507 Indian J. Dairy Sci. 66(6), 2013

INTRODUCTION

early all dairy farmers use antibiotics to treat

dairy cattle with respiratory conditions (97%)

and to a lesser extent mastitis (80%), metritis

(80%) and foot problems (83%). For mastitis,

penicillin (42%), ampicillin (26%) and tetracycline's

(18%) were commonly used antibiotics for treatment.

Penicillin (43%) and ceftiofur (41%) were the

antibiotics of choice for metritis or retained placentas

as well as foot problems (Zwald et al. 2004). The

current FDA approved antibiotic classes include β-

lactams, macrolides (erythromycin), coumarines

(novobiocin), and lincosamides (pirlimycin) (Pol and

Ruegg 2007). Of these four classes, β-lactams are

the most common antibiotics used for intramammary

treatment of mastitis on conventional dairy farms

(Pol and Ruegg 2007). The six main antimicrobials

that make up the β-lactam class are ampicillin,

amoxicillin, ceftiofur, cephapirin, cloxacillin, and

penicillin G (Holstege et al. 2002). The substantial

excretion of these residues in milk is attributed to

the indiscriminate use of antibiotics, lack of

medication records, use of unapproved drugs,

contaminated milking equipment, purchase of treated

cows, failure to observe withdrawal period in

lactation animals. Public concerns about drug use

in production, resistance to antibiotics and residues

in food as well as in the environment have generally

increased during the last decades (Refsdal 2000) and

awareness has risen especially in consumers of organic

products (Stolz et al., 2009; van Wijk-Jansen etal., 2009). These residues have been linked to allergic

reactions (in sensitive individuals), emergence of

resistant bacterial strains, and the impairment of

bacterial fermentation processes (Sierra et al. 2009).

Antibiotic residues must be monitored to prevent

contaminated milk from entering the human food

supply. The best way to monitor milk is through

the use of residue screening tests. These tests are

rapid, qualitative, and can detect a broad range of

antibiotic residues (Navrátilová 2008).

The analysis to detect antimicrobial residues in milk

is usually performed in two steps: first, a microbial

inhibition assay, enzymatic or receptor-based method

is used as a screening tool. Second, the samples

found positive are confirmed by a chemical method.

Spore Based Chromogenic Assay forDetection of βββββ-lactam Antibiotic in Milk

* Corresponding Author : Food Safety and Microbial Biosensors Lab, Dairy Microbiology Division, National Dairy Research Institute,Karnal, Haryana-132001. Email: nrshgoyal@yahoo.com/ raghuforever121@gmail.com

2013-081 Received:February 2013; Accepted:December 2013

Dormant spores of B. cereus 13061 used as a indicator strain for real time detection of βββββ-lactam antibiotic residues basedon βββββ-lactamase enzyme using nitrocefin as chromogenic substrate. Spore production of indicator strain was significantlyimproved by modifying the sporulation medium with an increase of 47. 86 % sporulation fold compared to conventionalmethod. A spore based chromogenic assay, where enzyme induction was optimized at 1.0 mL of milk sample, 500 μL ofspores suspension and 50 μL of nitrocefin after 20 min of incubation. The efficiency of the chromogenic assay was evaluatedthrough surveillance study on 94 samples of raw, pasteurized and dried milks samples in comparison with AOAC approvedCharm 6602 system and spore based assay. The presence of βββββ-lactam antibiotic was 8.51% at Codex MRL with falsepositive result only in 1.06 % of the samples. The assay can detect specific βββββ-lactam group of antibiotic within 15-20 min atregulatory codex limits with negligible sensitivity towards non βββββ-lactam groups. Output of the present study suggest thatdeveloped assay can be a viable option for dairy industry as a routine test for screening of βββββ-lactam groups in milk receivedat collection point, chilling centre, manufacturing unit, independent testing and R & D centre etc.

Keywords: βββββ-Lactam, Bacillus cereus ATCC 13061, Sporulation, Induction, nitrocefin Assay

Research Article

Kumar S., Raghu H. V., Kumar N .*, Singh N. A. and Malik R. K.

Food Safety and Microbial Biosensors Lab, Dairy Microbiology Division, National Dairy Research Institute, Karnal-132001

N

508

A confirmatory method has to detect which

molecule is present in the sample and quantitative

it. Liquid chromatography coupled with UV

detection (HPLC-UV) is the technique usually

adopted as a confirmatory method for antibiotic

residues (Ghidini et al. 2010). This technique has

some limitations: mainly it has a low sensitivity

and selectivity, therefore many purification steps

are needed (Faria Reyes et al. 2000). Sometimes, in

order to detect the analytes through a fluorescence

detector, a derivatization step is used to achieve

higher sensitivity (Marchetti et al. 2002). In any

case, the methods are quite time consuming and

unsuitable to process a great number of samples.

