Naresh Kumar

Antimicrobial resistance (AMR) is increasingly becoming a major global public health concern, as antibiotics are losing their effectiveness at an alarming rate due to drug resistance. The ß-lactam group of antibiotics are widely used in dairy farms to treat animal infections, and their presence in the food chain is a significant concern. Addressing this issue requires the development of effective analytical tools for the rapid detection of antibiotics. In this work, a miniaturized Biosensor-on-Pillar platform was developed for detecting ß-lactam antibiotics in milk, which operates in a rapid, cost-effective, and user-friendly format, making it particularly suitable for resource-limited settings. The platform employs an enzyme induction-based approach, wherein Bacillus cereus spores germinate in the presence of β-lactam antibiotics, leading to the production of β-lactamase enzyme, which is then recognized using a chromogenic substrate functionalized on paper associated with the pillar platform. The developed biosensor can detect 12 β-lactam antibiotics with limits of detection (LODs) ranging from 1 to 1000 ppb, achieving sensitivity at or below the maximum residue limits (MRLs) set by regulatory bodies (FSSAI/CODEX) for the majority of the tested antibiotics. The performance of the platform, including the design, fabrication, and working principle, was further evaluated by analyzing six blind milk samples, yielding significant results compared to the commercially available AOAC-approved gold-standard method. Hence, the developed biosensor demonstrates promising potential for the rapid, cost-effective and high-throughput screening of milk samples for β-lactam antibiotics, benefiting the dairy industry and ensuring food safety.

The consumption of milk contaminated with antibiotic-resistant bacteria poses a significant health threat to humans. This study aimed to investigate the prevalence of Enterobacteriaceae producing β-lactamases (ESBL, MBL, and AmpC) in cow and buffalo milk samples from two Indian states, Haryana and Assam. A total of 401 milk samples were collected from dairy farmers and vendors in the specified districts. Microbiological assays, antibiotic susceptibility testing, and PCR-based genotyping were employed to analyze 421 Gram-negative bacterial isolates. The overall prevalence of β-lactamase genes was 10% (confidence interval (CI) (7–13)), with higher rates in Haryana (13%, CI (9–19)) compared to Assam (7%, CI (4–11)). The identified β-lactamase genes in isolates were bla_CMY, bla_MOX, bla_FOX, bla_EBC, and bla_DHA, associated with AmpC production. Additionally, bla_CTX-M1, bla_SHV, and bla_TEM were detected as ESBL producers, while bla_VIM, bla_IMP, bla_SPM, bla_SIM, and bla_GIM were identified as MBL producers. Notably, Shigella spp. were the dominant β-lactamase producers among identified Enterobacteriaceae. This study highlights the presence of various prevalent β-lactamase genes in milk isolates, indicating the potential risk of antimicrobial-resistant bacteria in dairy products. The presence of β-lactam resistance raises concern as this could restrict antibiotic options for treatment. The discordance between genotypic and phenotypic methods emphasizes the necessity for comprehensive approaches that integrate both techniques to accurately assess antibiotic resistance. Urgent collaborative action incorporating rational and regulated use of antibiotics across the dairy value chain is required to address the global challenge of β-lactam resistance.

Bovine milk and milk products may contain pathogens, antimicrobial resistant bacteria, and antibiotic residues that could harm consumers. We analyzed 282 gram-positive isolates from milk samples from dairy farmers and vendors in Haryana and Assam, India, to assess the prevalence of methicillin-resistant staphylococci using microbiological tests, antibiotic susceptibility testing, and genotyping by PCR. The prevalence of genotypic methicillin resistance in isolates from raw milk samples was 5% [95% confidence interval, CI (3–8)], with 7% [CI (3–10)] in Haryana, in contrast to 2% [CI (0.2–6)] in Assam. The prevalence was the same in isolates from milk samples collected from farmers [5% (n = 6), CI (2–11)] and vendors [5% (n = 7), CI (2–10)]. Methicillin resistance was also observed in 15% of the isolates from pasteurized milk [(n = 3), CI (3–38)]. Two staphylococci harboring a novel mecC gene were identified for the first time in Indian dairy products. The only SCCmec type identified was Type V. The staphylococci with the mecA (n = 11) gene in raw milk were commonly resistant to oxacillin [92%, CI (59–100)] and cefoxitin [74%, CI (39–94)], while the isolates with mecC (n = 2) were resistant to oxacillin (100%) only. All the staphylococci with the mecA (n = 3) gene in pasteurized milk were resistant to both oxacillin and cefoxitin. Our results provided evidence that methicillin-resistant staphylococci occur in dairy products in India with potential public health implications. The state with more intensive dairy systems (Haryana) had higher levels of methicillin-resistant bacteria in milk.

