Overcoming Resistant Infections (Salmonella Tiphymurim) by formulating Antimicrobials (Streptomycin) with Medicinal Synthetic Aluminum magnesium silicate {Al4 (SiO4)3 + 3Mg2SiO4 → 2Al2 Mg3 (SiO4)3}.

I. INTRODUCTION

Antimicrobial resistance (AMR) is a Global health challenge. Use of antimicrobials as growth promoters in animal farming is a major cause of AMR in humans (Agyare et al., 2018). In poultry, treatment failures due to AMR result in great economic losses. AMR in poultry is also a big risk to human health (Van et al., 2012; Okorie-kanu et al., 2016).

Antimicrobial resistance means loss of sensitivity by microorganisms to drugs which were effective (Sinyangwe et al., 2004). Mechanisms for Antimicrobial resistance include loss of surface specific receptors or transporters for drugs, rapid metabolism of drugs and alteration of specific targets for drugs. There are many reports of increases in resistance involving bacteria of Veterinary and public health importance, including Salmonella typhimurium, against commonly used drugs which include Streptomycin (Bortolaia et al., 2016, Nguyen et al., 2017).

Apart from wrong use of antimicrobials in animal farming which causes development of resistance by infections in animals before they are transmitted to human beings, use of high dosages in treating animals leads to high concentrations of residues of the drugs in human foods of animal origin which leads to development of antimicrobial resistance in humans (Reig and Toldra, 2008, Goetting et al., 2011; Okorie-kanu et al., 2016; Agyare et al., 2018). Despite these public health concerns, antimicrobials are essential in poultry-production. So, effort should be to find treatment-strategies that would prevent and cure AMR.

Salmonella species are the most important zoonotic bacterial food-borne pathogens (Addis et al., 2015) being the most frequently isolated bacteria in food-borne disease-outbreaks (Balakrishnan et al., 2018), accounting for around 93.8 million food-borne illnesses and 155,000 deaths per year, worldwide (Heredia and Garcia, 2018). Salmonella species have also been associated, with increasing concern for emergence and spread of antimicrobial-resistance (Ejo et al., 2015).

Antibiotic-resistant Salmonella infections of both humans and animals are universal concerns, particularly in developing countries (Ejo et al., 2015). Apart from morbidity and mortality which they cause in humans and animals, restrictions to trade and discarding contaminated food due to them are important socioeconomic problems (Tadesse and Tessema, 2014). Of zoonotic Salmonella species, S. typhimurium and S. enteritidis are more common (Dhama et al., 2013; Tadesse and Tessema, 2014; Tegegne, 2019)

Molecules of Aluminum magnesium silicate (AMS), a WHO approved medicine/stabilizing agent, consist of Nanoparticles with negative electrical charges on their surfaces and positive charges on their edges (Cristina et al., 2007, Vanderbilt, 2012). Presence of the two charges on AMS Nanoparticles makes them to hydrate to form three dimensional colloidal structures in solutions. The colloidal structures stabilize other drugs that are in formulation with AMS (Vanderbilt, 2012). For this effect, AMS is an approved pharmaceutical stabilizing agent.

Meanings for stabilize include protecting a substance from being destroyed. Drug metabolism destroys drugs and renders them no longer effective. So, stabilizing drugs reduces rate at which the body metabolizes (destroys) them so that they remain at high concentrations for extended periods. When drugs remain at high concentrations in blood for longer periods, their effectiveness improves. Also, AMS consists of Nanoparticles and Nanoparticles enhance delivery of drugs to their effect-targets which also improves efficacy.

Antioxidants (Vitamins A, C and E) reduce oxidative stress and so, protect the immune system to enhance its response to challenges from bacteria, viruses and parasites. The Vitamins are required in small dosages for the immune system to function optimally. Vitamin C supplementation, in particular, has been reported to reduce duration and severity of COVID-19.

It was therefore postulated that treatment strategy of enhancing immunity of patients with antioxidants, enhancing efficacy of antimicrobials by stabilizing them with AMS and reducing side effects of the Antimicrobials by using their lower doses for treatments could terminate treated infections so that none remains to become AMR. Infections that have already become resistant could become curable by same antimicrobials they are resisting.

Nigeria does not have natural deposits of AMS still there are two other solid minerals, Aluminum silicate and Magnesium silicate which are also approved medicines (Galindo and Cereso 2006), found in the country and in many other countries. The two medicines were used to formulate a form of AMS, named, Medicinal synthetic Aluminum magnesium silicate . To overcome the challenge of AMS, Aluminum silicate and Magnesium silicate being un-absorbable, Dextrose monohydrate was incorporated in MSAMS so that the simple sugar conveys the electrically charged Nanoparticles across mucous membranes into blood by the principle of active transport (Murray, 2000) so that antimicrobials stabilized with MSAMS would have systemic effects. A formulation of 10% MSAMS and 5% Streptomycin, supported with Vitamin C was used to treat Streptomycin-resistant Salmonella tiphymurim infected chicks.

