Detection of ciprofloxacin residues in cow milk: A novel and rapid optical β-galactosidase-based screening assay

    Detection of ciprofloxacin residues in cow milk: A novel and rapid optical β-galactosidase-based screening assay Raviraj M. Kalunke, Gerardo Grasso, Renato D’Ovidio, Roberto Dragone, Chiara Frazzoli PII: DOI: Reference:

S0026-265X(16)30790-1 doi:10.1016/j.microc.2016.12.014 MICROC 2643

To appear in:

Microchemical Journal

Received date: Revised date: Accepted date:

16 September 2016 22 December 2016 25 December 2016

Please cite this article as: Raviraj M. Kalunke, Gerardo Grasso, Renato D’Ovidio, Roberto Dragone, Chiara Frazzoli, Detection of ciprofloxacin residues in cow milk: A novel and rapid optical β-galactosidase-based screening assay, Microchemical Journal (2016), doi:10.1016/j.microc.2016.12.014

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ACCEPTED MANUSCRIPT DETECTION OF CIPROFLOXACIN RESIDUES IN COW MILK: A NOVEL AND RAPID OPTICAL β-GALACTOSIDASE-BASED SCREENING ASSAY

Department of Science and Technology for Agriculture, Forestry, Nature and Energy

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Raviraj M. Kalunke1,4*, Gerardo Grasso2*, Renato D’Ovidio1, Roberto Dragone2, Chiara Frazzoli3

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(DAFNE), University of Tuscia, Via S. Camillo de Lellis snc, 01100 Viterbo, Italy Institute of Nanostructured Materials, Consiglio Nazionale delle Ricerche, P.le Aldo Moro 5,

00185, Rome, Italy

External Relations Office, Istituto Superiore di Sanità, V. Giano della Bella 34, 00162,

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Rome, Italy 4

Present address: Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-

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National Chemical Laboratory, Pune 411008, Maharashtra, India

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E-mails: RMK - [email protected]; GG - [email protected];

Corresponding authors:

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RD [email protected]; RoD - [email protected]; CF - [email protected]

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E-mail address: [email protected] (R. M. Kalunke)

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[email protected] (G. Grasso)

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ACCEPTED MANUSCRIPT Abstract Ciprofloxacin (a member of the fluoroquinolone class) is one of the most widely used antibacterial agents for the treatment of bacterial infections in livestock. The improper use of

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such antibacterial agents could lead to the presence of residues in animal origin foods

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(including milk) and consequently harmful effects for health of consumers, together with the

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spread of antibiotic-resistant bacterial strains. Therefore, in order to support good farming practices and to ensure food safety, antimicrobial (in particular fluoroquinolones) residues surveillance through improved monitoring techniques is crucial. However, commercial available kits for the detection of fluoroquinolones residues in food samples are time-

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consuming and still fail to detect MRL concentrations for fluoroquinolones (e.g. 0.1 mg/kg for the sum of enrofloxacin and ciprofloxacin residues). Here a novel and rapid assay for

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ciprofloxacin residual detection through optical microbiological screening in commercially pasteurized cows' milk samples is described. Escherichia coli ATCC 11303 cell proliferation was optically monitored by measuring endogenous β-gal activity that was determined through

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colorimetric assay in the presence of a chromogenic β-gal artificial substrate. Optical density

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of E. coli cell culture (linked to cell proliferation) was positively correlated with endogenous β-gal activity. As the presence of ciprofloxacin residues inhibits the E. coli cell proliferation

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in tested samples, β-gal levels decreased more in exposed samples than in control. The essential step of β-gal induction (usually obtained by IPTG) was obtained by exploiting the lactose present in the milk. Our findings show a detection of ciprofloxacin residues at 1 MRL

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concentration in 1 hour using ONPG as chromogenic β-gal artificial substrate and lactose as β-gal inducer. Compared to previously described methods, this assay proved to be a rapid, proficient and more eco-friendly (i.e. minimizing the use of additional reagents) system that could be potentially employed as screening method for detection ciprofloxacin residues in cow's milk.

