Identification of Pathogenic Germs and Antibiotics Residues in the Raw Milk and Their Effects on Human Health

Milk is a food of great value, it provides more essential nutrient than any other natural food. The presence of pathogenic bacteria and antibiotic residues in milk can cause a real danger to consumers. Effectively, the milk consumption contaminated by bacteria can have an immediate impact which means a toxi-infection. Therefore, the presence of antibiotics residues in milk can constitute an important risk at the allergic and antibiotic resistance cases on the consumer. The present study concerning the pathogens germs identification and Antibiotic residues seeking in milk and their impact on the human health, has been realized on a total number of 80 samples of raw milk resulted from direct sale channel （dairies） throughout Blida different regions localities. The Microbiological analysis has shown only three conform samples to JORA Standards. Really, milk non-conformity results to the microbiological standards consisting on total aerobic mesophilic flora total count, total coliforms, Thermotolerant coliforms E. coli, Faecal streptococcus, Staphylococcus aureus have shown the following contamination rate： 61.25%, 93.75%, 86.25%, 55%, 93.75% and 50%. Salmonella is characterized by a total absence in all analyzed milk samples. Moreover, the antibiotics residues research by Delvotest SP make plainly visible 33 positive samples. Further, two samples of the three which were judged conform to the bacteriology standards has been found contaminated by the antibiotic residues. The analyzed milk quality can be considered as a real danger to the consumption.

Antibiotic residues in milk and milk products:A momentous challenge for the pharmaceutical industry and medicine

Dairy products are nutritious food items that contain various essential nutrients,however,it has been proven that residual antibiotics have contaminated such products.These residues can cause several side effects on human health.They increase antimicrobial resistance against several threatening microorganisms,as well as significant growth in allergenic reactions.Various methods,including heat treatments,have been applied to alleviate and reduce the effect of antibiotic residue level in milk and milk products.Changes in drug levels were not sig-nificantly remarkable,obliging researchers to find new approaches to prevent or reduce their risk and limit their complications on human health.

Determination of Tetracycline and Oxytetracycline Residues in Honey by High Performance Liquid Chromatography

Honey is being used as a pure natural and as an ingredient in many foods, pharmaceuticals and cosmetics than ever before, so honey testing has become essential to maintain its healthful characteristics and protect public health. Tetracyclines （TCs） are used for the treatment or prevention of American and European foul broad in bee colonies which are caused especially by two species of bacteria--Paenibacillus larvae and Melissococcus pluton. The present study aimed to determine the tetracycline residues in different types of local and imported honey. The reversed-phase high performance liquid chromatography （HPLC） with ultraviolet detection （UV） was used. Tetracycline and oxytetracycline were extracted from honey with 0.01 M sodium succinate buffer. Of the examined samples, 31.25% and 12.50% had these oxytetracycline and tetracycline residues, respectively. It was observed that all the samples studied have been reported residues of tetracycline and oxytetracycline more than international standards of maximum residue limit （MRLs）.

Prevalence and risk assessment of antibiotics in riverine estuarine waters of Larut and Sangga Besar River, Perak

Antibiotics released into the environment through anthropogenic activities exert selective pressure,driving bacteria towards increasing antimicrobial resistance.The prevalence of antibiotics and the ecological risks posed in the riverine estuarine of Larut River and Sangga Besar River,which included wastewater effl uents from hospital,zoo,and poultry slaughterhouse sources were investigated.Solid phase extraction(SPE)followed by high-performance liquid chromatography tandem mass chromatography(HPLC-MS/MS)were used to extract and quantify the antibiotic residues from 22 antibiotics belonging to six major antibiotic classes(sulfonamide,macrolide,fl uoroquinolone,phenicol,trimethoprim,and tetracycline).Sixteen antibiotic residues were detected with concentrations ranging from limit of detection(LOD)to 1262.3 ng/L.Fluoroquinolones and macrolides were the most frequently detected compounds.Erythromycin,clarithromycin,and ofl oxacin detected in hospital and zoo effl uents posed a high risk to algae while tetracycline had low to medium ecological risks toward all the relevant organisms from aquatic environments(algae,invertebrate Daphnia magna,and fi sh).