Spores based biosensors detection systems are cost

effective, rapid, and easy to perform and require

almost negligible infrastructural facilities. Using

dormant spores as a biosensor, an analytical system

for broad spectrum antibiotics (Kumar et al. 2006;

Kumar et al. 2012), Aflatoxin M1 (Kumar et al.2010; Singh et al. 2013) and bacterial contaminants

like Enterococci (Kaur, 2011; Deshmukh, 2013) in

milk has been developed & patented (Kumar et al.2012). Further, efforts were made for specific

detection of β-lactam antibiotics based on β-

lactamase enzymes which decolorize starch iodine

complex by hydrolyzing penicillin into penicilloic

acid. This reaction was exploited in detection of

β-lactamase activity in our laboratory using Bacillus

cereus 66 as biosensor (Kumar et al. 2009; Das etal. 2011) and further transformed into a kit

prototype for its commercial application (Gaare etal. 2012). Therefore, detecting β-lactamase (Buynak

2006) in biological samples by using chromogenic

substrate (such as the well known nitrocefin and

PADAC indicators) (O'Callaghan et al. 1972). The

spore based chromogenic assay is very specific and

sensitive to most of β-lactamse enzymes produced

by various group of bacteria. Therefore, the current

investigation has been designed to develop a

chromogenic assay for checking the presence of β-

lactam group in milk using nitrocefin as color

substrate/indicator and measurement of color

intensity developed in the test liquid in comparison

with a control taken as criteria for detection of β-

lactam antibiotic residues in milk at codex MRL.

MATERIALS AND METHODSMaterials

Fresh Benzyl penicillin (10, 00, 000 units) was

procured from CDH. 30 mg (50000 units) was

dissolved in 1000 μL of phosphate buffer. Dissolve

1 mg Nitrocefin in 100 μL dimethylsulfoxide (DMSO)

and vortex. Add 1.9 mL phosphate buffer (100

mM, pH 7) to produce 2 mL total volume. This

yields a working Nitrocefin solution of 500 mg.

mL-1 (approx. 1 mM). B. cereus ATCC 13061

procured from American type culture collection

(ATCC) was obtained as a freeze dried pellet.

Growth and sporulation

Single pure colony of indicator B. cereus ATCC

13061 cultures was grown on nutrient agar plates

followed by incubation at 35±2oC by using

microprocessor based incubator (Metrex scientific

instrument) for overnight. After overnight growth

it was transferred into 5 mL nutrient broth (NB)

(HiMedia Labs, Mumbai, India). Hundred mL of

NB was inoculated with indicator strain of B. cereus

ATCC 13061 at a rate of 1 mL. 100 mL-1 and

incubated at 35 ± 2oC for 24-48 hrs. The cell

suspension was centrifuged at 8944 g for 10 min

using eppendorf centrifuge 2801R, Germany and

pellet was washed, recentrifuged twice to eliminate

all cell bound enzymes with normal saline (0.85%

NaCl) (Kumar et al. 2012; Das et al. 2011; Gaare

et al. 2012; Singh et al. 2013). The cell pellet was

inoculated at the rate of 35±5 mL. 100 mL-1 in

optimized sporulation medium containing 0.15%

beef extract, 0.25% peptone, 0.025 % NaCl and

0.1% of tryptone followed by incubation at 35±2ºC

for 48 hrs. Total viable count (TVC) and spore

count (SC) (Downes and Ito 2001) was enumerated

after sporulation. After sporulation, spores were

checked for iodine decolouration time by using

iodometric assay developed by Das et al. (2011).

Optimization of spore production, spore suspension,

quantity of milk and nitrocefin solution was designed

using CRD (Critical random design) with six

replicates (n=6) (Snedecor and Cochran 1980).

Development of spore based chromogenic assayand kit prototype

The heat activated Spores B. cereus ATCC 13061

Kumar S. et al.

509 Indian J. Dairy Sci. 66(6), 2013

spores in the form of suspension (200-500 μL),

quantity of pre-treated milk sample (0.25-1.25 mL),

quantity of 1mM nitrocefin solution (25-50 μL)

were optimized for the development of SBCA and

incubate at 35±2ºC for different time periods (10-

35 min). The reduction in colour of the milk medium

from dark red to light red was taken as criteria for

enzyme induction in presence of β-lactam antibiotic

residues in milk. The SBCA was optimized and its

prototype was assembled into a commercial kit

prototype for its industrial application. The assay

was screened for limit of detection (LOD) against

4 different β-lactam antibiotic residues such as

penicillin, ampicillin, cefalexin and ceftriaxone for

assessing the performance of indicator strain B. cereusATCC 13061 by inoculating 1.00±0.25 μL of

reconstituted milk (10 gm of SMP in 9.0 mL of

distilled water at 40°C) spiked with β-lactam antibiotic

residues at EU/ Codex maximum residual limit

(MRL).

