a, and 4b; respiratory syncytial virus A and B; human rhinoviruses; human enteroviruses; human coronaviruses OC43 and 229E; severe acute respiratory syndrome coronavirus; human metapneumoviruses; Mycoplasma pneumoniae; Chlamydophila pneumoniae; Legionella pneumophila; and adenoviruses (A to F). These multiplex nested PCRs adopted fast PCR technology. The high speed of fast PCR (within 35 min) greatly improved the efficiency of these assays. The results show that these multiplex nested PCR assays are specific and more sensitive (100- to 1,000-fold) than conventional methods. Among the 303 clinical specimens tested, the multiplex nested PCR achieved an overall positive rate of 48.5% (95% confidence interval [CI], 42.9 to 54.1%), which was significantly higher than that of virus isolation (20.1% [95% CI, 15.6 to 24.6%]) and that of direct detection by immunofluorescence assay (13.5% [95% CI, 9.7 to 17.4%]). The improved sensitivity was partly due to the higher sensitivity of multiplex nested PCR than that of conventional methods in detecting cultivatable viruses. Moreover, the ability of the multiplex nested PCR to detect noncultivatable viruses, particularly rhinoviruses, coronavirus OC43, and metapneumoviruses, contributed a major gain (15.6%) in the overall positive rate. In conclusion, rapid multiplex nested PCR assays can improve the diagnostic yield for respiratory infections to allow prompt interventive actions to be taken.

Genetic and phenotypic characterization of drug-resistant Mycobacterium tuberculosis isolates in Hong Kong.

OBJECTIVES: To characterize 250 drug-resistant Mycobacterium tuberculosis (MTB) isolates in Hong Kong with respect to their drug susceptibility phenotypes to five common anti-tuberculosis drugs (ofloxacin, rifampicin, ethambutol, isoniazid and pyrazinamide) and the relationship between such phenotypes and the patterns of genetic mutations in the corresponding resistance genes (gyrA, rpoB, embB, katG, inhA, ahpC and pncA). METHODS: The MIC values of the aforementioned anti-tuberculosis drugs were determined for each of the 250 drug-resistant MTB clinical isolates by the absolute concentration method. Genetic mutations in the corresponding resistance genes in these MTB isolates were identified by PCR-single-stranded conformation polymorphism/multiplex PCR amplimer conformation analysis (SSCP/MPAC), followed by DNA sequencing of the purified PCR products. RESULTS: Resistance to four or five drugs was commonly observed in these MTB isolates; such phenotypes accounted for over 34% of the 250 isolates. The most frequently observed phenotypes were those involving both rifampicin and isoniazid, with or without additional resistance to the other drugs. A total of 102 novel mutations, which accounted for 80% of all mutation types detected in the 7 resistance genes, were recovered. Correlation between phenotypic and mutational data showed that genetic changes in the gyrA, rpoB and katG genes were more consistently associated with a significant resistance phenotype. Despite this, however, a considerable proportion of resistant MTB isolates were found to harbour no detectable mutations in the corresponding gene loci. CONCLUSIONS: These findings expand the spectrum of potential resistance-related mutations in MTB clinical isolates and help consolidate the framework for the development of molecular methods for delineating the drug susceptibility profiles of MTB isolates in clinical laboratories.

High-level gentamicin resistance mediated by a Tn4001-like transposon in seven nonclonal hospital isolates of Streptococcus pasteurianus.

We report on the first occurrence of high-level gentamicin resistance (MICs > or = 512 microg/ml) in seven clinical isolates of Streptococcus pasteurianus from Hong Kong. These seven isolates were confirmed to be the species S. pasteurianus on the basis of nucleotide sequencing of the superoxide dismutase (sodA) gene. Epidemiological data as well as the results of pulse-field gel electrophoresis analysis suggested that the seven S. pasteurianus isolates did not belong to the same clone. Molecular characterization showed that they carried a chromosomal, transposon-borne resistance gene [aac(6')Ie-aph(2'')Ia] which was known to encode a bifunctional aminoglycoside-modifying enzyme. The genetic arrangement of this transposon was similar to that of Tn4001, a transposon previously recovered from Staphylococcus aureus and other gram-positive isolates. Genetic linkage with other resistance elements, such as the ermB gene for erythromycin resistance, was not evident. On the basis of these findings, we suggest that routine screening for high-level gentamicin resistance should be recommended for all clinically significant blood culture isolates. This is to avoid the inadvertent use of short-course combination therapy with penicillin and gentamicin, which may lead to the failure of treatment for endocarditis, the selection of drug-resistant Streptococcus pasteurianus and other gram-positive organisms, as well as the unnecessary usage of gentamicin, a drug with potential toxicity.

Multiplex PCR amplimer conformation analysis for rapid detection of gyrA mutations in fluoroquinolone-resistant Mycobacterium tuberculosis clinical isolates.

A new strategy known as multiplex PCR amplimer conformation was developed for detection of mutation in the gyrA gene of 138 clinical isolates of Mycobacterium tuberculosis. The method generated a single-stranded and heteroduplex DNA banding pattern of multiplex PCR amplimers of the region of interest that was extremely sensitive to specific mutations, thus enabling much more sensitive and reliable mutation analysis compared to the standard single-stranded conformation polymorphism technique. The genetic profiles of the gyrA gene of the 138 isolates as detected by MPAC were confirmed by nucleotide sequencing and were found to correlate strongly with the in vitro susceptibilities of the mutant strains to six fluoroquinolones (ofloxacin, levofloxacin, sparfloxacin, moxifloxacin, gatifloxacin, and sitafloxacin). All 32 isolates that contained gyrA mutations exhibited cross-resistance to the six fluoroquinolones (ofloxacin MIC for 90% of strains > 16 mg/liter), although moxifloxacin, gatifloxacin, and sitafloxacin (MIC for 90% of strains /==" BORDER="0"> 16 mg/liter). All gyrA mutations were clustered in codons 90, 91, and 94, and aspartic acid 94 was most frequently mutated. Twenty-three isolates without gyrA mutations were also found to exhibit reduced susceptibility to ofloxacin (MIC for 90% of strains = 4 mg/liter), but largely remained susceptible to other drugs (MIC for 90% of strains