Abstract
Antibiotic resistance of bio-threat agents holds major concerns especially in light of advances in methods for engineering pathogens with antibiotic resistance. Preparedness means for rapid identification and prompt proper medical treatment are of need to contain the event and prevent morbidity and spreading of the disease by properly treating exposed individuals before symptoms appearance. Herein, we describe a novel, rapid, simple, specific, and sensitive method named Micro-Agar-PCR-test (MAPt), which determines antibiotic susceptibility of bio-terror pathogens, directly from environmental samples, with no need for any prior isolation, quantification, or enrichment steps. As proof of concept, we have used this approach to obtain correct therapeutic antibiotic minimal inhibitory concentration (MIC) values for the Tier-1 select agents, Bacillus anthracis, Yersinia pestis, and Francisella tularensis, spiked in various environmental samples recapitulating potential bioterror scenarios. The method demonstrated efficiency for a broad dynamic range of bacterial concentrations, both for fast-growing as well as slow-growing bacteria and most importantly significantly shortening the time for accurate results from days to a few hours. The MAPt allows us to address bioterror agents-contaminated environmental samples, offering rational targeted prophylactic treatment, before the onset of morbidity in exposed individuals. Hence, MAPt is expected to provide data for decision-making personal for treatment regimens before the onset of symptoms in infected individuals.
Highlights
COVID-19 pandemic is a wake-up call for bio-threats preparedness
Micro-Agar-PCR-test is based on the combination of two assays: (a) an agar-dilution method that uses dilutions of antibiotics in agar media to determine minimal inhibitory concentration (MIC) values by visual examination of the growth inhibition of bacterial colonies, and (b) a quantitative PCR (qPCR) step which substitutes the visual examination by quantification of the bacterial DNA content of the bacteria
QPCR increases sensitivity allowing quantification of relatively low amounts of bacteria (~104 cfu/ml). This permitted us to perform the MAPt with relatively low-concentration bacterial samples, which is not applicable by the standard antimicrobial susceptibility testing (AST) and to shorten the time of incubation that is need for an answer
Summary
Tier-1 select pathogens are defined as bio-threat microorganisms possessing a high probability of causing a severe consequence event. These agents, which may cause mass casualties, constitute a matter of public health concern either in scenarios of natural outbreaks or their malicious use as weaponized agents which under certain circumstances may transit to an epidemic or even a pandemic. Bacillus anthracis is the causative agent of anthrax, a highly contagious and fatal disease that can infect humans by the cutaneous, gastrointestinal, or respiratory routes (Dixon et al, 1999; Chitlaru et al, 2011). Prompt diagnosis and rapid appropriate antibiotic treatment were shown to lower the rates dramatically (Holly et al, 2006). The Amerithrax incidents in October 2001, where 22 cases of anthrax were recorded, of which five ended in death, are an example of the bioterror potential of B. anthracis
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