Restoring Efficacy to Cotrimoxazole, against Resistant Salmonella pullorum,  with Medicinal Synthetic Aluminum-Magnesium Silicate ® [MSAMS: Al4 (SiO4)3 + 3Mg2SiO4 → 2Al2 Mg3 (SiO4)3]

To restore efficacy (≥95% infection-reduction) to drugs against resistant infections (now, posing a bigger medical challenge, globally, than even HIV/AIDS), Cotrimoxazole was formulated with a synthetic Aluminum-magnesium silicate (Medicinal mineral which molecules are made of Nanoparticles, that is already in use as pharmaceutical-stabilizing agent) and used with antioxidants to treat Cotrimoxazole-resistant Salmonella pulorum infected chicks. Chick-groups A, B and C were fed antioxidants-fortified feed while groups D, E, F, G and H were not. The treatment-groups and their Cotrimoxazole doses/formulations were: A and D (100%/MSAMS-Cotrimoxazole); B and E (75%/MSAMS-Cotrimoxazole); C and F (50%/MSAMS-Cotrimoxazole) and G (100%/Cotrimoxazole). Infection-reductions (96.23% and 94.98%) of the groups of 75%/MSAMS-Cotrimoxazole/antioxidants and 75%/MSAMS-Cotrimoxazole were significantly (P ≤ 0.05) better than 76.99% (100%/Cotrimoxazole); 10.04% (50%/MSAMS-Cotrimoxazole/antioxidants); 1.60% (50%/MSAMS-Cotrimoxazole); −212.60% (100%/MSAMS-Cotrimoxazole/antioxidants) and −230.96% (100%/MSAMS-Cotrimoxazole).


Introduction
Drug resistance has become a major medical challenge, not only in poultry but in other livestock and even in human medicine. Salmonellae and other microorganisms exhibit resistance to many antimicrobial agents like tetracycline, ciprofloxacin, chloramphenicol, penicillin G, amoxicillin, trimethoprin, sulphamethaxazole, gentamycin [1] [2] [3]. Penicillin, ampicilin, chloramphenicol, tetracycline and nitrofurantoin have been reported to be completely resisted by Salmonella pullorum while gentamycin, cotrimoxazole and nalixidic acid have been moderately resisted [3]. Fluoroquinolone is said to be less resisted [4]. Development of antimicrobial resistance by avian adapted Salmonella strains is attributed to chromosomal mutation or genetic recombination [2]. Resistance may also result from the irrational or wrong use of antimicrobials. Bacteria may also acquire resistance by transformation, conjugation or transduction [5].
Aluminum-magnesium silicate (AMS) has been in use, both as a drug [6] [7] and as a pharmaceutical raw material for drug formulations. It is used as a stabilizing agent, carrier, adsorbent, viscosity-enhancing agent, anti-caking agent, tablet binder, tablet and capsule des-integrant because of its indefinite stable property, ability to be used both in acidic and alkaline P H [6] [8] [9]. As a stabilizing agent, it prolongs time of high bioavailability of drugs by protecting such drugs against rapid degradation (metabolism). Also, molecules of AMS are made of particles that are only 0.96 nm thick (Nanopaticles) [10] and Nanoparticles enhance delivery of drugs to targets. Prolonging time of high bioavailability and enhancing delivery to targets improve efficacy of drugs.
When ampicilin trihydrate, chloroquin phosphate, piperazine citrate and sulphadimidine were stabilized with a synthetic AMS (Medicinal synthetic Aluminum-magnesium silicate: MSAMS), 75% of their doses terminated the infections by achieving ≥ 95% clearance of each [6] [11]- [16]. Stabilizing drugs with MSAMS and using them and antioxidants (vitamins A, C, E and/or selenium) to treat infected animals also made them regain effects against resistant infections [15]. This study is a test of effects of the MSAMS on Cotrimoxazole against resistant Salmonella infections.

Materials and Methods
Forty eight day-old cockerels were randomly assigned to eight groups (A, B, C, D, E, F, G and H) of six (6) each. The chicks were screened by the method of Roch-Silva et al. [17] to ensure they were free of Salmonella species. Each chick was infected with 1.2 × 10 5 CFU of a Cotrimoxazole-resistant Salmonella pullorum isolate. From six days post infection, they were treated for 7 days as follows: Groups A, B and C were fed with feed fortified with additional levels of Vitamin During the experiment, clinical signs were recorded daily. Individual cloacal swabs were collected from all the chicks on Day 4, post infection (PI) and processed as described by Wigley et al. [18], with some modifications as follows: Individual samples were inoculated into buffered peptone water, incubated at 37˚C for 24 hours and then sub cultured on Salmonella-Shigella agar, brilliant green agar and MacConkey agar respectively before incubating at 37˚C for 24 hours. The cultures were then examined to confirm growth of Salmonella pullorum as a confirmation of establishment of infection in the chicks. Temperature and weight of each chick were taken daily.
Two chicks from each group were euthenased on Days 1, 3 and 5, post treatment and their gall bladders were aseptically removed into sterile sample bottles. To 0.1 ml of bile, 0.9 ml of sterile normal saline was added to get 1:10 dilution of the bile. Again, 0.1 ml of the 1:10 bile dilution was diluted with 0.9 ml of sterile normal saline to get 1:100 dilution. Finally, 0.05 ml of the 1:100 diluted bile was plated on nutrient agar and incubated at 37˚C for 24 hours. Salmonella pullorum colonies (x) were counted (using hand lens), and calculated as colony forming units per ml of bile by the formula: CFU ml 10, 000 5 CFU per ml of bile for the 8 groups of chicks were compared for statistical differences by Analysis of variance (ANOVA).  This suggestion is supported by reports of Mubarak et al. [20], Sanda et al. [21] and Ibrahim and El-Sayed [22] who also observed that vitamins A, C and E (antioxidants) stimulate the immune system of animals. The results also agree with the report of Rajput et al. [23], that supplementary vitamins A, C and E in feed ensure better efficacy for treatments. Clearance of ≥95% of infections means termination for infections as immunity is able to complete elimination of ≤5% infection-load, left after treatments [24].  [15].

Discussion
When the dose of Cotrimoxazole was reduced to 75% and it was stabilized in MSAMS (with antioxidants in feed) best reduction of CFU/ml, best reduction in body temperature and best weight-gain were achieved. The reduction in dose may have reduced the side effects while prolongation of high bioavailability and enhanced delivery to effect-targets improved the desired effects such that the overall result became termination of even the resistant infection (≥95% infection-reduction).

Conclusion
By clearing ≥95% of Cotrimoxazole-resistant Salmonella pullorum infection, the 75% dose of Cotrimoxazole stabilized in the MSAMS, terminated the resistant infection and cured salmonellosis (reduced temperature and improved weight gain).