Spore germination based assay for monitoring antibiotic residues in milk at dairy farm

Abstract

Spore germination based assay involves the transformation of dormant spores of Bacillus stearothermophilus 953 into active vegetative cells. The inhibition of germination process specifically in presence of antibiotic residues was used as a novel approach for monitoring target contaminants in milk. The indicator organism i.e., B. stearothermophilus 953 was initially allowed to sporulate by seeding in sporulation medium and incubating at 55 °C for 18 ± 2 h. The spores exhibited a typical chain behavior as revealed through phase contrast microscopy. The minimal medium inoculated with activated spores was incubated at 64 °C for 2-3 h for germination and outgrowth in presence of specific germinant mixture containing dextrose, whey powder and skimmed milk powder added in specific ratio along with reconstituted milk as negative control and test milk samples. The change in color of the medium from purple to yellow was used as criteria for detection of antibiotic residues in milk. The efficiency of the developed assay was evaluated through a surveillance study on 228 samples of raw, pasteurized and dried milks and results were compared with AOAC approved microbial receptor assay. The presence of antibiotic level was 10.08 % at Codex maximum residual limit having false positive result only in 0.43 % of the samples. The results of the present investigation suggest that developed spore based assay can be a practical solution to dairy industry for its application at farm level, milk processing units, independent testing and R & D centres in order to comply with the legal requirements set by Codex.

A Novel Microbiological Method in Microtiter Plates for Screening Seven Kinds of Widely Used Antibiotics Residues in Milk, Chicken Egg and Honey

“…Therefore, improvement of the detection capability of the kit in present study for macrolides was operated by lowering CAP concentration in kit’s medium. G. stearothermophilus var C953 is only sensitive to β-lactam and lincomycin (Kumar et al, 2012). As a result, in this kit, TMP and CAP was used to improve the sensitivity of the kit to sulfonamides, and tetracyclines separately.…”

“…Geobacillus stearothermophilus is the most widely used indicator bacterium in microbiological inhibition methods in terms of test tubes, as it is not easily contaminated, demands high incubation temperature (55°C) and grows faster in a short time (less than 4 h) than other bacteria. Moreover, it is more sensitive to antimicrobial agents, particularly, β-lactam (Kumar et al, 2012). Additionally, spores of G. stearothermophilus are more resistant to adverse factors than vegetative cells and show stable activity for a long time.…”

Lateral Flow Assay–Based Rapid Detection of Cephalexin in Milk

“…), microbial inhibition (Kumar et al . ), receptor binding assay (Beltrán et al . ) and microbial receptor (Diserens et al .…”

“…Although these methods are sensitive, specific and reliable for quantification, they are time-consuming, expensive and are not applicable under field conditions. Among the rapid methods, enzyme-linked immunosorbent assay (ELISA) Bremus et al 2012), microbial inhibition (Kumar et al 2012), receptor binding assay (Beltrán et al 2014) and microbial receptor (Diserens et al 2010) based tests are widely used to detect CFX and other antibiotic residues in milk. However, these tests also have limitations as they require a laboratory setup and cannot be treated as field tests.…”

Development of “Field Level” Chromogenic Assay for Aflatoxin M1 Detection in Milk

“…After germination de novo enzyme was released from the core of the spore which act upon substrate and the signal can be captured using optical device to quantify the presence of target analyte [19]. This process of sporulation and germination can be used as biosensing mechanism for monitoring of broad spectrum antibiotic, specific β-lactam residues and Enterococci in milk at dairy farm level, reception dock and manufacturing unit of dairy industry [20][21][22]. Bacillus spp.…”