Aqueous two-phase system (ATPS) is a liquid-liquid fractionation technique and has gained an interest because of great potential for the extraction, separation, purification and enrichment of ...proteins, membranes, viruses, enzymes, nucleic acids and other biomolecules both in industry and academia. Although, the partition behavior involved in the method is complex and difficult to predict. Current research shows that it has also been successfully used in the detection of veterinary drug residues in food, separation of precious metals, sewage treatment and a variety of other purposes. The ATPS is able to give high recovery yield and is easily to scale up. It is also very economic and environment friendly method. The aim of this review is to overview the basics of ATPS, optimization and its applications.
Food contamination with antibiotic residues has become a worldwide problem due to the heavy and improper use of antibiotics. The development of fast, high-throughput, easy, and economical screening ...methods for the detection of antibiotic residues is an important requirement as an alternative to the traditional assays. Immunoassays are widespread promising methods that can achieve the conditions of an analytical method in the evaluation of food and environmental protection. This review offers an overview of the preparation of antibodies and the currently established immune-assays for the antibiotic residues detection, including enzyme-linked immunosorbent assay, fluorescence immunoassay, radioimmunoassay, colloidal gold immunoassay, and chemiluminescence immunoassay. Furthermore, it will elaborate on the future perspective on the performance and improvement of these assays for the determination of antibiotic residues in food samples. These assays include a simple sample preparation and high-sensitive detection that offer a significant assurance for the screening of antibiotic residues.
The development of antibiotic resistance in bacteria is a major public health threat. Infection rates of resistant pathogens continue to rise against nearly all antimicrobials, which has led to ...development of different strategies to combat the antimicrobial resistance. In this review, we discuss how the newly popular CRISPR-cas system has been applied to combat antibiotic resistance in both extracellular and intracellular pathogens. We also review a recently developed method in which nano-size CRISPR complex was used without any phage to target the mecA gene. However, there is still challenge to practice these methods in field against emerging antimicrobial resistant pathogens.
A broad-spectrum microbiological inhibition method has been developed for rapidly screening different kinds of antibiotics such as β-lactam, aminoglycosides, tetracyclines, sulfonamides, macrolides, ...lincosamides and quinolones in milk, chicken egg and honey by using an easy sample preparation. The microbiological system in microtiter plates consists of an agar medium, a mixture of nutrients, test bacteria (
C953), bromocresol purple, and other supplements such as trimethoprim, chloramphenicol, streptomycin and enrofloxacin which helps to improve the detection capability of the microbiological system toward the chosen antibiotics. It was observed that the limit of detection of the kit used in present study for all kinds of antibiotics in milk were lower than or close to maximum residue limits determined by EU or CODEX. For chicken egg and honey, the detection capability of the kit was similar to that determined in milk. Moreover, it was revealed that the kit in present study was more sensitive to aminoglycosides, macrolides and quinolones in various matrixes than internationally available commercial kits. The false-positive and false-negative rates for both were 0%. The coefficient of variations among various factors was all less than 4%. Additionally, the quality guarantee period of the kit was more than 6 months at 4°C. A good correlation between the kit results and the LC-MS/MS results for milk was also observed, which revealed that the kit was reliable to screen antibiotics residues in incurred samples.
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•This assay was developed for antibiotic detection in muscle and kidney of swine.•The method was sensitive to 26 antibiotics from seven different groups.•The other performance ...characteristics of the assay were all up to the standard requirements.•It might be a platform for clinical, environmental, food residual analyses.
In present study, a new ATP bioluminescence assay with Geobacillus stearothermophilus ATCC12980 as an indicator bacterium was developed to screen multiclass antibiotic residues in animal derived foods. The development experiments includes the optimization on the usage of D-luciferin, bacterial ATP extraction condition, incubation condition of detection system and sample pre-treatment. Additionally, the validation experiments of sensitivity, specificity, accuracy, precision and stability were performed. This method can detect β-lactams (6), aminoglycosides (4), tetracyclines (4), sulfonamides (4), macrolides (4), lincosamides (1) and quinolones (3) in muscle and kidney of swine at or close to maximum residue limits (MRLs) established by European Union (EU). The false-positive rate was 0%, and the false negative rates were < 5%. The recovery rate of antibacterial drugs were between 60% and 120%, and the daily coefficients of variation were less than 15%, which meets the criteria in Commission Decision 2002/657/EC. The shelf life of the detection system was more than 5 months. Accordingly, the proposed ATP bioluminescence assay shows great potential for rapid, sensitive and high throughput detection of multiple antibiotic residues in animal foods, which can provide a preliminary screening informations of antibiotic residues for the following chemical confirmation experiments and expedite disposal decisions of unqualified food for enhanced food safety inspection.