Evaluation of spore based chromogenic assay

Working efficacy of spore based chromogenic assay

(SBCA) was tested with reconstituted skimmed milk

spiked with different groups of antibiotics residues

namely penicillin, ampicillin, cefalexin, ceftriaxone,

tetracycline, streptomycin, kanamycin,

sulphamethazine, erythromycin, ofloxacin,

polymyxin B, neomycin, bacitracin &

chloramphenicol. The antibiotic free milk (25 nos)

collected from NDRI cattle yard spiked with benzyl

penicillin G (HiMedia Labs, Mumbai, India) at

>MRL in accordance with council regulation 37/

2010/EU (EC, 2010). Ceftriaxone belonging to

cephalosporin group was injected intravenously to

different farm animals (5 nos) & milk was drawn

at different intervals i.e. 12, 36, 48, 60 and 72 hrs

for checking its withdrawal period by using

developed assay. While antibiotic free milk served

as negative control. A total of 94 samples including

raw, pasteurised milk and dried milk products were

randomly collected from different sources for

analysis. The result of chromogenic assay results

were compared with microbial receptor based Charm

6602 analyzer (Charm Science Inc. USA) and spore

based assay (Kumar et al. 2012).

RESULTS AND DISCUSSIONSpore Production

In current investigation, due to modification of

conventional spore production process (Das et al.2011) with some modifications pertaining to

incubation time and temperature, composition of

sporulation medium, inoculum levels etc. A

significant increase in spore counts from 6.64 Log

CFU to 8.348 Log CFU with (35.48 to 53.70 folds)

meanwhile viable counts were decreased significantly

from 10.23 Log CFU to 8.57 Log CFU was observed

in modified sporulation medium. As a result the

germination and outgrowth period was found

decreased significantly from 6.81 hrs to 4.66±0.129

hrs. In our present study, enzyme action and iodine

decolourization step were combined and as a result

the iodometric assay time was reduced from 8.83

hrs to 5.128±0.311 hrs (Table 1). This reduction

in assay time was attributed to improved sporulation,

faster iodine decolourization (31 min compared to

94 min) in conventional iodometric assay. The iodine

decolourization time was 0.333±0.091 hrs. The work

pertaining to spore production in indicator strain

was initiated and the process was optimized by

Das et al. (2011). However, the level of conversion

of vegetative cells in spores was not significant. An

increase of 4.39 to 27.11 folds in spore count was

observed in sporulation medium. Induction of β-

lactamase enzyme in optimized medium with

supplementation of whey powder in germinant

mixture was achieved by Gaare et al. (2012). In

another study carried out by He et al. (2008) found

that spore production in B. cereus ATCC 13061,

B. cereus ATCC 10876, B. subtilis and B.

Table 1 : Iodine decolourization time of B. cereus ATCC 13061spores grown in modified sporulation medium

Germination Iodine IodometricTime (hrs) Decolourisation assay time

time (mins) (> MRL) (hrs)

4.5 0.4166 5.114.75 0.2500 5.214.75 0.2500 5.234.75 0.4166 5.354.75 0.4166 5.354.5 0.2500 4.52

4.666 ± 0.129 0.333 ± 0.091 5.128 ± 0.311(Mean ± SD) (Mean ± SD) (Mean ± SD)

Spore Based Chromogenic Assay for Detection of β-lactam Antibiotic in Milk

510

stearothermophilus by transferring vegetative cells

from refrigerated slant to tryptic soy agar (TSA),

followed by incubating at 37°C for 48 hrs.

