Synthesis, Structure Elucidation, Antibacterial Activities, and Synergistic Effects of Novel Juglone and Naphthazarin Derivatives Against Clinical Methicillin-Resistant Staphylococcus aureus Strains.

Infections caused by drug-resistant bacteria are a serious threat to human and global public health. Moreover, in recent years, very few antibiotics have been discovered and developed by pharmaceutical companies. Therefore, there is an urgent need to discover and develop new antibacterial agents to combat multidrug-resistant bacteria. In this study, two novel series of juglone/naphthazarin derivatives (43 compounds) were synthesized and evaluated for their antibacterial properties against various clinical and reference Gram-positive MSSA, clinical Gram-positive MRSA, and clinical and reference Gram-negative bacteria E. coli and P. aeruginosa. These strains are of clinical importance because they belong to ESKAPE pathogens. Compounds 3al, 5ag, and 3bg showed promising activity against clinical and reference MSSA (MIC: 1-8 µg/ml) and good efficacy against clinical MRSA (MIC: 2-8 µg/ml) strains. 5am and 3bm demonstrated better activity on both MSSA (MIC: 0.5 µg/ml) and MRSA (MIC: 2 µg/ml) strains. Their MICs were similar to those of cloxacillin against clinical MRSA strains. The synergistic effects of active compounds 3al, 5ag, 5am, 3bg, and 3bm were evaluated with reference antibiotics, and it was found that the antibiotic combination with 3bm efficiently enhanced the antimicrobial activity. Compound 3bm was found to restore the sensitivity of clinical MRSA to cloxacillin and enhanced the antibacterial activity of vancomycin when they were added together. In the presence of 3bm, the MIC values of vancomycin and cloxacillin were lowered up to 1/16th of the original MIC with an FIC index of 0.313. Moreover, compounds 3al, 5ag, 5am, 3bg, and 3bm did not present hemolytic activity on sheep red blood cells. In silico prediction of ADME profile parameter results for 3bm is promising and encouraging for further development.

Determination of a Tentative Epidemiological Cut-Off Value (ECOFF) for Dalbavancin and Enterococcus faecium.

Dalbavancin is a lipoglycopeptide antibiotic that shows potent activity against Gram-positive bacteria. It circumvents vanB-type glycopeptide resistance mechanisms; however, data on the in vitro activity of dalbavancin for Enterococcus faecium (E. faecium) are scarce, and thus, no breakpoints are provided. In recent years, there has been a continuing shift from vanA-type to vanB-type vancomycin-resistance in enterococci in Central Europe. Therefore, we aimed to investigate the in vitro activity of dalbavancin against different van-genotypes, with particular focus on vanB-type E. faecium. Dalbavancin susceptibility was determined for 25 van-negative, 50 vanA-positive, and 101 vanB-positive clinical E. faecium isolates (typed by cgMLST). Epidemiological Cut-Off Values (ECOFFs) were determined using ECOFFinder. For vanB-type E. faecium isolates, dalbavancin MICs were similar to those of vancomycin-susceptible isolates reaching values no higher than 0.125 mg/L. ECOFFs for van-negative and vanB-positive isolates were 0.5 mg/l and 0.25 mg/L respectively. In contrast, E. faecium possessing vanA predominantly showed dalbavancin MICs >8 mg/L, therefore preventing the determination of an ECOFF. We demonstrated the potent in vitro activity of dalbavancin against vancomycin-susceptible and vanB-type E. faecium. On the basis of the observed wildtype distribution, a dalbavancin MIC of 0.25 mg/L can be suggested as a tentative ECOFF for E. faecium.

Revisiting colistin susceptibility testing: will adding calcium to Mueller-Hinton agar improve the detection of colistin resistance?

OBJECTIVE: The aim was to investigate whether adding calcium to Mueller-Hinton agar for gradient MIC or disc diffusion tests could improve separation between colistin-susceptible and -resistant populations of Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa and Acinetobacter spp. and if this method could provide a reliable screening test for colistin resistance in routine laboratories. METHODS: An isolate collection of 57 E. coli, K. pneumoniae, P. aeruginosa and Acinetobacter spp. was tested. Ca2+ in concentrations from 2.5 to 40 mM was added to the Mueller-Hinton agar plates used for gradient MIC and disc diffusion tests. Broth microdilution (ISO 20776-1) MIC determination was used as reference. Escherichia coli and K. pneumoniae were investigated for colistin resistance genes. RESULTS: Results were similar for gradient tests and disc diffusion for all species. Correlation between phenotypic expression of resistance and resistance genes was not absolute. Addition of Ca2+ to Mueller-Hinton agar improved separation between colistin-susceptible and -resistant isolates for E. coli. For K. pneumoniae, separation was improved for isolates with mcr genes, but not for isolates harbouring other colistin resistance mechanisms. To further increase the concentrations of Ca2+ did not improve the separation between susceptible and resistant isolates of E. coli and K. pneumoniae. For P. aeruginosa and Acinetobacter species, addition of Ca2+ did not improve separation between susceptible and resistant populations. DISCUSSION: The results from this study show that addition of Ca2+ to the Mueller-Hinton agar does not sufficiently improve detection of colistin resistance by gradient MIC or disc diffusion tests for use in a routine laboratory.