II. MATERIALS AND METHODS

Eighty (80) broiler-chicks aged 3 weeks (age beyond which S. typhimurium may, no longer cause much mortality) which were infected with Streptomycin-resistant Salmonella typhimurium, were used for trial of the new drug-formulation and the suggested treatment-strategy. The treatment-groups (20 chicks each) were:

Treated with Streptomycin at its 100%-dosage,

Treated with Streptomycin-MSAMS at Streptomycin's 100%-dosage,

Treated with Streptomycin-MSAMS at Streptomycin's 75%-dosage + Vitamin C,

Untreated (control).

The treatment lasted 5 days. Before the treatment and on day-3 of the treatment, day-1 post treatment (PT) & day-4 PT, any first two (2) chicks caught on entering pen of each treatment-group were randomly selected, for bile which was used to determine Salmonella tiphymurim colony-forming units (CFU). Of bile from each chick, 0.1 ml was added to 0.9 ml of normal saline to get a 1:10 dilution. Again, 0.1 ml of the 1:10 bile dilution was added to 0.9 ml of normal saline to make a 1:100 bile dilution. Finally, 0.05 ml of the 1:100 bile dilution was plated on MacConkey agar and on SSA agar before incubating at 37°C for 24 hours. Salmonella colonies (X) were counted and expressed as colony-forming units per ml (CFU), using the formula: CFU = x/5 × 10,000. Infection-reduction rate for each group on each day of assessment was calculated as percentage of the mean-CFU before treatment, by which infection of that group reduced (Mean-CFU before treatment – CFU of the group on the day of assessment divided by Mean-CFU before treatment, multiplied by 100). Negative results indicate increase in load of the infection while positive results show reduction in the infection-load. The infection-reduction rates were compared by plotting them as a graph.

III. RESULTS

Mean colony forming units (CFU) in bile, of the Streptomycin-resistant Salmonella tiphymurim-infected chicks before treatment was 8.75. For, the untreated group of chicks the infection-reduction rates were: -482.86% (increase), -682.86% (increase) and -716.86% (increase) for day-3 on the treatment, day-1 PT and day-4 PT. For the group treated at recommended dosage of Streptomycin (25 mg/kg), the CFU-reduction rates for the three days were -1.83%(increase), -12.57% (increase) and -782.29% (increase) respectively but for the group treated at same recommended dosage of Streptomycin with Streptomycin-MSAMS formulation, the CFU-reduction rates were 14.29%, 26.40% and -855.42% (increase) respectively while for the group treated at 75% of recommended dosage of Streptomycin (18.75mg/kg) with the Streptomycin-MSAMS formulation plus Vitamin C, the reduction rates were 100%, 100% and 100% respectively (infection-termination). S. typhimurium CFUs per/ml of bile of the treated chicks are as presented on Table 1 while their infection reduction rates are as presented on Table 2 while their comparison is as on Figure 1.

Table 1: Colony-Forming Units ( ) of Streptomycin-Resistant Salmonella tiphymurim in Bile of Chicks Treated With Streptomycin-Medicinal Synthetic Aluminum Magnesium Silicate Formulation and Vitamin C

Table 2: Reduction-Rates (%) of Streptomycin-Resistant Salmonella tiphymurim Infections, in Chicks Treated With Streptomycin-Medicinal Synthetic Aluminum Magnesium Silicate Formulation and Vitamin C

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IV. DISCUSSION

Discovery of drugs that inhibit pathogenic microorganisms (antimicrobials) was a major revolution in medicine. So, development of resistance against the drugs is also a major setback to Global health. Current increase in drug-resistant pathogenic microorganisms is a big challenge to medical researchers, world over. Efforts to keep developing new drugs to replace those that disease-agents overcome is faced with many difficulties, including cost and time it takes to design new drugs. One strategy being tried to overcome AMR is development of adjuvants that could enhance efficacy of antimicrobials (Erin et al., 2015).

As a stabilizing agent, AMS protects drugs against rapid degradation by metabolic processes thus prolonging time of their high bioavailability. Prolonging time of high bioavailability enhances efficacy and so, dosages of the drugs needed to achieve desired effects are reduced. When lower dosages are used for treatments, immune responses of patients enhance. Synergy between enhanced efficacy and enhanced immunity could clear 95% of treated infections so that AMR is prevented (Brent et al, 2001). Even already resistant infections could be cured. Also, since AMS-units are Nanoparticles, they enhance delivery of drugs to targets and across physiological barriers, including blood-brain barrier.