Risk Assessment of Veterinary Drug Residues： The Dietary Exposure Assessment of Benzylpenicillin and Tetracycline Residues in Pork Consumed on Filipino Market

Biological, physical and chemical agents, recognized as food safety hazards, cause potential risks in modem food productions. Although the use of antibiotics in intensive animal productions is impossible to avoid the presence and remaining amount of their residues in food, it is possible to reduce them by using the principles of good veterinary, good manufacturing practices, and continuous control of food and risk assessment approach. This study was conducted to determine exposure risk to antibiotic drug residues in pork consumed in the Philippines and aimed to estimate dietary exposure of benzylpenicillin and tetracycline residues by age and gender groups. Dietary modeling, validated method used, combines food consumption data with food chemical concentration data to estimate dietary exposure to food chemicals. The results had shown that the dietary exposure of benzylpenicillin and tetracycline residues for children from 1 to 〈 3 years old had the highest exposure followed by adolescents, adults and infants while males were significantly lower than females （P 〈 0.05）. This is the initial study to determine risk assessment of dietary exposure to antibiotic residue of different population of Filipino groups.

Preliminary Research Study for Antibiotics in Eggs Produced and Sold in Bamako, Mali

The quality of agri-food products, in particular those of poultry origin, has now become an imperative. Because of the antibiotics used in poultry production, we are seeing the presence of antibiotic residues in products from poultry farming, especially eggs. Residues in eggs do not always comply with regulatory requirements, which can have harmful consequences for consumers such as: risk of allergies or risk of antibiotic resistance. This study was carried out with the aim of evaluating the presence of residues of veterinary antibiotics in eggs produced and marketed in the district of Bamako and surroundings. All samples were analyzed using the Premi ®Test which is a rapid detection kit for antibiotic residues in different materials. We collected 900 eggs from 10 farms and 10 markets in Bamako. Out of 900 eggs, 228 were tested. One hundred and eighty-nine (189) eggs tested negative for the presence of antibiotic residues, i.e. 82.9%;on the other hand, 39 were positive, i.e. 17.1%. This study clearly shows the presence of antibiotic residues in eggs from farms and various markets in Bamako and the surrounding area. This will allow us to continue our study at a later date to identify the types of antibiotic and to dose them.

Metagenomic insights into microorganisms and antibiotic resistance genes of waste antibiotic fermentation residues along production,storage and treatment processes

Antibiotic fermentation residue(AFR)is nutrient-rich solid waste generated from fermentative antibiotic production process.It is demonstrated that AFR contains high-concentration of remaining antibiotics,and thus may promote antibiotic resistance development in receiving environment or feeding farmed animals.However,the dominate microorganisms and antibiotic resistance genes(ARGs)in AFRs have not been adequately explored,hampering understanding on the potential antibiotic resistance risk development caused by AFRs.Herein,seven kinds of representative AFRs along their production,storage,and treatment processes were collected,and multiple methods including amplicon sequencing,metagenomic sequencing,and bioinformatic approaches were adopted to explore the biological characteristics of AFRs.As expected,antibiotic fermentation producer was found as the predominant species in raw AFRs,which were collected at the outlet of fermentation tanks.However,except for producer species,more environment-derived species persisted in stored AFRs,which were temporarily stored at a semi-open space.Lactobacillus genus,classified as Firmicutes phylum and Bacilli class,became predominant bacterial taxa in stored AFRs,which might attribute to its tolerance to high concentration of antibiotics.Results from metagenomic sequencing together with assembly and binning approaches showed that these newly-colonizing species(e.g.,Lactobacillus genus)tended to carry ARGs conferring resistance to the remaining antibiotic.However,after thermal treatment,remaining antibiotic could be efficiently removed from AFRs,and microorganisms together with DNA could be strongly destroyed.In sum,the main risk from the AFRs was the remaining antibiotic,while environment-derived bacteria which tolerate extreme environment,survived in ARFs with high content antibiotics,and may carry ARGs.Thus,hydrothermal or other harmless treatment technologies are recommended to remove antibiotic content and inactivate bacteria before recycling of AFRs in pharmaceutical industry.