Keywords: Fluoroquinolones, antibiotic resistance, One Health, primary production, HACCP, farm animals, risk assessment; risk management Abbreviations: ATCC, American Type Culture Collection; β-gal, β-galactosidase; IPTG, isopropyl- β-D-1-tiogalattopiranoside; LB, Luria Bertani; CFU, Colony Forming Unit; MRL, Maximum Residual Limit; OD: Optical Density; ONPG, O-nitrophenyl-β-Dgalactopyranoside; X-gal, 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside.

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ACCEPTED MANUSCRIPT 1. Introduction The use of antibiotics and antimicrobials in veterinary practices is widespread not only for the therapeutic treatment of livestock but also for the prevention and etiology of microbial

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diseases. Possible fraudulent (e.g. as dietary growth promoters) or non-controlled uses of

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antibiotics and antimicrobials in zootechny require the development of transferable

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surveillance and monitoring technologies. Quinolones (in particular the subgroup of fluoroquinolones) are among the most commonly used antimicrobial drugs for the treatment of bacterial infections in livestock. Fluoroquinolones possess several useful characteristics that make them suitable for veterinary uses, like a rapid and broad-spectrum antimicrobial

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activity. They are well tolerated by livestock and can be administered by different routes. In particular, ciprofloxacin is the best molecule of the fluoroquinolone group for the treatment

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of E. coli, Salmonella, Pasteurella and Mycoplasma infections in poultry, cattle, pigs and aquaculture livestock [1, 2]. Ciprofloxacin is the active metabolite of fluoroquinolone

period than enrofloxacin [4].

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enrofloxacin and it can be found in milk (as about 90% of the total residue) [3] over a longer

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Residues in animal origin foods are closely monitored by the European Commission Regulation (EU) No 37/2010 [5] (and the previous European Council Regulation (EEC) N

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2377/90) [6] and established MRLs for residues (including fluoroquinolones) (expressed as mg/kg or µg/Kg, e.g. 0.1 mg/kg for the sum of enrofloxacin and ciprofloxacin residues in cow milk) legally permitted and recognized as acceptable in a food [6]. Screening methods as

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defined by European Commission Decision 2002/657 [7] should provide semiquantitative/quantitative results with high throughput, low costs, ease use and rapid analysis. Recently, there has been increase in availability of commercial screening tests or kits for the determination of veterinary drugs that can be divided into microbiological and immunological methods. However, these methods are generally time consuming and do not cover the full spectrum, while immunological methods are antibody based detection that might interfere due to non-specific interactions. Availability of accurate field technologies is a key aspect for food safety from farm to fork. For instance, a rapid and continuous monitoring of the dairy supply chain using appropriate transferable-technology for quick determination of antibiotic residues in milk would represent a real revolution in the dairy livestock sector [8]. Therefore, here we report a novel colorimetric based assay on endogenous β-gal activity as an indirect measure of the cell proliferation of E. coli ATCC 11303 strain in presence of different concentrations of ciprofloxacin in spiked pasteurized 3

ACCEPTED MANUSCRIPT cows' milk samples. This assay was performed comparing X-gal and ONPG as a chromogenic artificial β-gal substrates for enzyme activity estimation. Colorimetric tests were also conducted to assess the possibility of using lactose (naturally present in the tested

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milk samples) as an inducer of lacZ gene and was also tested in ciprofloxacin-spiked cow

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assay for ciprofloxacin residues detection in cow milk.

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milk samples for pre-validation. Our final aim was to develop a rapid and efficient screening

2. Materials and methods

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2.1 Test microorganism culture conditions

The E. coli ATCC 11303 (VA, USA) was grown for 16h in 10 mL LB broth media (Sigma–

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Aldrich, Italy) at 37˚C in a shaking incubator (180 rpm). High-purity deionised water (MilliQ system, Merck Millipore, Billerica, MA, USA) was used for all the solutions prepared for

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this study.

