Antibiotic-Resistant Bacteria, Antimicrobial Resistance Genes, and Antibiotic Residue in Food from Animal Sources: One Health Food Safety Concern.

Antibiotic-resistant bacteria causing foodborne serious illnesses can be found in contaminated food. Therefore, this study aimed to identify the pathogens, genes, and antimicrobial residues present in raw milk and meat. We collected 40 raw milk and 40 beef samples using the aseptic method from various parts of the Faisalabad metropolis, Pakistan. The samples were cultured on blood, MacConkey, and UTI chrome agar. The VITEK 2 compact system was used for microbial identification and determination of minimum inhibitory concentrations. Antimicrobial resistance genes for extended-spectrum ß-lactamases, methicillin resistance in Staphylococcus aureus, and carbapenem resistance were identified using molecular techniques. ELISA was used to determine the tetracycline residue level in each sample. The beef samples showed polymicrobial contamination with 64 bacterial isolates, with Escherichia coli (29; 45.3%) and Klebsiella pneumoniae (11; 17.1%) predominating. The milk samples showed polymicrobial contamination with 73 bacterial isolates, with E. coli (22; 30%), K. pneumoniae (12; 16.4%), and S. aureus (10; 13.6%) forming the majority. Twenty-eight (43.7%) isolates from beef harbored tet genes, nineteen (29.6%) blaCTX-M, and fourteen (21.8%) blaNDM-1, and twenty-six (35.6%) isolates from milk harbored tet genes, nineteen (26%) blaTEM and blaCTX-M, and three (4%) blaNDM-1. Twenty-two (55%) each of the beef and milk samples exceeded the maximum residue limit for tetracycline. Polymicrobial contamination by bacteria possessing blaCTX-M, blaTEM, blaNDM-1, blaOXA, mecA, and tet genes was identified in food samples. The high tetracycline residue levels pose a serious health risk to consumers.

Radiotracer studies to isolate in-house receptors from poultry liver for multi-chemical hazard analysis in selected food and feed.

In-house receptors (IHRs) were isolated from non-immunized poultry liver to analyze selected contaminants and residues in targeted food and feed using 14C- and 3H-labeled radiotracers. Matrix (2 g) was homogenized and centrifuged with the resultant pellet used as IHRs. These were characterized for total protein contents (6.1 mg mL-1) and compared with commercial receptors for aflatoxins (0.28 mg tablet-1) and chloramphenicol (0.12 mg tablet-1). Gel electrophoresis of the IHRs showed a mixture of polypeptides-an important attribute for multi-residues analysis-compared with commercial receptors that presented specific protein bands at 65 kDa (chloramphenicol) and 70 kDa (aflatoxins). The inhibition index of IHRs for aflatoxins B1 and B2 in wheat and bovine feed and chloramphenicol in bovine tissue at, above, and below maximum limits or minimum required performance limits, revealed an inverse relationship between radiotracer and analyte concentrations. Saturation with increased radioligand concentration up to 5.5 kBq indicated higher holding potential. However, increasing incubation time to 30 min did not significantly increase analyte-binding. The IHRs performance was comparable to commercial receptors with control point averages of 348, 410, 555, and 307 counts per minute determined for gentamicin, chloramphenicol, oxytetracycline, and aflatoxin M1, respectively in local milk samples.

Development of in-house ELISA for detection of chloramphenicol in bovine milk with subsequent confirmatory analysis by LC-MS/MS.

This study was undertaken to develop and validate direct competitive ELISA for the determination of chloramphenicol residues in bovine milk. Antisera and an enzyme-tracer for chloramphenicol were prepared and used to develop an ELISA with inhibition concentrations, IC20 and IC50, of 0.09 and 0.44 ng mL-1, respectively. Milk samples were spiked with standards equivalent to 0, 0.2, 0.3, 0.5, 1.0 & 1.5 ng mL-1 and extracted in methanol. The mean recoveries were found to be 73-100% with coefficient of variance 7-11%. The decision limit (CCα) and detection capability (CCß) were calculated as 0.10 and 0.12 ng mL-1, respectively. The results were found comparable with the commercial ELISA, having recoveries of 87 to 100%, CCα 0.09 ng mL-1 and CCß 0.12 ng mL-1. As per Commission Decision 2002/657/EC, in-house ELISA was further validated by using LC-MS/MS. Mass spectral acquisition was done by using electrospray ionization in the negative ion mode applying single reaction monitoring of the diagnostic transition reaction for CAP (m/z 152, 194 and 257). The calibration curve showed good linearity in concentrations from 0.025 to 1.6 ng mL-1 with correction coefficient 0.9902. The mean recoveries were found to be 88 to 100%. The CCα was calculated as 0.057 ng mL-1 and CCß 0.10 ng mL-1. Since CCα and CCß are less than half of the MRPL (0.15 ng mL-1), the test was found suitable for screening and quantification of CAP residues in bovine milk samples. Results of surveillance studies indicated that out of 31 analyzed milk samples, 12.9% samples were found with CAP residues but only 3.2% samples were declared positive with maximum concentration 0.31 ng mL-1, slightly above the MRPL.