Ultra-rapid flow cytometry assay for colistin MIC determination in Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii.

OBJECTIVES: Both EUCAST and CLSI recommend broth microdilution for antimicrobial susceptibility testing of colistin, but this method is cumbersome and takes 16-24 h to give results. Our objective was to evaluate a rapid quantitative colistin MIC susceptibility assay based on flow cytometry analysis (FASTcolistin MIC) in comparison with standard broth microdilution assay. METHODS: One hundred and sixteen Gram-negative bacilli (78 Enterobacterales, 28 Pseudomonas aeruginosa and 10 Acinetobacter baumannii) were studied in parallel using standard broth microdilution following EUCAST recommendations and FASTcolistin MIC kit. In the last one, a bacteria suspension (0.5 MacFarland) was prepared, diluted in Muller-Hinton broth, incubated in the susceptibility panel containing different colistin concentrations (range 0.125-64 mg/L) with a fluorescent probe and incubated 1 h at 35ºC. After that, a flow cytometry analysis using CytoFLEX (Beckmam) was performed. Using a dedicated software (BioFAST) an automated MIC result was obtained after 1.5 h. Performance evaluation was performed according to the ISO standard 20776-2. Reproducibility and repeatability, categorical (CA) and essential agreement (EA), and lot-to-lot variation and operator-to-operator variability, as well as time to results were determined. RESULTS: Overall, 100% CA (CI 97-100%) and 95.7% EA (CI 90-98%) was obtained with high repeatability (100%; CI 80-100%)and reproducibility (97%; (CI 83-99%)). Absence of lot-to-lot variations or differences in the operators' performance was observed. CONCLUSIONS: FASTcolistin MIC is an accurate, reliable and ultra-rapid method (1 h incubation versus 24 h) for susceptibility testing of colistin of common Gram-negative bacilli recovered in clinical laboratories.

Burkholderia pseudomallei multi-centre study to establish EUCAST MIC and zone diameter distributions and epidemiological cut-off values.

OBJECTIVES: Melioidosis, caused by Burkholderia pseudomallei, requires intensive antimicrobial treatment. However, standardized antimicrobial susceptibility testing (AST) methodology based on modern principles for determining breakpoints and ascertaining performance of methods are lacking for B. pseudomallei. This study aimed to establish MIC and zone diameter distributions on which to set epidemiological cut-off (ECOFF) values for B. pseudomallei using standard EUCAST methodology for non-fastidious organisms. METHODS: Non-consecutive, non-duplicate clinical B. pseudomallei isolates (9-70 per centre) were tested at eight study centres against eight antimicrobials by broth microdilution (BMD) and the EUCAST disc diffusion method. Isolates without and with suspected resistance mechanisms were deliberately selected. The EUCAST Development Laboratory ensured the quality of study materials, and provided guidance on performance of the tests and interpretation of results. Aggregated results were analysed according to EUCAST recommendations to determine ECOFFs. RESULTS: MIC and zone diameter distributions were generated using BMD and disc diffusion results obtained for 361 B. pseudomallei isolates. MIC and zone diameter ECOFFs (mg/L; mm) were determined for amoxicillin-clavulanic acid (8; 22), ceftazidime (8; 22), imipenem (2; 29), meropenem (2; 26), doxycycline (2; none), tetracycline (8; 23), chloramphenicol (8; 22) and trimethoprim-sulfamethoxazole (4; 28). CONCLUSIONS: We have validated the use of standard BMD and disc diffusion methodology for AST of B. pseudomallei. The MIC and zone diameter distributions generated in this study allowed us to establish MIC and zone diameter ECOFFs for the antimicrobials studied. These ECOFFs served as background data for EUCAST to set clinical MIC and zone diameter breakpoints for B. pseudomallei.

