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Synergistic Effect of Third Generation Cephalosporin and Ciprofloxacin Towards Multi-drug Resistant (mdr) Salmonella Typhimurium Isolated from Undercooked Beef

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ABSTRACT

Antibiotic resistance is a major issue in Salmonella enterica serovar Typhi resulting in innumerable cases of typhoid disease (enteric fever) causing severe infection in humans and animals. The adaptive ability of bacterial pathogens to acquire resistance to conventional antimicrobials compounds are of universal concerns and remains as significant public threat in past and present era, Therefore, there is an ongoing need to develop more advanced techniques and methodologies for reprisal of this ever increasing threat. The primary goal of the project is the augmentation of two novel, third generation antimicrobials, cephalosporin and ciprofloxacin, to counter the MDR S. typhi which have acquired resistance towards individual impacts of multiple drugs over a wide range and to elucidate the mechanism which plays an essential role towards inhibition and elimination of MDR S. typhi isolates from undercooked beef which will provide future insight towards antimicrobial synergism.

Key word: Drug resistance, Salmonella typhi, synergism, enteric fever, disease, undercooked beef.

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INTRODUCTION

Multidrug-resistant bacterial infections are an ever-growing threat because of the shrinking arsenal of efficacious antibiotics (Courtney et al. ,2016). New resistance mechanisms are constantly being described, and new genes and vectors of transmission are identified on a regular basis (Blair et al. ,2015). Salmonella typhi, the causative agent of typhoid fever, a gram-negative, motile, rod-shaped, facultative anaerobe is a particular Salmonella serovar serving as a major public health problem in developing countries (Qamar et al. ,2014). Salmonella is considered as one of the most notorious and prevalent food borne pathogen. The serotypes serving as causative agents for typhoid include Typhi, Paratyphi A, Paratyphi B, Paratyphi C. Consumption of MDR Salmonella isolates along with undercooked meat dishes is directly relevant to the global public health crisis of antimicrobial resistance. The factors which majorly contribute to the cause for such emergence of food borne outbreak are uncleaned utensils, contaminated and raw food items, inappropriate food storage, poor personal hygiene during food handling, improper preparation, inadequate cooling and reheating of food items and time lapse between food preparation and its consumption.

The contributing factors for prevalence of MDR strains in developing nations include misuse of antibiotics, drug prescription without susceptibility testing, self-medication, incomplete drug course and long term hospitalization. Antimicrobial-resistant Salmonella species have been isolated from different foods of animal origin around the world, which is attributed to inappropriate use of antimicrobials and therapeutic or prophylactic agents in human and veterinary medicine, as well as the use of growth promoters in animal production (WHO,2012). Typhoid remains a major cause of morbidity and mortality especially in countries which are overpopulated and have poor sanitation system and is endemic in most parts of Central America, Southeast Asia and Indian subcontinent also increasing number of cases have been reported in Africa. S. typhi causes up to 22 million cases of typhoid fever a year. Acute gastroenteritis is one of the leading causes of illness and death in infants, children, immuno-compromised and aged individuals throughout the world (Mengistu et al. ,2014). Early symptoms include high fever, headaches and stomach pain. Left untreated, typhoid fever can lead to intestinal hemorrhage and perforation of the bowel, and kill up to 15% of infected people. Despite the availability of antibiotics, about 200,000 people die annually. The emergence of multidrug resistance to the commonly used antibiotics has further complicated the treatment and management of enteric fever and this is recognized as one of the greatest challenges in the management of this disease (Sehra et al. ,2013). Early treatment failures with antibiotics was not considered as significant clinical problem because other classes of agents, with different cellular targets were available.

The major problem in the clinic today is the emergence of multiple-drug resistance, i. e. resistance to several types of antimicrobial agent (Amenu,2014). S. typhi which has developed resistance to first line antibiotics for typhoid including ampicillin, chloramphenicol, trimeth-methoprim sulfamethoxazole with additional resistance to streptomycin, sulfamethoxazole and tetracycline are considered as multi-drug resistant (MDR), Such isolates were first observed in late 1970s to early 1980s. Ceftriaxone, a third generation cephalosporin is used to treat typhoid fever when other options are ineffective, However, sporadic cases of ceftriaxone resistant S. typhi have been recently reported. Usually, microorganisms such as S. typhi can quickly acquire new resistance genes within and even between different species of bacteria and more importantly, S. typhi can transform from MDR to XDR (extremely drug resistant) strains by acquiring a plasmid, horizontal acquisition of resistant genes, mobilization via insertion sequences, transposons and conjugative plasmids, recombination of foreign DNA into the chromosome, or by mutations in different chromosomal loci. Hence, despite the emergence of newer antimicrobial drugs, enteric fever has continued to be a major health problem (Zaki and Karande,2011). The origin of antibiotic resistance has been around for as long as antibiotics have been used to treat infections, it extends much further back in evolutionary terms and reflects the attack and counter attack of complex microbial flora in order to establish ecological niches and survive (Denyer et al. ,2011).

OBJECTIVES

The objective of this study is twofold: 1- To determine the synergistic effect of third generation antibiotics, Cephalosporin and Ciprofloxacin towards multi-drug resistant (MDR) Salmonella typhimurium. 2- To understand the mechanism opted by the augmented antimicrobials for inhibition and elimination of multi-drug resistant S. typhi.

HYPOTHESIS

It is hypothesized that low doses of the augmented, third generation antimicrobials will be super effective towards the multi-drug resistant (MDR) S. typhi strains and will result in successful elimination of the resistant pathogen and disease.

MATERIALS AND METHODS

A total of 120 samples can be collected randomly from local food vendors and restaurants aseptically placed in sterile bags and delivered to the research facility in ice box. The beef samples will then be mixed with 10ml of peptone (0. 1%) water and homogenized suspension can be prepared using pestle and mortar. Homogenized meat suspension can then be inoculated into nutrient broths, nutrient agar and EMB agar and can be incubated at 37C for 24 hours, culture positive samples can then be sub cultured. The objectives can then be answered by performing multiple study techniques including Kirby-Beur disk diffusion technique for antibiogram study, Pulse field gel electrophoresis (PFGE) and polymerase chain reaction (PCR).

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