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2.2 Colorimetric assay conditions

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For preliminary colorimetric assays using X-gal as artificial substrate, samples were prepared by inoculating 200 µL of 16h grown culture into 1790 µL of LB broth (10% of inoculum at the concentration of about 1×104 CFU/mL. Preliminary test were performed to calculate

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CFU/mL for a given percentage of E. coli 16h grown culture. Samples preparation was completed by addition of 5 µL of X-gal (≥98%, powder, Sigma–Aldrich, Italy) 50 mg/mL solution and 5 µl (of IPTG (≥99% (TLC), ≤0.1% Dioxane, Sigma–Aldrich, Italy) 1M solution. For preliminary colorimetric assays using ONPG as artificial substrate, 100 µL (400µg) ONPG (stock solution of 4 mg/mL) was used instead of the X-gal solution and incubated for 10 min before spectrophotometric measurements. For E. coli ATCC 11303 culture concentrations vs. relative β-gal activity (using ONPG as artificial substrate) correlation, same condition were applied but different percentage of 16h grown bacterial culture (10%, 20%, 30%, 40% and 50% that correspond to about 1×104, 2×104, 3×104, 4×104 and 5×104 CFU/mL) were tested. Naturally present lactose in cow milk was tested using about 1×104 CFU/mL from E. coli 16h grown culture: IPTG-control samples were prepared as above (final concentration 2.5 mM), 4

ACCEPTED MANUSCRIPT whereas for lactose-test samples no IPTG solution was added, and blank pasteurized cow milk samples were added (30% of final mix volume). For the tests with milk (commercially pasteurized cows' milk, Centrale del Latte di Roma),

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the colorimetric assays were performed under the same conditions, except the following: no

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IPTG solution was added to the mix, and milk samples (both blank and ciprofloxacin-spiked milk samples) at 30% of final mix volume were added. Ciprofloxacin-spiked milk samples

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were prepared using 100 MRL (10 mg/L) ciprofloxacin [≥98.0% (HPLC), Sigma–Aldrich, Italy] stock solution in high-purity deionised water. Ciprofloxacin stock solution was stored in an amber glass bottle at 4 °C when not in use. The concentration of the stock solution was

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checked periodically to ensure no changes in concentration by spectrophotometric measurements (Unicam UV2 UV/Visible spectrophotometer, λmax of 271nm, against high-

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purity deionised water in quartz cuvettes with a 1-cm optical path length). In order to obtain the proper dilution in the final assay mix (1, 10 and 20 MRL), aliquots of ciprofloxacin solution were added to milk samples for all the three tested concentrations.

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For all the colorimetric assays performed, assays were incubated at 37 °C in shacking

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incubator (170 rpm) for respective time (1h, 2h, 3h, or 18 h). Finally, assays were transferred in 96-well microplates and OD was measured using a Multiskan™ GO Spectrophotometer

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(Thermo Scientific, NH, USA) at 600nm (for E. coli culture concentration), 420nm (for β-gal activity using ONPG), 612nm and 654nm (for β-gal activity using X-gal).

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3. Results and discussion

3.1 Comparison between artificial substrates ONPG for β-gal activity determination The β-gal is the 464-kDa homotetrameric enzyme, which plays a key role in central carbon metabolism in E. coli (hydrolysis of β-galactosides like lactose into monosaccharides like glucose and galactose). In E. coli, the lacZ gene of β-gal is part of an inducible genetic system lac operon, a functional unit that basically consists of the lac promoter and three structural genes (coding for a protein that has no regulatory function) lacZ, lacY and lacA, that encode for the enzymes β-galactosidase, β-galactoside permease and β-galactoside transacetylase respectively [9]. Lac-operon is controlled through a negative regulation mechanism [10], and gene transcription is activated in response to the presence of Lactose which act an inducer. Another artficial inducer of Lac-operon is IPTG, a non-metabolizable analogue of lacose. X-gal is widely used as artificial substrate in artifical β-gal assays. This 5

ACCEPTED MANUSCRIPT colorless organic compound is a structural analogue of lactose artificial substrate and can be hydrolyzed by β-gal producing 5,5'-dibromo-4,4'-dichloro-indigo, that is a blue insoluble compound. This exhibits a maximum absorption at 612nm, that can be detected by

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spectrophotometry. Hence, the presence of blue colored product can be used to assess the

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presence of the active β-gal in various applications in the field of molecular biology, genetics, embryology and bacteriology as reporter gene and/or as marker enzyme [11,12]. Another

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alternative artificial β-gal substrate is ONPG which is The ONPG more economical than Xgal. Like X-gal, ONPG normally is a colorless substrate, which on hydrolyzes by β-gal convert in to ortho-nitrophenol (yellow color compound that can be detected by

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spectrophotometric reading with maximum absorption at 420 nm) [11].