Quinolone Susceptibility in Salmonella Isolates Based on Minimum Inhibitory Concentration Determination.

Introduction Typhoid fever, caused by Salmonella typhi and paratyphi , is a generalized infection with case fatality of about 10%. The symptoms may be severe, with life threatening sequelae of infection in a proportion of cases. Antimicrobial agents are the mainstay of therapy in enteric fever so as to prevent the complications associated with severe illness and mortality in the patients. Fluoroquinolones (e.g., ciprofloxacin) are very effective against completely susceptible Salmonella bacteria. However, their efficacy is doubtful once any resistance is detected. Pefloxacin testing has ultimately helped in the accurate identification of quinolone susceptibility for a better therapeutic success rate. In the present study we have tried to evaluate the quinolone susceptibility in Salmonella isolates based on minimum inhibitory concentration (MIC) determination. Materials and Methods The method used in the study is quinolone susceptibility in Salmonella isolates based on MIC determination. Salmonella isolates show intermediate susceptibility to ciprofloxacin using disk diffusion. Both ciprofloxacin and pefloxacin MIC evaluation has been done to corroborate the results with pefloxacin disk diffusion testing. Results There was a positive correlation between the susceptibility to ciprofloxacin and pefloxacin. However, the isolates with intermediate susceptibility had variations in terms of susceptibility to pefloxacin. MIC values for pefloxacin and our findings suggested that pefloxacin susceptible on disk diffusion as per Clinical and Laboratory Standards Institute guidelines showed lower values for MIC using Pefloxacin HICOMB test and pefloxacin resistant isolates showed higher MIC values.

Antimicrobial susceptibility testing of colistin - evaluation of seven commercial MIC products against standard broth microdilution for Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Acinetobacter spp.

OBJECTIVE: Both EUCAST and CLSI recommend broth microdilution (BMD) for antimicrobial susceptibility testing of colistin, but BMD is rarely used in routine microbiology laboratories. The objective of this study was to evaluate five commercially available BMD products and two brands of gradient tests for colistin MIC determination using BMD according to ISO standard 20776-1 as reference. METHODS: Colistin MIC determination was performed according to the manufacturer's instructions on five commercially available BMD products (Sensititre, MICRONAUT-S, MICRONAUT MIC-Strip, SensiTest, and UMIC) and two gradient tests (Etest and MIC Test Strip). Colistin reference MICs were determined using frozen panels according to ISO standard 20776-1. An international collection of Gram-negative bacteria (n=75) with varying levels of colistin susceptibility was tested. RESULTS: The colistin BMD products correlated well with reference tests, in particular for Sensititre and the two MICRONAUT products (essential agreement ≥96%: 66/69 (96%, CI 88-99%), 72/75 (96%, CI 88-99%) and 74/75 (99%, CI 92-100%)). The results were somewhat poorer for the BMD products SensiTest and UMIC: EA 88% (51/58, CI 77-95%) and 82% (61/74, CI 72-89%), respectively), and considerably poorer for the gradient tests (EA 43-71% depending on gradient test and Mueller-Hinton agar manufacturer). The gradient tests generally underestimated colistin MICs, resulting in a significant number of false susceptible results (9-18 of total 75 tests, compared with 1-3 for the BMD products). CONCLUSIONS: Based on the results of this study, we advise laboratories not to trust gradient tests for colistin susceptibility testing and to use broth microdilution methods for this purpose. There are several commercial broth microdilution tests available and in principle they perform well.

¿Se debe revisar la determinación de la CIM de colistín en Acinetobacter baumannii? / The colistin MIC determination in Acinetobacter baumannii, Should it be reviewed?

Currently, there is a controversy in how to determine the minimal inhibitory concentration (MIC) of colistin against Acinetobacter baumannii. We compared three methods, concluding that the addition of Tween-80 (0.002%) to Müller-Hinton broth in the microdilution method could improve MIC determination and it could reduce false resistance.

Comparison of Clostridium difficile minimum inhibitory concentrations obtained using agar dilution vs broth microdilution methods.

Due to increasing antibiotic resistance among anaerobic bacteria, routine antimicrobial susceptibility testing is recommended by the Clinical and Laboratory Standards Institute (CLSI). This study compared the minimum inhibitory concentrations (MICs) from 920 Clostridium difficile isolates tested against seven antimicrobial agents using the two current CLSI reference methodologies, agar dilution method, vs broth microdilution method. A subset of isolate testing was performed independently by two laboratories to evaluate reproducibility. A negative bias was noted for MICs generated from broth microdilution compared to agar dilution and the reproducibility was variable and drug dependent. Therefore, broth microdilution is not recommended as an alternative to agar dilution for C. difficile antimicrobial susceptibility testing.

