Vol. 64, No 4/2017 609–613 https://doi.org/10.18388/abp.2016_1495 Regular paper Effects and time-kill assessment of amoxicillin used in combination with chloramphenicol against bacteria of clinical importance Olufunmiso O. Olajuyigbe1,2*, Roger M. Coopoosamy1 and Anthony J. Afolayan2 1Department of Nature Conservation, Mangosuthu University of Technology, Durban, KwaZulu-Natal, South Africa; 2Medicinal Plants and Eco- nomic Development Research Centre, University of Fort Hare, Alice, 5700, South Africa With the emergence of multidrug-resistant organisms in INTRODUCTION an era when drug development faces challenges caus- ing pharmaceutical companies to curtail or abandon In the late twentieth century the availability and suc- research on anti-infective agents, the use of combined cess of antibiotics and vaccinations resulted in a confi- existing antimicrobial agents may be an alternative. dence that technology and modern medicine would be This study evaluated the effects of combining amoxicil- victorious against infectious diseases. During this early lin and chloramphenicol, to which many bacteria have period of the antibiotic usage bacterial infections were become resistant, in vitro against Gram positive and considered tamed as potentially lethal infections were be- Gram negative bacteria by agar diffusion, checkerboard ing cured with antibiotics. However, while the introduc- and time-kill assays. The test isolates were susceptible tion of antimicrobial agents was accompanied by nega- to amoxicillin with minimum inhibitory concentrations tive rather than positive impact on the patients, the num- (MICs) ranging between 0.448 and 500 µg/ml and be- ber of individuals to be treated with antibiotics increased tween 1.953 and 31.25 µg/ml for chloramphenicol. Upon with enhanced pathogenicity and invasiveness. The wide- combining these agents, there was a drastic reduction spread use of antibiotic, therefore, resulted in the emer- in their MICs indicating an increased antibacterial activ- gence of outbreaks and epidemics of antibiotic-resistant ity that showed synergistic interaction against all the pathogens including multidrug resistant strains (Normak bacteria. At the highest concentrations, the inhibition & Normak, 2002). Today, resistant pathogens, an under- zones ranges were 20.33–38.33±0.58 µg/ml for amoxi- appreciated threat to public health throughout the globe cillin, 27.67–37.67±0.58 µg/ml for chloramphenicol and (Zhang et al., 2006), are rapidly growing problems lead- 31.67–39.33±0.58 µg/ml for the combined agents. The ing to an urgent need for novel antimicrobial agents fractional inhibitory concentration indices (FICIs) showed (Kumar & Schweizer, 2005; Edgar et al., 2012). synergy ranging from 0.129 to 0.312 while FICIs for ad- Although there is a continued effort into seeking new ditive interaction were between 0.688 and 1.0. There therapies in response to the consequences of the pres- 1 was no antagonistic interaction. At the /2MICs of the sure on the widespread use of antibiotics or problems combined antibiotics, all the tested bacteria, except for associated with increasing drug resistance (Cameron et Klebsiella pneumoniae ATCC 4352, Proteus vulgaris CSIR al., 2004), bacteria have, also, continued to develop dif- 0030 and Enterococcus cloacae ATCC 13047 were elimi- ferent resistance mechanisms to virtually all antibiotics in nated before 24 h. At the MICs, all the tested bacteria general clinical practices (Clatworthy et al., 2007). These were eliminated except Enterococcus cloacae ATCC 13047 resistance mechanisms may include altered penicillin- which was almost totally eliminated. Post-antibiotic as- binding proteins, presence of various β-lactamases and sessment after 48 h showed that all the cultures were loss of porins (Bou & Martïnez-Beltran, 2000). While sterile except for that of Enterococcus cloacae ATCC active efflux and enzymatic inactivation are the mecha- 13047. The lack of antagonism between these antibacte- nisms responsible for resistance to aminoglycosides rial agents in checkerboard and time-kill assays suggest- (Smith et al., 2007), the most common mechanisms of ed that combining amoxicillin with chloramphenicol can resistance to chloramphenicol are decreased outer mem- provide an improved therapy in comparison to the use brane permeability (Burns et al., 1989), enzymatic inac- of each antibiotic individually. The study indicates the tivation by acetylation essentially by acetyltransferase or potential beneficial value of combining amoxicillin and by chloramphenicol phosphotransferases (Schwartz et al., chloramphenicol in the treatment of microbial infections 2004; Aakra et al., 2010), target site modulation (Montero in clinical settings. et al., 2007) and presence of efflux pump (Daniels & Ra- mos, 2009). To overcome various resistance mechanisms Keywords: drug-drug interactions; fractional inhibitory concentra- and dissemination of antibiotic resistance genes, explor- tions; multidrug resistance; time-kill assessment ing the possible synergy between conventional antibiot- Received: 31 December, 2016; revised: 13 February, 2017; accepted: ics becomes necessary. This study, therefore, aimed at 11 August, 2017; available on-line: 30 November, 2017 investigating the combinatory effects and time-kill assess- *e-mail: [email protected] ment of amoxicillin and chloramphenicol against bacteria Abbreviations: FICIs, fractional inhibitory concentration indices; of clinical importance. These antibiotics have long been MDR, multidrug resistance; MICs, minimum inhibitory concentra- used for the treatment of enteric fever. However, most tions enteric organisms including Salmonella typhi causing ty- phoid fever have become highly resistant to them (Ka- bra, 2000; Das & Bhattacharya, 2000). 610 O. O. Olajuyigbe and coworkers 2017 1 MATERIALS AND METHODS Hinton broth at /2MIC and MIC respectively. Controls consisting of Mueller Hinton broth with the respective Bacterial culture and preparation of antibiotic so- antibiotic added alone at the test concentrations were in- lutions. The bacteria used in this study included Shigella cluded in each experiment. The experimental and control sonnei ATCC 29930, Salmonella typhi ATCC 13311, Staphy- flasks were inoculated with each test organism to a final 9 lococcus aureus OK2a, Acinetobacter calcoaceuticus UP, Ente- inoculum density of approximately 10 cfu/ml. Immedi- rococcus cloacae ATCC 13047, Proteus vulgaris CSIR 0030, ately after inoculation, aliquots (100 μl) of the negative Pseudomonas aeruginosa ATCC 19582, Bacillus cereus ATCC control flasks were taken, serially diluted in sterile dis- 10702, Klebsiella pneumoniae ATCC 4352 and Staphylococcus tilled water and plated on nutrient agar in order to deter- aureus ATCC 6538. Antibiotic powders of Amoxicillin mine the zero h counts. The test flasks were incubated (Duchefa) and Chloramphenicol (Duchefa) were used. at 37°C with shaking on an orbital shaker at 120 rpm. Stock antibiotic solutions were prepared and dilutions A 100 µl aliquot was removed from the culture medium made according to the manufacturer’s recommendations. at 0, 24 and 48 h for the determination of cfu/ml. The Antibiotic susceptibility testing-Agar diffusion problem of antibiotics carryover was addressed by dilu- method. Each bacterial strain’s colony suspension was tion. After incubation, emergent bacterial colonies were matched with 0.5 McFarland standards to give a resultant enumerated, the mean count (cfu/ml) for each test and 7 concentration of 1.5×10 cfu/ml. The antibiotic suscep- controls was calculated and expressed as log10. The in- tibility was determined by swabbing the Mueller-Hinton teractions were considered synergistic if there was a de- agar (MHA) (Oxoids UK) plates with the adjusted bacte- crease of >2 log10 cfu/ml in colony counts after 24 h rial strains. Agar wells were made with heat sterilized 6 for the antibiotics combination compared to the activity mm cork borer before being filled with 100 µl of differ- of each antibiotic used alone. Additivity or indifference ent solutions (62.5 µg/ml, 125 µg/ml and 250 µg/ml) of was described as a <2 log10 cfu/ml change in the av- each of the antibiotics. These concentration ranges were erage viable counts after 24 h for the combination, in chosen to cover the maximum serum therapeutic range comparison to the activity of each antibiotic used alone. that could be reached in vivo when 100 µl of each anti- Antagonism was defined as a >2 log10 cfu/ml increase in biotic was dispensed. Different solutions containing the colony counts after 24 h for the combination compared same concentrations of amoxicillin and chloramphenicol to the activity of each antibiotic used alone (Pankey et were prepared and used to determine the effect of com- al., 2005). bining the antibiotics with care taken to prevent spillage Statistical analysis. All the data were subjected to of the solutions onto the agar surface. The plates, in one way analysis of variance (ANOVA) and the mean triplicate, were allowed to stand for 1 h before being in- values were separated at p<0.05 using Duncan’s Multiple cubated at 37°C for 24 h. After incubation, the diameter Range Test. The one way ANOVA test was used to de- of the inhibition zones produced by each antibiotic alone termine if there was any statistically significant difference and their combinations were measured with a transpar- in the size of inhibition zones for each bacterial isolate ent ruler. Synergism was stated when inhibition zones of exposed to each antibiotic