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A rapid method for estimation of the efficacy of potential antimicrobials in humans and animals by agar diffusion assay.

ABSTRACT: Drug resistance continues to challenge traditional antimicrobial drugs and limit their clinical utility. This requires us to continue our search for new drug candidates with novel mechanisms of action against infectious diseases. We now describe a simple agar diffusion assay, which can be used as a general method for the rapid detection of antimicrobial activity of drug candidates in animal or human blood plasma for the ultimate prediction of the efficacy of potential drugs prior to clinical trials. We present an example for a clinical candidate against Mycobacterium tuberculosis.

SUBMITTER: Salina EG

PROVIDER: S-EPMC6533168 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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A rapid method for estimation of the efficacy of potential antimicrobials in humans and animals by agar diffusion assay.

Salina Elena G EG Ekins Sean S Makarov Vadim A VA

Chemical biology & drug design 20181123 6

Drug resistance continues to challenge traditional antimicrobial drugs and limit their clinical utility. This requires us to continue our search for new drug candidates with novel mechanisms of action against infectious diseases. We now describe a simple agar diffusion assay, which can be used as a general method for the rapid detection of antimicrobial activity of drug candidates in animal or human blood plasma for the ultimate prediction of the efficacy of potential drugs prior to clinical tri ...[more]

PMID: 30468306

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Project description:Current methods for screening small molecules that inhibit the plasmid pCD1-encoded Yersinia pestis type III secretion system (T3SS) include lengthy growth curves followed by multistep luminescence assays or Western blot assays to detect secretion, or lack thereof, of effector proteins. The goal of this research was to develop a novel disk diffusion assay on magnesium oxalate (MOX) agar as a simple way to evaluate the susceptibility of Y. pestis to type III secretion system inhibitors. MOX agar produces distinct Y. pestis growth characteristics based on the bacteria's ability or inability to secrete effector proteins; small, barely visible colonies are observed when secretion is activated versus larger, readily visible colonies when secretion is inhibited. Wild-type Y. pestis was diluted and spread onto a MOX agar plate. Disks containing 20 μl of various concentrations of imidocarb dipropionate, a known Y. pestis T3SS inhibitor, or distilled water (dH2O) were placed on the plate. After incubation at 37°C for 48 h, visible colonies of Y. pestis were observed surrounding the disks with imidocarb dipropionate, suggesting that T3S was inhibited. The diameter of the growth of colonies surrounding the disks increased as the concentration of the T3SS inhibitor increased. Imidocarb dipropionate was also able to inhibit Y. pestis strains lacking effector Yops and Yop chaperones, suggesting that they are not necessary for T3S inhibition. This disk diffusion assay is a feasible and useful method for testing the susceptibility of Y. pestis to type III secretion system inhibitors and has the potential to be used in a clinical setting. IMPORTANCE Disk diffusion assays have traditionally been used as a simple and effective way to screen compounds for antibacterial activity and to determine the susceptibility of pathogens to antibiotics; however, they are limited to detecting growth inhibition only. Consequently, antimicrobial agents that inhibit virulence factors, but not growth, would not be detected. Therefore, we developed a disk diffusion assay that could detect inhibition of bacterial virulence factors, specifically, type III secretion systems (T3SSs), needle-like structures used by several pathogenic bacteria to inject host cells with effector proteins and cause disease. We demonstrate that magnesium oxalate (MOX) agar can be used in a disk diffusion assay to detect inhibition of the T3SS of Yersinia pestis, the causative agent of bubonic plague, by small-molecule inhibitors. This assay may be useful for screening additional small molecules that target bacterial T3SSs or testing the susceptibility of patient-derived samples to drugs that target T3SSs.

Project description:Beta-lactam and quinolone antimicrobials are commonly used for treatment of infections caused by non-typhoidal Salmonella (NTS) and other pathogens. Resistance to these classes of antimicrobials has increased significantly in the recent years. However, little is known on the genetic basis of resistance to these drugs in Salmonella isolates from Ethiopia.Salmonella isolates with reduced susceptibility to beta-lactams (n?=?43) were tested for genes encoding for beta-lactamase enzymes, and those resistant to quinolones (n?=?29) for mutations in the quinolone resistance determining region (QRDR) as well as plasmid mediated quinolone resistance (PMQR) genes using PCR and sequencing.Beta-lactamase genes (bla) were detected in 34 (79.1%) of the isolates. The dominant bla gene was blaTEM, recovered from 33 (76.7%) of the isolates, majority being TEM-1 (24, 72.7%) followed by TEM-57, (10, 30.3%). The blaOXA-10 and blaCTX-M-15 were detected only in a single S. Concord human isolate. Double substitutions in gyrA (Ser83-Phe?+?Asp87-Gly) as well as parC (Thr57-Ser?+?Ser80-Ile) subunits of the quinolone resistance determining region (QRDR) were detected in all S. Kentucky isolates with high level resistance to both nalidixic acid and ciprofloxacin. Single amino acid substitutions, Ser83-Phe (n?=?4) and Ser83-Tyr (n?=?1) were also detected in the gyrA gene. An isolate of S. Miami susceptible to nalidixic acid but intermediately resistant to ciprofloxacin had Thr57-Ser and an additional novel mutation (Tyr83-Phe) in the parC gene. Plasmid mediated quinolone resistance (PMQR) genes investigated were not detected in any of the isolates. In some isolates with decreased susceptibility to ciprofloxacin and/or nalidixic acid, no mutations in QRDR or PMQR genes were detected. Over half of the quinolone resistant isolates in the current study 17 (58.6%) were also resistant to at least one of the beta-lactam antimicrobials.Acquisition of blaTEM was the principal beta-lactamase resistance mechanism and mutations within QRDR of gyrA and parC were the primary mechanism for resistance to quinolones. Further study on extended spectrum beta-lactamase and quinolone resistance mechanisms in other gram negative pathogens is recommended.

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A rapid method for estimation of the efficacy of potential antimicrobials in humans and animals by agar diffusion assay.

Publications

A rapid method for estimation of the efficacy of potential antimicrobials in humans and animals by agar diffusion assay.

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