Microbial inhibition tests

A variety of biological approaches for the rapid detection of antibiotics in food exist, including traditional microbial inhibition tests, immunoassays, and... 

Microbial inhibition tests are extremely sensitive for -lactam antibiotics, primarily penicillin, but mostly are more than 100-fold less sensitive for other commonly used antibacterials such as macrolides, sulfonamides, tetracyclines, or chloramphenicol (4, 5). Therefore, inhibition tests usually classify residues as belonging to the -lactam group. Antibiotics other than -lactams and sulfonamides can be detected by use of the enzyme penicillinase and aminobenzoic acid, respectively (1, 6). 

The CHARM II test for tissues is relatively fast, easy to perform, and requires limited laboratory equipment. However, for antibacterials with established tolerance levels, it can serve only as a screening test because the results are not quantitative and therefore should be supported by additional quantitative chemical methods. The microbial receptor assay, with its broad-spectrum capability, can enhance any existing monitoring system as a first-line monitoring test or as a confirmation for any program using microbial inhibition tests. 

A new analytical method was based on the treatment of SAs with p-aminobenzoic acid, forming derivatives suitable for UV detection. The SAs were extracted from the kidney and liver samples, with recoveries ranging from 55% to 100%. The reaction yield was tested by microbial inhibition tests sensitive to low concentrations of SAs. After the incubation with / -aminobenzoic acid, none of eight SAs produced an inhibition zone on the assay medium, which showed 100% conversion to the appropriate derivatives (162). 

The basic microbial inhibition assay format involves a standard culture of a test organism, usually Bacillus stearothermophilus, Bacillus subtilis, Bacillus cereus, Micrococcus luteus, Escherichia coli, Bacillus megatherium, or Strepto-793... 

The CHARM inhibition assay (CIA), Charm farm test (CFT), and Valio TlOl test are all simple multiresidue screening tests based on microbial inhibition (34, 35). The CIA test is actually a disc assay using Bacillus stearothermophilus and specially formulated agar media to increase the sensitivity to sulfonamides. The CFT is a tube assay using the same test organism in a specific formulation, which, along with the nutrients, is in a tablet form. To roughly identify penicillins and sulfonamides with the CIA and CFT tests, positive samples should be reanalyzed after the addition of penicillinase and p-aminobenzoic acid. 

This research quantified the enzymatic digestibility of the solid component and the microbial inhibition of the liquid component of pretreated aspen wood and com stover hydrolysates. Products of liquid hot water and carbonic acid pretreatment were compared. Pretreatment temperatures tested ranged from 180 to 220°C/ and reaction times were varied between 4 and 64 min. Both microbial inhibition rates and enzymatic hydrolysis rates showed no difference between pretreatments containing carbonic acid and those not containing no carbonic acid. Microbial inhibition increased as the reaction severity increased, but only above a midpoint severity parameter of 200°C for 16 min. Both the rates and yields of enzymatic hydrolysis displayed an increase from the lowest tested reaction severity to the highest tested reaction severity. 

Both microbial inhibition rates and enzymatic hydrolysis rates showed no differences between pretreatments containing carbonic acid and those not containing carbonic acid. Additionally, when the microbial inhibition and enzymatic hydrolysis rates were tested at varying reaction severities and between different substrates, this remained true. 

No evidence of genotoxicity was observed in vitro with or without metabolic activation in the following assays microbial mutagen tests using mutant strains of Salmonella typhimurium or Escherichia coli, Chinese hamster ovary forward mutation assay, unscheduled DNA synthesis in rat primary hepatocytes, chromosome aberrations in Chinese hamster ovary cells, and spindle inhibition in human lymphocytes. In addition, there was no evidence of genotoxicity in vivo in a mouse micronucleus test there was equivocal evidence of mutagenicity in a mouse domi nant lethal test. 

Japanese workers are at the forefront in the search for physiologically active microbial metabolies. Their detection methods are worthy of note for lack of satisfactory testing methods has long hampered this search. Umezawa and his co-workers used in vitro enzyme inhibition tests to detect fusaric acid, pepstatin, chymostatin, and the leupeptins. Nigrifactin was discovered by Terashima and co-workers by seeking in experimental fermentations, materials having the chemical properties of alkaloids. 

Furthermore, what is considered active is subjective. While one researcher might consider microbial inhibition at 1% as active, another would only consider activities of less than 0.025% to be active. Since test methods vary greatly, it is difficult to compare the results directly. In addition, the different methods tend to lead to different results due to many issues including, but not limited to, solubility and solvent choice. When standards are used, it is important to consider structurally related standards with similar solubilities in water. 

