Hypothesis on Function of Autotrophs and Wild Types of Salmonella Typhimurium and Escherichia Coli Strains

The Ames assay early developed the test to screening of potential carcinogens drugs, toxins... The Ames tester strains are S. typhimurium and E. coli strains which have been used for more than 60 years to detect of mutagenic compounds. All carcinogenic compounds are mutagen but all mutagenic compounds haven’t carcinogenic effects. Point mutations were made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. Assay used to evaluate professions and environmental samples of carcinogenicity and is between the assays of mutagenicity testing in environmental and pharmaceutical chemical regulatory agencies. All of these engineered commensal microbes are weak and morphology of them exhibited gram negative and sensitive to temperature and UV light. It seems that wild types of S. typhimurium and Escherichia coli strains were susceptible to some of antimicrobial agent.That might be also used to determine Dose/Response and Susceptible/Resistance. Afterwards these types make pathogenic effects and cause disease. Ames tests results showed that the chemical or pharmaceutical compound has directly carcinogenic effects in 66-90% cases of rodent animals.

Ames assay history

The Ames assay key role developments and its history established by Bruce Nathan Ames during the 1960s and 1970s, a professor at the University of California, Berkeley, and early developed the test to screening of potential carcinogens drugs, toxins.. while at the National Institutes of Health. The procedure was described in a series of papers published by Ames and his colleagues in 1970s. The Ames tester strains are S. typhimurium and E. coli strains which have been used for more than 60 years to detect of mutagenic compounds [1-3].

Ames assay microbe’s strains

The Ames assay uses specific strains of bacterias, primarily Salmonella typhimurium and Escherichia coli, which have been genetically engineered to be unable to produce an essential amino acid like histidine or tryptophane. The Ames assay uses specific standard set of strains includes S. typhimurium strains are included (TA98, TA100, TA102, TA104, TA1535, TA1537, TA1538), and either E. coli WP2 uvrA or E. coli (WP2 uvrA or WP2 uvrA (pKM101) plasmid [4]. These strains are autotrophs, meaning they will grow on a medium lacking the required amino acids only if the tested pharmaceutical and chemical compounds cause a reverse mutation that restores the ability to synthesize it in their medium special cultures. All of these engineered commensal microbes are weak and morphology of them exhibited gram negative andsensitive to temperature and UV light, and they can’t produce pathologic effects in hosts by themselves. Afterwards some of these salmonella typhimurium bacteria with involving plasmid (pKM101) are resistenced to ampicillin antibiotic. Not only These Salmonella typhimuriummicrobes are not typically associated with diarrhea, such as typhoid fever, febrile illnesses, but also are accompanied by nonspecific clinical features with self-treatment [5-7,15]. 

Ames assay applications 

The Salmonella mutagenicity test is the primary step assay used to evaluate professions and environmental samples of carcinogenicity and is between the assays of mutagenicity testing in environmental and pharmaceutical chemical regulatory agencies, such as the Environmental Protection Agency (EPA) and Food Drug Administration (FDA). The assay has a main role of playing in other regulatory activities, such as the European Registration, Authorization and Restriction of Chemicals (REACH) program and the Toxic Substances Control Act (TSCA) and it is also one of the cheapest and fast assays included in the Organization for Economic Cooperation and Development (OECD) [4,9-15].   

Ames assay protocol

All of histidine negative strains after adding trace amount .001mmol histidine and carcinogenic compounds in melt top agar in 38-420C will be converted to histidine positive wild types gram negative bacterias.so these microbes will grow with revertant colonies in glucose minimum agar medium incubated in 370C due 24-72hours. So, histidine positive wild types strains would have been pathogenic conditions. However, after exposing mutated bacteria’s with carcinogenic compounds, they will made revertant colonies in specific medium cultures. These colonies are included wild types microbes [4,9,10].

Metabolic activation

It was soon recognized that many chemicals mutagenic compounds are not directly carcinogenic but become so after being metabolized by the liver microsomal P450 enzymes. To address this, a system for metabolic activation was introduced, typically using a liver enzyme extract (S-9Mix) from male rodents to mimic mammalian metabolism by process of freeze ultra-centrifugation [4,9,10].