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For β-gal activity determination, both X-gal and ONPG (artificial substrates) were separately incubated with E. coli ATCC 11303 suspension culture in the same condition described above and scanned from 300nm to 700nm of OD range without preparing cell extract. The β-

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gal activity on ONPG gives a yellow colour with a maximum OD at 420nm and X-gal gives a

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blue colour (Supplementary Material Fig. S1) with a maximum OD at 612nm (data not shown). Furthermore, to determine more appropriate artificial substrate for β-gal activity, X-

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gal and ONPG were separately incubated with E. coli ATCC 11303 cell suspensions, and absorbance ratio of OD612nm/OD600nm and OD420nm/OD600nm were measured respectively. The results shows that ONPG resulted in 250% increase (p < 0.001) in the OD at 420nm (Fig. 1A), whereas X-gal resulted in only 8.33% (p< 0.001, Fig. 1B). Further, addition of ONPG to

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LB media does not interfere with LB media OD (Fig. 1C): no significant difference for OD at 420nm and 600nm was found. Hence, in our experimental conditions for preliminary tests, ONPG is a more appropriate artificial substrate for β-gal activity than X-gal. 3.2 Correlation between E. coli ATCC 11303 culture density and relative β-gal activity The experimental data showed an excellent linear correlation between the activity of β-gal using ONPG as substrate (100 µL of a 4 mg mL-1stock solution) and density of E. coli ATCC 11303 (expressed as OD600nm). In particular, the ratio of OD420nm/OD600nm remains constant (3.30 ± 0.01) with varying percentage of culture in LB media used. In addition to linear correlation experimentally established between the optical density of E. coli cultures (cell proliferation) and the induction of endogenous β-gal activity, these results confirmed the advantage in the use of ONPG as artificial substrate for β-gal activty (Table 1). 6

ACCEPTED MANUSCRIPT 3.3 Comparison between IPTG and lactose present as inducers of endogenous β-gal The β-gal activity was measured at OD420nm (i.e. using ONPG as artificial substrate) in

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IPTG-control samples (i.e. no milk added), and compared to lactose-test samples (i.e. no

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IPTG solution added).

In our experimental conditions, the endogenous lacZ gene of E. coli ATCC 11303 was found

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to be induced by lactose present in commercially pasteurized cows' milk for all the tested times (1 and 2 hours). Lactose was able to induce β-gal with an induction effect almost similar to IPTG with no significant difference (Fig. 2). Lactose average concentration in

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cows' milk is about 50 g/L [13] (thus presumptive final concentration in the assay mix was about 45 mM) whereas IPTG concentration is 2.5 mM. Contrary to lactose, IPTG is not

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metabolized by E. coli thus its concentration presumably remains constant. Probably, even if lactose is metabolized by E. coli during the assay, its relative high concentration compared to IPTG (about 20 fold higher than IPTG) is enough to keep constant the rate of expression of

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lacZ gene, at least for 2 hours (as running time of tests).

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3.4 Assay testing on cow milk samples

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The presence of antibiotic residues (including ciprofloxacin) in foods of animal origin like milk and dairy products is of high concern due the environmental spread of antibioticresistant bacterial strains and antibiotic resistance, depletion of the beneficial gut bacteria,

