Phenotypic detection of AmpC ß-lactamases, extended-spectrum ß-lactamases and metallo-ß-lactamases in Enterobacteriaceae using a resazurin microtitre assay with inhibitor-based methods.

Dissemination of antibiotic resistance in Enterobacteriaceae mediated by AmpC ß-lactamase, extended-spectrum ß-lactamase (ESBL) and metallo-ß-lactamase (MBL) is clinically significant. A simple and relatively quick method for the detection of these resistance phenotypes would greatly improve chemotherapeutic recommendation. This technology would provide valuable input in our surveillance of resistance on a global stage, particularly if the methodology could be applicable to resource-poor settings. A resazurin microtitre plate (RMP) assay incorporating cloxacillin, clavulanic acid and EDTA for the rapid phenotypic identification of AmpC, ESBL and MBL and the co-existence of ß-lactamases has been developed. A total of 47 molecularly characterized Enterobacteriaceae clinical isolates producing AmpCs, ESBLs, co-producers of ESBL and AmpC, MBLs and co-producers of ESBL and MBL were phenotypically examined using the RMP assay. The ceftazidime- and cefotaxime-based RMP assays successfully detected all 16 AmpC, 14 ESBL and 9 MBL producers, 6 ESBL-AmpC co-producers and 2 ESBL-MBL co-producers without false-positive results. The ceftazidime-based assay was more reliable in detecting AmpC alone, while the cefotaxime-based assay performed better in identifying co-producers of ESBL and AmpC. There was no difference in the detection of ESBL and MBL producers. The findings of the present study suggest that use of the RMP assay with particular ß-lactamase inhibitors explicitly detects three different ß-lactamases, as well as co-existence of ß-lactamases, within 6 h of initial isolation of the pathogen. This assay is applicable to carry out in any laboratory, is cost-effective and is easy to interpret. It could be implemented in screening patients and controlling infection and for surveillance purposes.

Synergistic activity and mechanism of action of Stephania suberosa Forman extract and ampicillin combination against ampicillin-resistant Staphylococcus aureus.

BACKGROUND: Ampicillin-resistant S. aureus (ARSA) now poses a serious problem for hospitalized patients, and their care providers. Plant-derived antibacterial that can reverse the resistance to well-tried agents which have lost their original effectiveness are the research objectives of far reaching importance. To this aim, the present study investigated antibacterial and synergistic activities of Stephania suberosa extracts (SSE) against ARSA when used singly and in combination with ampicillin. RESULTS: The majority chemical compounds of SSE were alkaloid (526.27 ± 47.27 mg/1 g of dried extract). The Minimum inhibitory concentration (MICs) for ampicillin and SSE against all ARSA strains were >512 µg/ml and 4 mg/ml, respectively. Checkerboard assay revealed synergistic activity in the combination of ampicillin (0.15 µg/ml) and SSE (2 mg/ml) at fractional inhibitory concentration index (FICI) <0.5. The killing curve assay had confirmed that the viability of ARSA was dramatically reduced from 5 x 10(5) cfu/ml to 10(3) cfu/ml within 6 h after exposure to SSE (2 mg/ml) plus ampicillin (0.15 µg/ml) combination. Electron microscopic study clearly revealed that these ARSA cells treated with this combination caused marked morphological damage, peptidoglycan and cytoplasmic membrane damage, and average cell areas significant smaller than control. Obviously, Immunofluorescence staining and confocal microscopic images confirmed that the peptidoglycan of these cells were undoubtedly disrupted by this combination. Furthermore, the CM permeability of ARSA was also increased by this combination. Enzyme assay demonstrated that SSE had an inhibitory activity against ß-lactamase in concentrations manner. CONCLUSIONS: So, these findings provide evidence that SSE has the high potential to reverse bacterial resistance to originate traditional drug susceptibility of it and may relate to three modes of actions of SSE: (1) inhibits peptidoglycan synthesis, resulting in morphological damage, (2) inhibits ß-lactamases activity, and (3) increases CM permeability. It is widely recognized that many types of drugs are derived from alkaloids. So, this SSE offers the prominent potential to develop a novel adjunct phytopharmaceutical to ampicillin for the treatment of ARSA. Further active ingredients study, toxicity of it, and the synergistic effect on blood and tissue should be performed and confirmed in an animal test or in humans.