Important Mutations

Point mutations were made in the or the tryptophan (Escherichia coli) histidine (Salmonella typhimurium) operon, rendering the bacteria incapable of producing the corresponding amino acid. These mutations result in his- or trp- organisms that cannot grow unless histidine or tryptophan is supplied. The Ames tester strains are S. typhimurium and E. coli strains which have been used for more than 60 years to detect carcinogenic pharmaceutical and chemical compounds. Point mutations were made in the histidine (Salmonella typhimurium) or the tryptophan (Escherichia coli) operon, rendering the bacteria incapable of producing the corresponding amino acid. Media lacking histidine or tryptophan are used for this selection which allow only those cells that have undergone the reversion to tryptophan / histidine prototrophy to survive and grow. A mutagenic event causing frameshift mutations or base substitutions and others within the gene may cause a reversion to amino acids prototrophy. Not only these reverted mutation bacterias will respectively grow in histidine - or tryptophan lacked glucose minimum agar, but also non reverted bacteria will not be able to grow in this culture, hereby these reverted microbes will have named wild types [4,9,10,12-15].

Wild types microbes antibiogram

So, the drug sensitivity pattern commercially available antibiotic discs from (Himedia, India, Oxoid, England) that might be also used to determine Dose/Response and Susceptible/Resistance. At the same time, it seems that wild types of S. typhimurium and Escherichia coli strains were susceptible to antimicrobial agents such Ciprofloxacin, Norfloxacin Streptomycin, Ampicillin, Penicillin, Gentamycin, ofloxacin, amikacin, chloramphenicol, tobramycin and trimethoprim, whereas most of these types were resistant to amoxicillin, erythromycin, azithromycin and tetracycline, kanamycin spectinomycin.... [5-8,14,15]. Some of studies revealed that presence of multidrug resistant (MDR) of Salmonella and E. coli are isolated in meat markets. So, the interpretation on susceptibility was done according to the guidelines of Clinical and Laboratory Standard Institute (CLSI, 2012) priory known as NCCLS [6-9].

Bacteriology tests on wild types

Some of biochemistry test e.g. Indole, Methyl Red, Voqes – prosquer and citrate for enterobacteriacea confirmation of these strains are requirement. It is probably some of these tests have been variation results for wild types [9-12].

The main Ames test studies data are Ratio = Plate test colony count/Plate Control colony count, and they collected from considering usage with +S-9Mix and without -S-9Mix. The ratio ≥2 of Ames tests results showed that the chemical or pharmaceutical compound has directly carcinogenic effects in 66-90

Regulatory agencies

By the late 1970s, the Ames test was being used by regulatory agencies like the Occupational Safety and Health Administration (OSHA), and International Agency research on cancer (IARC). This provided a simple and inexpensive method to screen for potential carcinogens by observing if the bacteria could grow on a histidine/Tryptophan free medium after exposing to the carcinogenic toxins and drugs in specific conditions. All carcinogenic compounds are mutagen but all mutagenic compounds haven’t carcinogenic effects [4,12-15].

Protection, prevention and prophylaxis management

Therefore, after passage, created the wild types pathogenic strains must be autoclaved and disposed under sanitary conditions. Employees must also take care of their personal hygienic when working with high frequency of contaminated microbes. Afterwards these types make pathogenic effects and cause disease. Immunocompromised individuals, including those with HIV and other infected disease, and infants and young adults and olds and pregnant women living in areas where malnutrition are common exposure to high risk [7-12]. Nowadays Cancers are currently one of the most problems between the world population, and the cancerous patients have encountered with treatment challenges. Therefore, new world of microbes and roles of them with other modern molecular and cellular sciences and technologies can be provide rapid and cost benefit easy ways to diagnosing and management of malignant disease [12,13,14,15].

We are grateful to the pharmacology department of pharmacy school of Tabriz-Iran university medical Sciences, and also pharmacology department of medicine school of university medical Sciences of Tehran-Iran for supporting of this study scientific background.