Dipstick format of an improved ELISA for on-site atrazine monitoring in water in Pakistan.

A dipstick format was developed for on-site atrazine monitoring in water samples of different origins. It was derived from an in-house-developed ELISA based on polyclonal antibodies that also cross-react with hydroxyatrazine (30%) and terbuthylazine (17%). Test reagents were evaluated for temperature and pH stabilities and rapidity for field applications. Reagents performed well within a temperature range of 20-30 degrees C and were tolerant to alkaline pH (up to 8.5) of the assay buffering system. Tracer incubation time could be reduced to 40 min. Bovine serum albumin addition (1%) in the assay buffer improved assay performance, giving 50% B/B0 (IC50) of 65 ng/L and the lowest LOD of 2 ng/L at 90% B/B0 (IC10). The dipstick ELISA format was standardized on a membrane support. Nylon membrane, positively charged, was superior to PVDF for qualitative or semiquantitative analysis regarding color intensity and stability. Tracer incubation time was further reduced to 30 min with a lowest LOD of 0.1 microg/L. For real sample screening with dipsticks, acceptable results were obtained for water. Significant correlation was found between dipstick and plate ELISA results. Validation using GC with a nitrogen-phosphorus detector and HPLC indicated that dipstick signals in aged water samples, which were mainly due to hydroxyatrazine, were significantly above European Commission regulations of 0.1 microg/L. However, dipsticks were superior, fast, and cost-effective.

Comparison of in-house-developed ELISA with HPLC techniques for the analysis of atrazine residues.

In-house developed ELISA was standardized to monitor atrazine residues in different environmental samples. The standard curve was linear, indicating an increase in log concentration with decrease in absorbance (%B/B(0)=1.075-0.042 Log C; r= -0.966). The middle of the test was at 75 ng/L and the lowest detection limit at 4 ng/L. ELISA significantly correlated with the high performance liquid chromatography (HPLC) (r=0.990). Internal validation showed good accuracy and precision. Maximum atrazine residues were present in Jehlum River water/sediments and maize/sugarcane plant roots. Most of the food samples were found to be contaminated. ELISA required less clean-up steps than HPLC, but showed matrix effect in soil/colored extracts.

Development of ELISA technique for the analysis of atrazine residues in water.

A highly sensitive enzyme immunoassay is described for the detection of atrazine residues in water. Atrazine derivative was conjugated to Bovine Serum Albumin (BSA) to obtain an immunizing antigen and to Horseradish Peroxidase enzyme (POD) to obtain a marker for immunoassay. The formation of these conjugations was confirmed by UV spectroscopy as well as by gel-electrophoresis. Polyclonal antibodies were raised in rabbits by immunization with an atrazine-BSA conjugate containing 29 atrazine residues per BSA molecule. An ELISA on microtitration plates was optimized with peroxidase-atrazine conjugate. The middle of the test (50% B/Bo) was found to be at 90 ng/l, which is well below the maximum concentration permitted by the EC guidelines for drinking water. Detection limits for atrazine of about 1 ng/l could be reached. The assay did not require concentration or cleanup steps for drinking or ground water samples. Validation experiments showed good accuracy and precision. No cross-reactivities were shown by other s-triazines like terbutryn, ametryn, terbuthylazine, des-isopropylatrazine, and de-ethylatrazine except hydroxyatrazine. The latter was present at very low levels that can be calibrated/standardized before analysis or it may be considered as leftover residues of atrazine. Based on these results, it is suggested that this test can be applied to obtain fairly accurate results for atrazine concentration in water samples from different sources.