Drugs have both desired effects and side effects. Most antimicrobial drugs cause immune-suppression when used at high dosages. Formulating antimicrobials with the MSAMS makes it possible for their lower dosages to achieve desired effects so that their side effects which occur with high dosages could be reduced. Reducing side effects of antimicrobials improves immune responses of patients. Improving immune responses of patients help to clear infections.

Trials of the MSAMS on efficacy of different antimicrobials, so far, have shown that it improves efficacy of the antimicrobials to help them clear enough infections so that AMR is prevented. Formulating other drugs and MSAMS has led to prevention of AMR against Ampicillin trihydrate by bacteria (Ezeibe, et al 2012 a). It also prevented AMR by Heligosomodes bakeri, (helminths) against Piperazine citrate (Ezeibe et al, 2012 b). Formulating MSAMS with Chloroquine sulphate also led to enough clearance of Plasmodium berghei such that development of AMR by the malaria parasite may no longer occur (Ezeibe 2020). That strategy of formulating MSAMS with antimicrobials and supporting their treatment with antioxidants has also restored efficacy to Ampicillin trihydrate against resistant E. coli (Ezeibe et al 2013) and to Cotrimaxazole against resistant Salmonella pulorum. (Ezeibe et al 2019) In current trial of MSAMS-Streptomycin formulation supported with Vitamin C against resistant Salmonella typhimurium infections, all that Streptomycin alone at its recommended dosage (25 mg/kg) could achieve was to slow rate at which the infection was increasing on day-3 of the treatment and on day-1 post treatment but by day-4 post treatment, the infection increased even more than that of the untreated group. That reduced effect of Streptomycin at the recommended dosage is a confirmation that the S. typhimurium infection was Streptomycin-resistant. The antimicrobial had lost efficacy against the infection and so could no longer cure its disease. Sudden rise in infection-rate in that group, 4 days after treatment, suggests that while Streptomycin had only negligible effect on the resistant infection, its side effects depressed immunity of the chicks which was the only defense left for the patients. So, when the treatment was withdrawn, there was nothing to hinder the infection.

In the group of chicks treated at same recommended dosage of the antimicrobial, stabilized with MSAMS, the treatment reduced (not just slowing multiplication of the infection) the resistant infection on both day-3 on the treatment and on day-1 PT but again following withdrawal of treatment, the infection flared up above any other group. That the infection actually reduced in this group while it did not reduce with Streptomycin alone, suggests that formulating Streptomycin with MSAMS improved its efficacy such that even the already resistant infection became sensitive. The sudden increase of the infection, higher than both the untreated group and the group treated with Streptomycin without MSAMS, suggests that MSAMS potentiated both desired effect of Streptomycin (efficacy) and its side effect (immune suppression) so that with withdrawal of the treatment, the infection (which treatment could not terminate), had the least resistance.

Reducing dosage of Streptomycin by 25% to use its 75%- dosage (18.75 mg/kg) for treatment with the Streptomycin-MSAMS formulation, terminated the resistant infection, after only 3-days on the treatment. The 100% infection load reduction achieved in that group of 75% Streptomycin-dosage in MSAMS plus Vitamin C remained both on day-1 PT and on day-4 PT, confirming that the treatment terminated the resistant infection.

Apart from terminating the resistant infection which means cure for AMR, that the treatment achieved such an effect with reduction both in dosage of the drug and in course of the treatment means reduction in cost of treatment and reduction in residues of the drug in human foods of animal origin. Such reduction in drug-residues in foods will reduce incidences of AMR in humans.

When same treatment-strategy (using 75% of recommended dosages of MSAMS-stabilized antimicrobials, supported with antioxidants) was applied to Ampicillin trihydrate (Ezeibe et al 2013) and Cotrimoxazole (Ezeibe et al 2019) against resistant infections, though they achieved enough infection-reduction that could lead to cure and prevention of AMR ( 95%), they could not terminate the infections. Some infections ( 5%) were left which immunity could clear with time (Brent et al, 2001). In this study with Streptomycin, the strategy terminated the resistant infection (100%-infection reduction). Level of resistance by isolate used in each trial and side effect of each antimicrobial being tested may be responsible for differences in rates (%) of clearance that result with that strategy of using 75% of recommended dosages of MSAMS-stabilized antimicrobials supported with antioxidants to treat resistant infections. However, that the treatment-strategy restores efficacy to antimicrobials has remained consistent.

Results of these trials of antimicrobials-MSAMS (Nano-stabilizing agent) formulations and antioxidants on both sensitive and resistant infections of different bacteria, protozoa and helminths suggest that formulating antimicrobials with Nano-stabilizing agents and supporting their treatments with antioxidants may be an effective treatment-strategy for prevention and treatment of AMR. Also, since a reduced dosage of Streptomycin, stabilized with MSAMS, achieved 100% clearance of the resistant infection after only three days, the strategy may also lead to reduction in course of treatments for Streptomycin and possibly for other antimicrobials, too.