Shortly after their development in the 1940s, antibiotics were used in veterinary medicine, first to prevent or treat mastitis in cows and later for the treatment of other diseases. Initial concern about antibiotic residues in milk was not a public health issue but came from dairy processors who noticed inhibition of starter cultures used in the production of cheese and yogurt, thus generating a need for screening tests to examine milk for antibiotic residues. " Since inhibition of starter cultures by penicillin in milk was the main problem, the earliest microbial inhibition assays were based on growth inhibition of lactic acid bacteria. Spores of Bacillus species were also utilized spores are easier to handle and far more stable than the vegetative cells. 

A limited range of microbial inhibition assays has also been developed and is commercially available for the analysis of tissue samples (muscle, kidney, liver), egg, fish, and honey. These assays include the PremiTest, produced by DSM Food Specialities Ltd. (Delft, The Netherlands) the Explorer test, developed by Zeu Inmunotec (Zaragoza, Spain) and the kidney inhibition swab (KIS test), produced by Charm Sciences Inc. (Massachusetts, USA). 

The KIS test is marketed for use at either abattoirs or laboratories for the analysis of kidney samples to identify animal carcasses with non-compliant concentrations of antimicrobial residues present in the edible tissues. The KIS reagents are supplied as self-contained and pre-measured for a single use presented in a disposable swab format. The swab is directly inserted into an incision made in the kidney test sample to collect a sample of the serum for analysis via microbial inhibition. The impregnated swap is then inserted into a device containing a tube diffusion MIA... 

Other tests based on the use of swabs and microbial inhibition assays were developed at the US Department of Agriculture and Food Safety and are widely used in the United States of America and Canada primarily by meat inspection agencies, including the swab test on premises (STOP), calf antibiotic screen test (CAST), and the fast antibiotic screen test (FAST)." 45 -j g STOP assay employs Bacillus subtilis, and the CAST and FAST assays use Bacillus megaterium. However, these tests will not be discussed further in the context of commercially available assays. 

The starting point in the technical development of a microbial inhibition assay is the sensitivity of the test microorganism to different antimicrobial compounds under different media conditions. Factors such as the pH and nutrient profile of the media can be varied to obtain the optimum performance. The sensitivity of MIAs may also be modulated by the addition of specific compounds to the test composition. For certain antimicrobial compounds an enhancement in sensitivity may be advantageous, whereas for others, a sensitivity decrease is required to avoid the number of false non-compliant results. For example, cysteine can reduce the overall sensitivity to the P-lactam class. Antifolates, such as ormethoprim or trimethoprim, are known to improve the sensitivity of the test organism to sulfonamide compounds. Sensitivity to sulfonamides can also be modulated by using the enzyme dihydropteroate synthetase that selectively inhibits the response of sulfonamides." Antibiotic receptors such as antibodies can also be employed to decrease the sensitivity of the test to specific compounds, as required. 

Okerman et al. compared the performance of a Tetrasensor with three microbial inhibition assays for the analysis of tetracycline antibiotics in tissue. The group concluded that when large numbers of samples have to be analyzed without the requirement for immediate results, classical agar diffusion tests with thin plates and performed as prescribed for the EPT still seem the most economical choice. However, the receptor-based test Tetrasensor was recommended for use in official surveys and also in cases when immediate results are required. Unlike the inhibition tests, this receptor test does not require a well-equipped laboratory for use and is more suited for the meat industry. ... 

To determine what microorganism types are susceptible to the antimicrobial soap product, as well as the rates of microbial inactivation, in vitro tests should be conducted. These include time-kill kinetic evaluations, minimum inhibition concentration evaluations, and microbial sensitivity tests. However, in order to determine the actual effectiveness, human-use studies must be conducted. There are three common ways of evaluating antimicrobial hand soaps (1) the health care personnel handwash evaluation, (2) the modified Cade handwash procedure, and (3) the general-use handwash evaluation. 

Similarly, there are also tests for microbial inhibition. In these tests, the snrface is pretreated with the cleaner and then inoculated with a mixture of nutrient and microorganism of interest. The surface is then incubated, and the amount of growth observed after incnbation indicates the inhibitory effect of the cleaner. In particular, the literature gives an example of this type of test to exhibit antifungal property [111]. 

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