Several factors are involved in the emergence of antibiotic-resistant bacteria and pose a serious threat to public health safety. Among them, clustered regularly interspaced short palindromic repeat- ...(CRISPR-) Cas system, an adaptive immune system, is thought to be involved in the development of antibiotic resistance in bacteria. The current study was aimed at determining not only the presence of antibiotic resistance and CRISPR-Cas system but also their association with each other in Salmonella enteritidis isolated from the commercial poultry. A total of 139 samples were collected from poultry birds sold at the live bird markets of Lahore City, and both phenotypic and genotypic methods were used to determine antimicrobial resistance. The presence of the CRISPR-Cas system was determined by PCR, followed by sequencing. All isolates of S. enteritidis (100%) were resistant to nalidixic acid, whereas 95% of isolates were resistant to ampicillin. Five multidrug-resistant isolates (MDR) such as S. enteritidis isolate (S. E1, S. E2, S. E4, S. E5, and S. E8) were found in the present study. The CRISPR-Cas system was detected in all of these MDR isolates, and eight spacers were detected within the CRISPR array. In addition, an increased expression of CRISPR-related genes was observed in the standard strain and MDR S. enteritidis isolates. The association of the CRISPSR-Cas system with multiple drug resistance highlights the exogenous acquisition of genes by horizontal transfer. The information could be used further to combat antibiotic resistance in pathogens like Salmonella.
Coronaviruses (CoVs) infect a wide range of domestic and wild mammals. These viruses have a potential and tendency to cross-species barriers and infect humans. Novel human coronavirus 2019-nCoV ...(hCoV-19) emerged from Wuhan, China, and has caused a global pandemic. Genomic features of SARS-CoV-2 may attribute inter-species transmission and adaptation to a novel host, and therefore is imperative to explicate the evolutionary dynamics of the viral genome and its propensity for differential host selection. We conducted an in silico analysis of all the coding gene sequences of SARS-CoV-2 strains (n = 39) originating from a range of non-human mammalian species, including pangolin, bat, dog, cat, tiger, mink, mouse, and the environmental samples such as wastewater, air and surface samples from the door handle and seafood market. Compared to the reference SARS-CoV-2 strain (MN908947; Wuhan-Hu-1), phylogenetic and comparative residue analysis revealed the circulation of three variants, including hCoV-19 virus from humans and two hCoV-19-related precursors from bats and pangolins. A lack of obvious differences as well as a maximum genetic homology among dog-, cat-, tiger-, mink-, mouse-, bat- and pangolin-derived SARS-CoV-2 sequences suggested a likely evolution of these strains from a common ancestor. Several residue substitutions were observed in the receptor-binding domain (RBD) of the spike protein, concluding a promiscuous nature of the virus for host species where genomic alternations may be required for the adaptation to novel host/s. However, such speculation needs in vitro investigations to unleash the influence of substitutions towards species-jump and disease pathogenesis.