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and problems of technological nature (e.g. impairment of technological characteristics of milk used to produce fermented foods like cheese). The results obtained by adding commercially pasteurized cows' milk samples (both blank and ciprofloxacin-spiked milk samples; 30% of final mix volume) to the assay mix are shown in Fig. 3. The β-gal colorimetric assay was able to detect the presence of ciprofloxacin 1 MRL in spiked milk within 1 h (P < 0.001, Fig. 3) A linear correlation between E. coli ATCC 11303 (measured at OD600nm) and relative β-gal activity (measured at OD420nm) expressed as OD420nm/OD600nm ratio was found in milk-spiked media as well as observed in LB. More specifically, OD420nm/OD600nm ratio was found to be 3.3 ± 0.01 in LB (Table 1) and 1.79 ± 0.01 in milk spiked media (Table 2). This indicated that detection of β-gal activity using ONPG as a substrate is feasible, and also able to increase the detection sensitivity of ciprofloxacin in spiked milk. E. coli ATCC 11303 strain was previously found to be a suitable for testing microorganism for the detection of fluoroquinolone residues; thus, it was exploited for the

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ACCEPTED MANUSCRIPT microbiological screening of enrofloxacin, ciprofloxacin and flumequine residues [14, 15, 16, 17, 18]. Previous studies showed that a bio-optical method based on the estimation of growth inhibition effect on E. coli ATCC 11303 cultures (measuring OD600nm) could detect the

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ciprofloxacin residues in milk after 3 hrs of inoculation [4]. Conversely, our current method

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proves to be effective in the detection of ciprofloxacin in spiked milk sample in 1 hr of incubation time. This improved method can be integrated in automated procedure to facilitate

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screening assays for the safety and quality control of the milk (and dairy products) production chain [19].

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4. Conclusion

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The proposed colorimetric assay provides improved and fast detection of ciprofloxacin in spiked commercially pasteurized cows' milk by exploiting the endogenous β-gal activity of E. coli ATCC 13303. The use of this culture has already been proven specificity for

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fluroquinolones detection without giving false positive results both when incubated with

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blank milk samples or spiked with milk using different kind of antibiotics such as β-lactams, sulfonamides, aminoglycosides and tetracyclines. This method proved to be effective in the

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detection of ciprofloxacin in spiked cow milk samples at 1 MRL concentration in 1 hr. In addition, the response time was comparable (about 1 hr) to those of the traditional routine microbial methods. The use of ONPG as endogenous β-gal artificial substrate and lactose as

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endogenous β-gal inducer could lead to additional cost cutting benefits. Concerning lactose naturally present in milk, it could be a suitable alternative to IPTG for the development of a cheaper and more eco-friendly (i.e. minimizing the use of additional reagents) screening assay for ciprofloxacin detection in milk Subsequent to full testing on raw cow milk and validation, the assay is simple enough for automation and integration in the patented technological platform BEST (PCT WO/2010/001432): (Bio) Sensors’ system in Food Safety [BEST] [20] as at-line monitoring systems for hazard analysis and management in the food chain and the environment.

5. Acknowledgments The work has been developed in the frame of the project ALERT “Integrated System of biosensors and sensors (“BEST”) for the monitoring of wholesomeness and quality, as well 8

ACCEPTED MANUSCRIPT as for traceability in the cow milk chain” funded by the Italian Ministry of economic development under the Call Industria 2015 News technologies for Made in Italy (www.alert2015.it) and thanks to the support provided by FILAS–Lazio, Italy, for the

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PROMILK project.

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6. References

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1. A.O. Coker, R.D. Isokpehi, B.N. Thomas, K.O. Amisu, L.C. Obi, Human campylobacteriosis in developing countries-synopsis-statistical data included, Emerg. Infect. Dis. 8 (2002) 237-243.

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2. M.B. Skirrow, M.J. Blaser, Clinical aspects of Campylobacter infection,Campylobacter, second ed, ASM Press, Washington, DC, 2008, pp. 69-88.

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3. O.R. Idowu, J.O. Peggins, R. Cullison, J. Von Bredow, Comparative pharmacokinetics of enrofloxacin and ciprofloxacin in lactating dairy cows and beef steers following intravenous administration of enrofloxacin, Res. Vet. Sci. 89 (2010) 230-235.

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4. B. Appicciafuoco, R. Dragone, C. Frazzoli, G. Bolzoni, A. Mantovani, A.M. Ferrini,

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Microbial screening for quinolones residues in cow milk by bio-optical method, J. Pharm. Biomed. Anal. 106 (2015) 179-185.

