Multiple drug resistance (MDR) in bacteria causes higher mortality and morbidity, complicates treatment and increases healthcare outlay. With no new-generation antibiotics in sight, its rapid spread through the environment poses grave danger. Therefore, rapid detection to identify effective antibiotics and to prevent their indiscriminate use is imperative. However, the widely used clinical method for antibiogram, the Kirby-Bauer disc-diffusion method (DDM), requires 2-3 days, has inherent shortcomings of solid-phase assays and is not suitable for high-throughput operations. In our research on MDR associated with childhood diarrhea, we determined the antibiogram of 73 clinical diarrheagenic Escherichia coli strains using both the DDM and the more reliable liquid turbidity method (LTM) performed in 96-microwell plates. The results were further correlated with a dye-exclusion efflux assay using fluorescein diacetate. Although LTM is apparently superior in saving critical time, suitability to highthroughput operations and reliability, we found that the serious shortcomings of DDM could be effectively countered by just doubling the dosage of antibiotics currently used in discs or by using two discs in place of one. With 48 of the 49 MDR strains being positive for efflux and the 12 strains 'susceptible' to all the antibiotics being negative, the efflux assay could be useful as an integral component of the antibiogram test or for additional confirmation. The presence of 65% of MDR strains among diarrheagenic E. coli is a matter of serious concern, although most of them could be treated with either Gentamycin or Amikacin, as is the practice by experience.

The presence of microorganisms in biological fluids like urine and blood is an indication of vulnerability to infections. Iron is one of the important micronutrients required for bacterial growth. In an iron-deficit environment, bacteria release high-affinity iron-chelating compounds called siderophores which can be used as non-invasive target molecules for the detection of such pathogens. However, only limited reagents and procedures are available to detect the presence of these organic molecules. The present study aims at detecting the presence of siderophores in the iron-depleted media, exploiting the reversible quenching of Calcein Blue and iron(III) complex. The fluorescence of Calcein Blue is known to be quenched in the presence of iron(III); if a stronger chelator removes this ion from the fluorophore, the fluorescence of the fluorophore is regained. This behaviour of the fluorophore was exploited to detect and quantify siderophores down to 50 and 800 nM equivalent of standard siderophore, deferroxamine mesylate (desferal) in Dulbecco's PBS and siderophore quantification (SPQ) medium, respectively. The siderophores released by pathogens, equivalent to standard desferal, were in the range of 1.29 to 5.00 μM and those for non-pathogens were below 1.19 μM. The simple, sensitive and cost-effective method performed in a 96-well plate was able to detect and quantify iron chelators within 7-8 h of incubation.

The objective of this study is to develop an antibiogram (AB) method superior to a disc diffusion method (DDM) with respect to rapidity, reliability, and accuracy especially in view of an increasing threat from multidrug resistance (MDR) of infectious bacteria. A high-throughput liquid-phase fluorescent antibiogram method capable of providing results within 6-8 hours has been developed. The AB method has been optimally designed for ease of operation, growth, and dye stability. This new method was more reliable than DDM in differentiating AB sensitivity as susceptible, intermediate, and resistant within 6-8 hours and providing evidence for efflux mechanism in the MDR phenotype. The superiority of this method even over the standard liquid turbidity method was evidenced by more accurate determination of intermediary resistance in a set of 23 clinical Escherichia coli strains against five common antibiotics. In view of the demand for the right choice of an antibiotic in short time, the newly developed AB method is clinically applicable and useful in the rational use of antibiotic and minimizing of MDR emergence.