To investigate the optimal dosage which can improve clinical efficacy and minimize resistance, pharmacokinetics/pharmacodynamics model of enrofloxacin was established. Effect of enrofloxacin ...treatments on clearance of Salmonella in experimentally infected chickens and simultaneously resistance selection in Salmonella and coliforms were evaluated in three treatment groups (100, PK/PD designed dosage of 4, 0.1 mg/kg b.w.) and a control group. Treatment duration was three rounds of 7-day treatment alternated with 7-day withdrawal. Results showed that 100 mg/kg b.w. of enrofloxacin completely eradicated Salmonella, but resistant coliforms (4.0-60.8%) were selected from the end of the second round's withdrawal period till the end of the experiment (days 28-42). PK/PD based dosage (4 mg/kg b.w.) effectively reduced Salmonella for the first treatment duration. However upon cessation of medication, Salmonella repopulated chickens and persisted till the end with reduced susceptibility (MIC
= 0.03-0.25 mg/L). Low frequency (5-9.5%) of resistant coliforms was selected (days 39-42). Enrofloxacin at dosage of 0.1 mg/kg b.w. was not able to eliminate Salmonella and selected coliforms with slight decreased susceptibility (MIC
= 0.25 mg/L). In conclusion, short time treatment (7 days) of enrofloxacin at high dosage (100 mg/kg b.w.) could be effective in treating Salmonella infection while minimizing resistance selection in both Salmonella and coliforms.
Since the emergence of COVID-19 pandemic in China in late 2019, scientists are striving hard to explore non-toxic, viable anti-SARS-CoV-2 compounds or medicines. We determined In vitro ...anti-SARS-CoV-2 activity of oral formulations (syrup and capsule)of an Iodine-complex (Renessans). First, cell cytotoxicity of Renessans on the Vero cells was determined using MTT assay. Afterwards, the antiviral activity of Renessans was determined using viral inhibition assays and TCID
50
. For this, nontoxic concentrations of the Renessans were used. The results showed that Renessans is nontoxic to the cells up to 50 µg/mL. At 1.5 µg/mL concentration, SARS-CoV-2 production was significantly reduced to 10
1.43
TCID
50
and 10
1.58
TCID
50
for the syrup and capsule, respectively, as compare to virus infected control cells 10
6.08
TCID
50
and we found the dose dependent inhibition of virus replication in the presence of Renessans. Renessans inhibited SARS-CoV-2 with an EC50 value of 0.425 µg/mL and 0.505 µg/mL for syrup and capsule, respectively. Furthermore, there was no virus detected at concentration of 50 µg/mL of Renessans. This study indicates that Renessans, containing iodine, have potential activity against SARS-CoV-2 which needs to be further investigated in human clinical trials.
Newcastle disease (ND), caused by virulent
Avian avulavirus
1 (AAvV 1), affects variety of avian species around the globe. Several AAvV 1 viruses of different genotypes have recently emerged with ...varying clinical impacts on their susceptible hosts. Although experimental infection with velogenic and mesogenic strains in chickens and pigeons is well-studied, nevertheless, there exists a paucity of data for comparative variations in serum biochemistry profile of susceptible hosts upon challenge with isolates of varying pathogenicities. With this background, a comparative assessment of a range of serum biochemical parameters was made following challenge with duck-originated velogenic strain (sub-genotype VIIi; MF437287) and pigeon-originated mesogenic strain (sub-genotype VIm; KU885949) in chickens and pigeons. For each of the isolate, commercial broiler chickens and wild pigeons were challenged (10
–6.51
EID
50
/0.1 mL for sub-genotype VIIi and 10
–6.87
EID
50
/0.1 mL sub-genotype Vim) separately via intranasal and intraocular route. Sera were collected on 0, 3rd, 5th, 7th, and 9th day post-infection (dpi), and processed for quantitative analysis of different biochemical parameters. By day 3 post-infection (pi), a substantial decrease (
p
< 0.0001) in serum alkaline phosphatase (ALP) was observed in chickens and pigeons challenged with velogenic isolate. On the other hand, from day 5 pi and onward, a significant increase (
p
< 0.001) in serum ALP and total protein concentration was observed exclusively in pigeons challenged with mesogenic isolate. For serum aspartate aminotransferase (AST), a significant increase (
p
< 0.05) in concentration was observed on day 3 pi which decreased from day 5 pi and onward in pigeons and chickens challenged with mesogenic isolate. Also, to reveal antigenic differences among homologous and heterologous vaccine and field-prevalent strains, cross-hemagglutination inhibition assay demonstrated antigenically diverse nature (
R
-value < 0.5) of both strains from vaccine strain (LaSota, genotype II). The study concludes antigenic differences among prevalent genotypes than vaccine strain and, although requires further studies to ascertain study outcomes, the serum biochemical profile may facilitate presumptive diagnosis of disease in their susceptible hosts.
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