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5. European Commission Regulation (EU) 37/2010, on pharmacologically active substances and their classification regarding maximum residue limits in foodstuffs of animal origin. Off. J. Eur. Commun. L15 (2009). 6. EU Council Regulation (EEC) No. 2377/90 laying down a Community procedure for the

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establishment of maximum residue limits of veterinary medicinal products in foodstuffs of animal origin. Off. J. Eur. Commun. 224 (1990) 1–8. 7. EC 2002. Commission Decision 2002/657/EC of 12 August 2002 implementing Council Directive 96/23/EC. Off. J. Eur. Commun. 221 (2002) 8-36. 8. C. Frazzoli, A. Mantovani, R. Dragone, Local role of food producers’ communities for a Global One-Health framework: the experience of translational research in an Italian dairy chain. J. Agri. Chem. Envir. 3(2014) 14-19. 9. S. Oehler, E.R. Eismann, H. Krämer, B. Müller-Hill, The three operators of the lac operon cooperate in repression, EMBO J. 9 (1990) 973–979. 10. M. Lewis, C. Geoffrey, C.H. Nancy, Horton, M.A. Kercher, Crystal structure of the lactose operon repressor and its complexes with DNA and inducer,Science 271 (1996) 1247-1254.

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ACCEPTED MANUSCRIPT 11. I. Bronstein, J. Fortin, P. E. Stanley, , G.S. Stewart, L.J. Kricka, Chemiluminescent and bioluminescent reporter gene assays,Anal. Biochem. 219(1994)169-181. 12. D. N.Arvidson, P. Youderian, T.D. Schneider, G. D. Stormo, Automated kinetic assay of

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β-galactosidase activity. BioTechniques 11 (1991) 733–737.

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13. Montreuil, J. "The glucides of milk." Bulletin de la Société de chimie biologique 42 (1960): 1399.

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14. L. Ellerbroek, The microbiological determination of the quinolone carbonic acid derivatives enrofloxacin, ciprofloxacin and flumequine, Fleischwirtschaft 71 (1991) 187189.

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15. A.M. Ferrini, V. Mannoni, P. Aureli, Combined Plate Microbial Assay (CPMA): A 6plate-method for simultaneous first and second level screening of antibacterial residues in

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meat, Food Addit. Contam. 23 (2006) 16-24.

16. L. Okerman, H. Noppe, V. Cornet,L. De Zutter, Microbiological detection of 10 quinolone antibiotic residues and its application to artificially contaminated poultry

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samples, Food Addit. Contam. 24 (2007) 252-257.

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17. L. Okerman, S. Croubels, S. De Baere, J.V. Hoof, P. De Backer, H. De Brabander, Inhibition tests for detection and presumptive identification of tetracyclines, β-lactam

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antibiotics and quinolones in poultry meat, Food Addit. Contam. 18 (2001) 385-393. 18. A.L. Myllyniemi, R. Rannikko, E. Lindfors, A. Niemi, C. Bäckman, Microbiological and chemical detection of incurred penicillin G, oxytetracycline, enrofloxacin and

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ciprofloxacin residues in bovine and porcine tissues, Food Addit. Contam.17 (2000) 991-

19. R. Dragone, G. Grasso, Biosensoristic devices: monitoring and diagnostics in agrozootechnical productions, in: Frazzoli, C., Asongalem, E. A., Orisakwe, O. E. (Eds.), Cameroon-Nigeria-Italy scientific cooperation: veterinary public health and sustainable food safety to promote “one health/one prevention”, Roma, Istituto Superiore di Sanità, Rapporti ISTISAN 12/49, 2012, pp. 70-77. 20. C. Frazzoli, A. Mantovani, L. Campanella, R. Dragone, Technological integrated bioelectronic system and relevant control charting for early intervention on food chain and the environment [BEST]. WIPO. WO DOI: 2010/001432 A1. 2010.

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ACCEPTED MANUSCRIPT Figure legends

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Fig. 1. The β-galactosidase activity of E. coli ATCC 11303 using (A) ONPG and (B) X-gal

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substrate. Effect of ONPG addition in LB media (C). LB, Luria broth; ONPG, orthoNitrophenyl- β-galactoside. Means of OD values based on triplicate for each sample.