The study's goal was to develop a spore-based paper strip biosensor for detecting ß-lactam antibiotics in milk using the enzyme induction principle. A new spore-based paper strip biosensor has been developed after important operating parameters such as spore volume, substrate volume, exposure time and temperature, and incubation time and temperature were optimised. The limit of detection for various ß-lactam antibiotics, including amoxicillin, penicillin, ampicillin, carbenicillin, cloxacillin, nafcillin, oxacillin, cephalothin, cefalexin, cefoxitin, cefazolin, and cefuroxime, was determined in milk with detection sensitivity of 1 ppb, 2 ppb, 2 ppb, 10 ppb, 10 ppb, 10 ppb, 20 ppb, 10 ppb 1000 ppb, 10 ppb 300 ppb and 100 ppb, respectively. It was also tested with other contaminants such non-ß-lactam antibiotics, pesticides, aflatoxin, heavy metals, and other chemical contaminants, and no interference was found, indicating that the created biosensor had a low rate of false positive and negative results. In comparison to the AOAC-approved CHARM-ROSA ß-lactam strip test, which identified 7 raw milk and zero pasteurised milk samples positive for ß-lactam antibiotics, the sensor was further analysed and verified using 200 raw milk and 105 pasteurised milk samples. This indicates a perfect match between our biosensor and the AOAC-approved CHARM-ROSA ß-lactam strip test. The developed spore-based paper strip biosensors are expected to be useful in the rapid and cost-effective detection of ß-lactam antibiotic residues in milk samples at the dairy farm, reception dock, and production units, respectively.

Antimicrobial resistance (AMR) is a major global public health issue. In India, access to medicines is poorly regulated and therefore antibiotics in dairy cattle are commonly used by farmers withou...

Spores of Bacillus megaterium were immobilized on a sensor disk by entrapment method followed by addition of pretreated milk and fluorogenic substrate, i.e., diacetate fluorescein with a response time of about 20 ± 5 min with ≥0.25 μg/L detection limit. Fluorescence was measured semiquantitatively using microbiological plate reader at specific excitation and emission spectra, i.e., 485 and 535 nm, respectively. High fluorescence within 20 ± 5 min is an indication of spore germination with the release of enzyme and its action on fluorogenic substrate. However, decrease in fluorescence indicates that milk is positive for AFM₁ at 0.25 ppb. Reliability of fluorescence‐based assay has been checked by spiking AFM₁ in milk, which shows that fluorescence is inversely proportional to AFM₁ concentration. The developed assay was evaluated with real food matrix such as raw, pasteurized and dried milk (n = 59), and it was compared with radioimmunoassay (Charm assay) and enzyme‐linked immunosorbent assay which showed correlation of 0.94 and 1.0, respectively. Fluorogenic assay based on spore germination principle on sensor disk has the potential for routine monitoring of AFM1 in dairy farm, reception dock and manufacturing unit of dairy industry.Practical Applications: Fluorescence‐based detection of AFM₁ in milk using immobilized spores on a sensor disk have a response time of 20 ± 5 min with a detection limit of ≥0.25 ppb. High fluorescence within 20 ± 5 min specifies germination of spores with the release of marker enzyme and its action on fluorogenic substrate, whereas decrease in fluorescence shows the presence of AFM₁ in milk at 0.25 ppb. The assay was validated with radioimmunoassay (Charm assay) and enzyme‐linked immunosorbent assay in real food matrix such as raw, pasteurized and dried milk. The assay has the potential for routine monitoring of AFM₁ in dairy farm, reception dock and manufacturing unit of dairy industry.

A spore germination-based concept and its transformation into a field level prototype for monitoring aflatoxin M₁ (AFM₁) in milk was developed. Initially, 15 strains of Bacillus spp. procured from different culture collection were screened for AFM₁ sensitivity using spot assay and marker strain showing inhibition at 0.5 ppb was selected based upon maximum zone of inhibition. The selected strain B. megaterium 2949 was further screened for different enzymes activities and subsequently its spores were produced to an extent of 73.13% ± 3.197% in newly developed sporulation medium containing beef extract (0.0075% ± 0.0004%), yeast extract (0.015% ± 0.001%), peptone (0.0375% ± 0.0016%), and sodium chloride (0.0375% ± 0.0018%). A spore germination-based concept/ assay was optimized by immobilizing spores in eppendorf with pretreated milk (80°C/15 min) containing germinant and chromogenic substrate followed by incubation at 37°C. The appearance of sky blue color within real time of 45 min indicated spores germination and release of specific marker enzyme such as acetyl esterase and its specific action on chromogenic substrate which demonstrates absence of AFM₁ in milk. However, if there was no color change, presence of AFM₁ at 0.5 ppb MRL was denoted by Codex. The developed concept on AFM₁ detection was validated and a correlation of 0.97 was established with AOAC approved Charm 6602 and ELISA at Codex MRL with minimal false positive and negative results. The cost effective test has potential application in dairy farms, manufacturing, and R&D units for routine monitoring of AFM₁ in milk.

The spore forming Bacillus cereus (66) was screened for the induction of β-lactamase in presence of an inducer using iodometric assay. A significant induction in marker enzyme was observed in B. cereus 66 at maximum residual limit (MRL) of penicillin, ampicillin, cloxacillin, amoxicillin, cefalexin, and cephazolin belonging to β-lactam group of antibiotics. A microbial based assay, where enzyme induction was optimized at pH 7.0, temperature 30°C, and whey powder (0.25%) after 4 h of incubation. The spore based assay was tested with milk samples spiked with 6 different β-lactam antibiotics. The results were 100 and 83.33% in correlation with microbial receptor and inhibition based assay, respectively. Overall, spore based assay can be a useful and cost effective tool for the specific detection of β-lactam group of antibiotics in milk.