Development of spore induction chromogenicassay and kit prototype

The cell suspension from the slant was inoculated

into optimized sporulation medium (35±5 mL. 100

mL-1) and incubated at 35±2oC for upto 48 hrs. A

continual increase in spore count, i.e., 6.64±0.1302,

7.52±0.215, and 8.348±0.1014 Log CFU count,

was observed with the increase of incubation period

from 0 to 24 and 48 hrs, respectively (CD =0.6925,

P<0.01). However, in cell biomass a regular

decreasing trend, i.e., 10.23±0.0895, 9.115±0.1211

and 8.57±0.0922 was observed with the change in

incubation period from 0, 24 and 48 hrs, respectively

(CD=0.4592, P< 0.01) (Fig. 1). The concentration

of milk, spore suspension, nitrocefin & iodine were

optimized by using clean & sterile test tube as

shown in Table 2. In present investigation, the

starch iodine mixture in iodometric assay was replaced

with the optimized nitrocefin solution (45±5.0 μL)

by reducing enzyme action time from 4 hrs to 20

min. A chromogenic substrate i.e., nitrocefin has

been used in competitive inhibition studies was

initially yellow in colour it changes to red colour

after enzyme action. Based on these observations,

a real time SBCA based on β-lactamase enzyme

using nitrocefin as chromogenic substrate has been

developed. The difference between control & test

samples spiked with penicillin G (>4 ppb) was

achieved in developed SBCA. The assay was found

extremely useful in specific detection of antibiotic

residues belonging to β-lactam group at codex/EU

regulatory limits with no false positive/negative

observations. It was also observed that the

chromogenic assay was extremely sensitive & can

detect drug residues at concentration lower than

recommended codex/EU regulatory limits. The assay

was equally good in specific detection of β-lactam

group with poor sensitivity towards non β-lactam.

The test was found working at codex/EU regulatory

limits as well as lower regulatory limits with

minimum false positive or negative results. The

sensitivity of different test namely microbial inhibitor

test, Penzym S, β star, SNAP test & ROSA test

was determined by adding the inhibitory substances

namely penicillin G, amoxicillin, ampicillin, oxacillin,

oxytetracycline, sulfamethazine, erythromycin,

neomycin & streptomycin, trimethoprim into milk

in quantities corresponding to MRL establish by

council regulation EEC No.2377/90 (Zvirdauskiene

Figure 1. Growth and sporulation pattern of indicator strains ofB. cereus ATCC 13061 in modified sporulation medium

Kumar S. et al.

511 Indian J. Dairy Sci. 66(6), 2013

and Salomskieneet 2007). A test based on similar

principle using pure penicillinase enzyme was

developed for detection of β -lactam group in

biological fluid such as milk or urine (Klein 1986).

Screening of SBCA for Limit of Detection

The limit of detection (LOD) of SBCA was

established by screening twenty five milk samples

(n=25) spiked with different groups of β-lactam

and non-β-lactam antibiotic residues at MRL level

set by Codex. Interestingly, SBCA showed sensitivity

at their respective MRL limits against four different

type of β - lactam, antibiotic residues namely

penicillin, ampicillin, cefalexin and ceftriaxone.

Further, ten other non-β-lactam antibiotic residues

namely tetracycline, streptomycin, kanamycin,

sulphamethazine, erythromycin, oflaxycill in,

polymyxin, neomycin, bacitracin and

chloramphenicol showed higher LOD, i.e., 2.5, 2.5,

10, 100, 1000, 100, 1000, 10, 100 and 250 ppb,

respectively (Fig. 2).

Evaluation of spore based chromogenic assay

Ceftriaxone belonging to cephalosporin group was

injected intravenously to different farm animals and

milk was analysed for its withdrawal for time interval

form 12 to 72 hrs by using developed assay, AOAC

approved microbial receptor assay and SBA. Three

out of five animals demonstrated the presence of

ceftriaxone with concentration varying from 5 to

250 ppb with withdrawal periods up to 48 hrs.

Other two animals showed the presence of

ceftriaxone (14 to 100 ppb) with withdrawal period

up to 36 hrs (Table 3). None of the sample showed

the presence of drug residues in samples drawn

after 60 and 70 hrs. All three assays i.e., developed

PEN - Penicillin; AMP-Ampicillin; CEF-Cefalexin; CEFT-Ceftriaxone; TET-Tetracycline;STR-Streptomycin; KAN-Kanamycin; SUL-Sulphamethazine; ERY-Erythromycin; OFL-Ofloxacin;

POL-Polymyxin, NEO-Neomycin; BAC-Bacitracin; CAP- ChloramphenicolFigure 2. Limit of detection (ppb) of βββββ-Lactam antibiotic residues in milk by

Spore based chromogenic assay (SBCA)

Table 2. Optimization of concentration of milk, spore suspension, nitrocefin & iodine for thedevelopment of spore based assay for detection of βββββ-lactam antibiotics in milk

Volume of milk Spore suspension Nitrocefin >MRLa Control

0.75 400 40 1.5 + 4 +1.00 450 45 1.5 + 4 +1.25 500 50 1.5 + 4 +

1.00 ± 0.25 450±0.25 45±0.25 1.5 + 4 +

a color gradation; ± (Mean ±error)

Spore Based Chromogenic Assay for Detection of β-lactam Antibiotic in Milk

512

assay, microbial receptor assay, SBA could detect

the presence of ceftriaxone in positive milk sample.