An integrated microfluidic device for the simultaneous detection of multiple antibiotics

Residual antibiotics in food pose a serious long-term threat to human health.Therefore,an on-site visualization method for antibiotic detection is required.However,the requirements of traditional antibiotic testing methods in terms of operator proficiency and equipment cost hinder the rapid point-of-caretesting detection of suspected samples.Herein,we reported an integrated microfluidic device combining a microfluidic chip containing cruciform valves with immunochromatographic strips for the rapid detection of multiple antibiotics in milk.The rapid qualitative and quantitative analysis of four types of antibiotics(sulfonamides,β-lactams,streptomycin,and tetracyclines)was performed using mobile phone photography and mobile phone application analysis.The detection time was maintained at 10 min.The limits of detection(LODs)for the four antibiotics were 0.15,0.12,0.25,and 0.29 ng/mL,respectively,and the selectivity for the different antibiotics was observed even in a highly complex matrix.This device successfully integrated separation and real-time detection onto a chip and might provide a promising perspective for the detection of multiple antibiotics in milk.

Monoclonal antibody-based ELISA and colloidal gold-based immunochromatographic assay for streptomycin residue detection in milk and swine urine

A protein conjugate of streptomycin （streptomycin-bovine serum albumin （BSA） conjugate） was prepared and used as immunogen to produce monoclonal antibodies （MAb）. One hybridoma secreting anti-streptomycin MAb was obtained and then used to produce MAb. The MAb named 13H5 showed the 50% maximal inhibitory concentra- tion （IC50） value of 4.65 ng/ml and the IC20value of 0.21 ng/ml in phosphate buffered saline （PBS）. At optimum con- ditions, an indirect competitive enzyme-linked immunosorbent assay （ELISA） and a colloidal gold-based immuno- chromatographic assay （CGIA） were developed and applied to detect streptomycin residues in milk and swine urine samples. The developed ELISA showed that the minimum detection limit was 2.0 and 1.9 ng/ml for milk and swine urine samples, respectively, without obvious cross-reactivity to other tested antibiotics except dihydrostreptomycin which gave a 118.32% cross reaction value. Milk and swine urine samples spiked with streptomycin at 10, 50, 100 and 200 ng/rnl were analyzed by the established ELISA. The mean recovery of streptomycin was from 81.9% to 105.5% and from 84.3% to 92.2% for milk and swine urine, respectively. The optimized CGIA showed that the minimum de- tection limit was 20.0 ng/ml for milk and swine urine samples. The results of spiked analysis and specific analysis demonstrate that the CGIA could be applicable for screening milk and swine urine samples for the presence of streptomycin residues on-site. The established ELISA and CGIA allow the rapid, low-cost, and sensitive determination of streptomycin residues in food samples.

Antibiotic residues from aquaculture farms and their ecological risks in Southeast Asia:a case study from Malaysia

Background and Objectives:One major source of antibiotic contamination in the sea is from aquaculture.We monitored the concentration of commonly used antibiotic classes and antibiotic resistance genes(tet(M),sul1,sul2 and sul3)in aquaculture farms in Peninsular Malaysia.Methods:Antibiotic residues and resistance genes were quantified using high-performance liquid chromatography and real-time PCR respectively.Risk quotients in European technical guidance document on risk assessment was used to assess the potential environmental risk.Results:We detected 23 antibiotics with tetracyclines,sulfonamides and quinolones were the most frequently detected classes,indicating a wide distribution of antibiotics in Malaysian aquaculture farms.The dendrogram and heatmap revealed three groups of antibiotic concentration patterns but with no differences in the types of antibiotics usage among aquaculture farms.The ARGs(10^(−3)copies/16S)were detected in>90%of the sites except for sul3.Ciprofloxacin,enrofloxacin,norfloxacin and lincomycin posed risks to cyanobacteria and algae in Kelantan,Perak and Pahang.Conclusion:Relative to Asian aquaculture farms,the residues detected here were at low or moderate levels except for quinolones.This study will be useful to develop effective management of aquaculture wastewater in order to mitigate antibiotic pollution and transmission of ARGs to humans through the food chain.

Determination of antibiotic residues in the pasteurized milk produced in West Azerbaijan province,North West of Iran

Objective:To monitor antibiotic residues in pasteurized milk in West Azerbatjan province,North West of Iran.Methods:In this study,848 pasteurized milk samples were collected from factories and tested for the presence of antibiotic residues using the Copan test kit based on the manufacturer's instructions.Results:Results indicated that 30.14%of samples were contaminated with a variety of antibiotics based on the detection of associated residues and 3.19%of these samples were suspected.Given the current rise of antimicrobial resistance among microbial pathogens,these findings amplify the need to ensure continuous monitoring of pasteurized milk that intended for human consumption.Conclusions:Continuous monitoring of pasteurized milk may improve human health but also limit the development and transmission of antibiotic resistant strains in the environment.