Activity of fluconazole and its Cu(II) complex towards Candida species.

Candida species, although they are present as commensal organisms in the digestive tract of healthy individuals, can produce a broad spectrum of serious illnesses in compromised hosts. Fluconazole, a water-soluble triazole with bioavailability greater than 90 %, has been extensively used to treat a wide range of Candida infections. However, a growing resistance of microorganisms in the treatment leads to the discovery of new drugs or modifications of existing ones. The aim of the present study was to investigate whether coordination of Cu(II) ions to fluconazole affects its antifungal activity. The in vitro susceptibility tests and antifungal studies were performed with two Candida spp.: Candidaglabrata and Candida albicans. Overall, 34 strains of the former and 16 strains of the latter were treated with fluconazole, its Cu(II) complex and free Cu(II) ions. The obtained MIC values in 16 cases of the C. glabrata and in 5 cases of the C. albicans were lower for the complex in comparison to the drug. This implies that the complex is more effective against particular strains than the parent drug. The most significant improvement in the complex drug efficacy was observed for fluconazole-resistant species.

Development of EUCAST disk diffusion method for susceptibility testing of the Bacteroides fragilis group isolates.

With the emergence of antibiotic resistance among Bacteroides fragilis group isolates the need of susceptibility testing in routine laboratories is increasing. The aims of the present study were to evaluate the disk diffusion method for susceptibility testing in case of different clinical isolates of Bacteroides spp by comparing zone diameter results with MICs obtained earlier during an Europe-wide antibiotic susceptibility surveillance, and to propose zone diameter breakpoints, which correlate for the EUCAST MIC breakpoints. We tested 381 clinical isolates of the B. fragilis group to amoxicillin/clavulanic acid, cefoxitin, clindamycin, imipenem, metronidazole, moxifloxacin, piperacillin/tazobactam, tigecycline by agar dilution method previously. The inhibition zones of the same antibiotics including meropenem disc were determined by the disc diffusion on Brucella blood agar supplemented with haemin and vitamin K1. Plates were incubated at 37 °C in an anaerobic atmosphere for 24 h. The zone diameters were read at 100% inhibition. In case of discrepant results MICs were determined by gradient test and compared with the inhibition zones on the same plate. We found a good agreement between the inhibition zone diameters and the MICs for imipenem, metronidazole, moxifloxacin and tigecyclin. The inhibition zone diameters of meropenem also separated clearly the isolates, which can be considered wild-type isolates. In case of amoxicillin/clavulanic acid and piperacillin/tazobactam intermediate and susceptible isolates according to the MIC determination, overlap during the zone diameter determination. Isolates with an inhibition zone <23 mm for amoxicillin/clavulanic acid and <25 mm for piperacillin/tazobactam should be retested by a MIC determination method. The 10 µg clindamycin disc clearly separated the resistant and the susceptible population of B. fragilis group strains. In the case of cefoxitin only resistant population could be separated with an inhibition zone <17 mm, intermediate and susceptible isolates overlap. In conclusion, we suggest that disk diffusion can be an option for susceptibility testing of B. fragilis group isolates for most relevant antibiotics in routine laboratories.

Comparative evaluation of the microplate nitrate reductase assay and the rezasurin microtitre assay for the rapid detection of multidrug resistant Mycobacterium tuberculosis clinical isolates

The microplate nitrate reductase assay (MNRA) and the rezasurin microtitre assay (REMA) were used for the susceptibility testing of 73 clinical isolates and the results were compared with those that were obtained using the Bactec 460 TB and Bactec MGIT 960 systems. The REMA and the MNRA were performed in 96-well plates. For the REMA, the concentrations of isoniazid (INH) and rifampicin (RIF) ranged from 1.0-0.01 µg/mL and 2.0-0.03 µg/mL, respectively. For the MNRA, the INH concentration was between 1.0-0.03 µg/mL and the RIF concentration was between 2.0-0.06 µg/mL. For the MNRA, the sensitivity, specificity, positive predictive value, negative predictive value and INH/RIF agreement were 100/95.6, 97.6/100, 96.8/100, 100/98 and 98.6/98.6, respectively, and for the REMA, they were 100/91.3, 90.4/100, 88.5/100, 100/96.1 and 94.5/97.2, respectively. Our data suggest that these two rapid, low-cost methods may be inexpensive, alternative assays for the rapid detection of multidrug resistant tuberculosis in low-income countries.