The assay based on chromogenic principle developed

in current investigation was also validated by

comparing with AOAC approved system and SBA.

In overall 94 milk samples including 48 raw milk,

31 pasteurized milk and 15 dried milk samples

analyzed using developed assay, microbial receptor

assay and SBA (Fig. 3). Performance of SBCA was

found parallel with AOAC approved charm 6602

milk and dried products. Whereas, Macaulay and

Packard (1981) reported 11 % false positive results

in milk samples by testing by using Delvotest may

be attributed to somatic cell counts in milk (Kang

and Kondo 2001). Kumar et al. (2012) also reported

10.08 % incidence of antibiotic residues at Codex

MRL in raw, pasteurized and dried milks in India

by using SBA and microbial receptor assays with

false positive results in 0.43 % of samples. Chung

et al. (2009) reported 7.8 % raw milk contaminated

Figure 3.Evaluation of developed SBCA with reference microbialreceptor assay, i.e., Charm 6602 assay (CA) and SBA for detection

of antibiotic residues in raw, pasteurized and dried milks

Table 3 : Testing of milk sample withdrawn from animal treated with ceftriaxone (Intravenous) using different assay

Withdrawal period (hrs) Animal No.

Method 6793 6377 6896 1778 6215

12 SBCA + + + + +SBA + + + + +

CharmAssay 250 120 100 24 250

24 SBCA + + + + +SBA + + + + +

Charm Assay36 SBCA + + + + +

SBA + + + + +Charm Assay 52 23 51 14 50

48 SBCA + + + + +SBA + + + + +

Charm Assay 5a 9 0 0 2560 SBCA - ve - ve - ve - ve - ve

SBA - ve - ve - ve - ve - veCharm Assay 0 0 0 0 0

72 SBCA - ve - ve - ve - ve - veSBA - ve - ve - ve - ve - ve

Charm Assay 0 0 0 0 0

MRL levela

system and SBA with minimum false

negative or positive results (1.06%).

Six out of forty eight sample of raw

milk and one out of thirty one

sample of pasteurized milk found

positive for β-lactam group at codex

MRL limits within 15 to 20 minutes.

In case of dried milks none of the

sample was found positive for β-

lactam group at Codex MRL level.

A significant correlation was

established between developed assay,

AOAC microbial receptor assay

(charm 6202) and SBA was established

in survey work with raw, pasteurized

Kumar S. et al.

513 Indian J. Dairy Sci. 66(6), 2013

with antibiotic residues collected from 13 cities of

Korea by using tetrazolium trichloride (TTC) and

STAR based protocols.

CONCLUSIONS

An innovative approach for detection of β-lactam

antibiotic residues in milk was attempted based on

induction and competitive enzyme action on β-lactam

antibiotic residues produced by B. cereus ATCC

13061. Because of modification sporulation medium

for sporulation of B. cereus there is significant decrease

in germination and outgrowth period. In our current

investigation, enzyme action and iodine

decolourization step were combined and as a result

the iodometric assay time was reduced. The starch

iodine mixture in iodometric assay was replaced with

nitrocefin as a chromogenic substrate by reducing

enzyme action time to 20 min with initial change of

colour from yellow to red has been developed for

its industrial application. The assay can detect specific

β-lactam antibiotic residues in milk within 20 min

at MRL level set by codex with negligible sensitivity

towards non β-lactam groups. The presence of

inhibitors other than antibiotic residues in milk did

not interfere with the working performance of the

developed assay. A significant correlation between

SBCA, SBA and AOAC approved microbial receptor

based assay (charm 6202) was established in survey

work with raw, pasteurized and dried milks with

minimum false positive/ negative results. The SBCA

can find immense application in dairy industry as

"ON FARM" milk screening test for β-lactam group

in milk at MRL codex limit.

ACKNOWLEDGEMENTS

The research work in this publication was carried

out under externally funded project awarded by

Ministry of Food Processing Industries (MoFPI),

Govt. of India with financial support to purchase

the desired equipments used in execution of proposed

research project. We acknowledge their immense

contribution in development of rapid bio-assay

prototype for its commercial application. The views

and conclusions contained in this document are those

of authors and should not be interpreted as

necessarily representing the official policies of MoFPI.

The Director NDRI is thankfully acknowledged

for Institute fellowship to Mr. Sunil Kumar.

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