A genetically engineered Escherichia coli that senses and degrades tetracycline antibiotic residue

Due to the abuse of antibiotics,antibiotic residues can be detected in both natural environment and various industrial products,posing threat to the environment and human health.Here we describe the design and implementation of an engineered Escherichia coli capable of degrading tetracycline(Tc)-one of the commonly used antibiotics once on humans and now on poultry,cattle and fisheries.A Tcdegrading enzyme,TetX,from the obligate anaerobe Bacteroides fragilis was cloned and recombinantly expressed in E.coli and fully characterized,including its Km and kcat value.We quantitatively evaluated its activity both in vitro and in vivo by UVeVis spectrometer and LC-MS.Moreover,we used a tetracycline inducible amplification circuit including T7 RNA polymerase and its specific promoter PT7 to enhance the expression level of TetX,and studied the dose-response of TetX under different inducer concentrations.Since the deployment of genetically modified organisms(GMOs)outside laboratory brings about safety concerns,it is necessary to explore the possibility of integrating a kill-switch.Toxin-Antitoxin(TA)systems were used to construct a mutually dependent host-plasmid platform and biocontainment systems in various academic and industrious situations.We selected nine TA systems from various bacteria strains and measured the toxicity of toxins(T)and the detoxifying activity of cognate antitoxins(A)to validate their potential to be used to build a kill-switch.These results prove the possibility of using engineered microorganisms to tackle antibiotic residues in environment efficiently and safely.

Environmental risk quotient of the antibiotic,phenotypic,and genotypic profiles for antibiotic resistance of Escherichia coli collected from manure and wastewater at swine farms in Prachinburi Province,Thailand

Currently,antimicrobial drug resistance is a problem that deserves thoughtful consideration.Especially in the swine production industry,many pig farms tend to release wastewater into natural water sources,which villagers use for their livelihood.This study aimed to demonstrate the occurrence of phenotypic and genotypic resistance characteristics of E.coli,as well as antibiotic residues in manure and wastewater on swine farms in Prachinburi Province,Thailand.Samples were collected in 2020 and 2021 during the rainy and summer seasons.The results of a questionnaire showed that farmers used antibiotics for disease treatment and prevention at a ratio of 2/4(50%).The most common antibiotic used was amoxicillin(4/4,100%),followed by Enrofloxacin(75%)and colistin(25%).The analysis of antibiotic residue in wastewater by Liquid Chromatography-Mass Spectrometry(LCMS,LC-MS)during the rainy season revealed that amoxicillin(54%)was the highest,followed by florfenicol(14%)and tiamulin(13%).While oxytetracycline(59%),amoxicillin(20%),and florfenicol(11%)were the highest in the summer.The study of Risk Quotient(RQ)indicated that water resources present a risk of antibiotic contamination by sulfonamides,trimethoprim,b-lactam,quinolones,and tetracycline at high levels(SRQ>1)during both seasons.The minimal inhibitory concentrations(MICs)were determined by an antibiotic susceptibility test(AST),analyzed by microdilution technique,and VITEK®2 Compact.The test result indicated that a total of 143 isolates were found in manure(45/143),pre-treatment(51/143),and final effluent(posttreatment,47/143).ESBL-positive resistance was detected at 13%.However,an immensely high percentage of antimicrobial resistance was found for ampicillin(90%),whereas imipenem did not show any drug resistance(0%).The average MIC value towards colistin in all samples was 2.71 mg/ml,while mcr1 was not found in any samples.Furthermore,MDR was expressed in E.coli at as high as 76.22%(109/143).The highest MDR pattern detected was AMP-TETeFFCeSXT.The PCR technique detected that the diversity and abundance of AMR genes were not significantly different from animal manure and wastewater at the swine farms.The bla-TEM was found more frequently than bla-PS.While the tetracycline group(tetA,tetB)and aminoglycoside(aadA1,aadA2,aadB)represented a major proportion,respectively.The PFGE study revealed the possibility of similar genetic morphology from the different isolates found in this study.