Biospeckle laser digital image processing for quantitative and statistical evaluation of the activity of Ciprofloxacin onEscherichia coliK-12

Abstract

47Antibiotic susceptibility testing is a necessary step prior to the treatment of clinical 48 infections. A major concern is the time required to obtain a fast and reliable result. The aim 49 of this work is to use Biospeckle laser in a 15min assay for an antimicrobial susceptibility 50 test of Ciprofloxacin in serial two-fold dilutions on Escherichia coli K-12 using Venereal 51 Disease Research Laboratory (VDRL) plates. Analysis of images by video edition is 52 performed on a quantitatively selected region of interest, and processed with ImageJ-53 ImageDP; and by the construction of time series and analysis with either statistical 54 diagnostics tests or Autoregressive Integrated Moving Average (ARIMA) models. 55 Antimicrobial susceptibility tests are also performed for the purpose of quantitative 56 comparison, showing a profile that is comparable to the result obtained with ImageJ-57 ImageDP processing after 15min of antibiotic action. Only the time series of the least 58 affected bacteria (low Ciprofloxacin concentration) behaves in an expected manner, being 59 non-independent and mainly non-linear, non-normal, and heteroscedastic. The most 60 affected bacteria (higher Ciprofloxacin concentration) are non-independent and tend to be 61 linear, normal and heteroscedastic. Adjustment to a linear regression identifies both, the 62 culture medium without bacteria and the most affected bacteria, normality identifies the 63 most affected bacteria and heteroscedasticity-homoscedasticity distinguishes the presence-64absence of bacteria, respectively. ARIMA models () 11 fit the 65 time series of the most affected bacteria while the latter also fits the culture medium 66 without bacteria. The time series of the least affected bacteria are identified by a 67 (7,1,2)(1,0,1) 11 model. The non-linear, non-normal and heteroscedastic behavior of this 68 group is probably responsible for its adjustment to a model with a relatively high 69 parameter. The four methods: diagnostic statistical tests, fitting of ARIMA models, ImageJ-70 ImageDP and antimicrobial susceptibility tests, show similar results, being able to 71 distinguish among the groups of assays with bacteria and Ciprofloxacin below and above 72 the Minimal Inhibitory Concentration. 74 Author Summary 75Biospeckle laser patterns occur when a dynamic surface is illuminated. This research 76 describes its application to the activity of Escherichia coli bacteria and the effect of 18 77 different concentrations of an antibiotic (Ciprofloxacin) in a 15min assay, using VDRL 78 plates where the sample has a relatively small volume and is flat shaped. The assay is 79 performed on an anti-vibration table in a dark room with a laser that sequentially 80 illuminates each of the wells of the plate. A camera takes short 30sec videos with 81 approximately 750 frames and sends them to a computer where image processing takes 82 place. In order to select a segment of 80 successive frames to analyze, the region with the 83 higher variation was identified, punched out and edited as a "fl...

A prototype biospecklemeter for microbiological analysis: a starting point for a potential digital-image laser antibiotic susceptibility test

“…In this case 20 s videos were taken. In order to process the videos with the method of generalized differences, new videos were edited (Grassi et al 2019) with 40 frames each a A positive result corresponds to a value of 110% or higher with respect to the control value. ND, not determined.…”

“…It has been possible to evaluate the effect of ciprofloxacin on cells of Escherichia coli K-12, using the wells of VDRL plates as containers, a biospeckle laser and data processing (Grassi et al 2019), searching for a potential digital-image laser antibiotic susceptibility test (DILAST). However, new software such as ImageDP and R/SAGA Script (Grassi et al 2016) and new processing methods such as Qxi determination and flip-book animation (Grassi et al 2019) must be evaluated to improve these developments quantitatively and statistically.…”