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*indicates that the data sets are significantly different (p<0.05) followed by t-student test. Fig. 2. Comparison of β-galactosidase activity using IPTG and milk lactose as inducer in E.

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coli ATCC 11303 at different time. The β-galactosidase activity was measured at OD420nm using ONPG as artificial substrate. Means values of OD420nm are based on triplicate analysis

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for each sample and plotted versus time in hours (h).

Fig. 3. The β-galactosidase activity using ONPG as artificial substrate of E. coli

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ATCC11303 in commercially pasteurized cows' milk spiked with ciprofloxacin (Cipro) 1, 10

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and 20 MRL. The β-galactosidase activity was measured at OD420nm (mean values, 5 replicates) are plotted versus times in hours (h). *indicates that the data sets are significantly

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different (p<0.05) followed by t-student test.

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ACCEPTED MANUSCRIPT Table 1. Correlation between the β-galactosidase activity in presence of ONPG and density of E. coli ATCC11303 measured using OD420nm and OD600nm, respectively. Means of OD values based on five replicates for each sample. OD420nm

Ratio (OD420nm / OD600nm)

0.085 ± 0.01 0.116 ± 0.01 0.148 ± 0.01 0.190 ± 0.01 0.220 ± 0.01

0.281 ± 0.01 0.382 ± 0.01 0.485 ± 0.01 0.627 ± 0.01 0.743 ± 0.01

3.28 3.29 3.27 3.30 3.30

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OD600nm

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E. coli ATCC 11303 culture (CFU/mL) 1×104 2×104 3×104 4×104 5×104

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E. coli ATCC 11303 + Milk + ONPG + 10MRL Cipro

0.612 ± 0.01 1.290 ± 0.01 1.450 ± 0.01 0.612 ± 0.01 1.247 ± 0.01 1.365 ± 0.01 0.612 ± 0.01 1.064 ± 0.01 1.093 ± 0.01 0.612 ± 0.01 1.065 ± 0.01 1.083 ± 0.01

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E. coli ATCC 11303 + Milk + ONPG + 20MRL Cipro

0 1 2 0 1 2 0 1 2 0 1 2

OD600nm

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E. coli ATCC 11303 + Milk + ONPG + 1MRL Cipro

OD420nm

0.612 ± 0.01 0.726 ± 0.01 0.829 ± 0.01 0.612 ± 0.01 0.687 ± 0.01 0.686 ± 0.01 0.612 ± 0.01 0.593 ± 0.01 0.580 ± 0.01 0.612 ± 0.01 0.590 ± 0.01 0.570 ± 0.01

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E. coli ATCC 11303 + Milk + ONPG

Time (h)

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Culture

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Table 2 The β-galactosidase activity of E. coli ATCC11303 in commercially pasteurized cows' milk samples spiked with ciprofloxacin (Cipro) 1, 10 and 20 MRL. The βgalactosidase activity in presence of ONPG and density of E. coli ATCC11303 measured using OD420nm and OD600nm, respectively. Means of OD values based on five replicates for each sample.

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Ratio (OD420nm/ OD600nm) 1.76 1.76 1.76 1.76 1.76 1.76 1.76 1.76 1.76 1.76 1.76 1.76

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ACCEPTED MANUSCRIPT Highlights

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 This study reports the findings of a new colorimetric assay based on endogenous βgalactosidase (β-gal) activityas an indirect measure of the cell proliferation of E. coliATCC 11303 strain in presence of different concentrations of ciprofloxacin in spiked milk samples.  This screening assay was operated at three different concentrations of ciprofloxacin in spiked cow milk samples with two different chromogenic artificial substrates for β-gal; Onitrophenyl- β-D-galactopyranoside (ONPG) and 5-Bromo-4-chloro-3-indolyl βDgalactopyranoside (X-gal).  This screening assay successfully detected ciprofloxacin residues at 1 MRL concentrations within 1 hour using ONPG, that is considerably cheaper than X-gal and proved to be more proficient than previous described assays.

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