BackgroundResistance to extended-spectrum cephalosporins among Enterobacteriaceae has been reported yet they serve as the last line treatment for severe infections in Uganda and other countries. This resistance often leads to nosocomial infection outbreaks and therapeutic failures from multidrug resistant bacteria. The main objective of this study was to determine the prevalence of extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae in clinical samples of patients in various wards of Mulago Hospital; Uganda’s main national referral and teaching hospital.MethodsThis cross-sectional study was conducted between January-April, 2014. Purposive consecutive sampling was used to collect pus swab, urine, blood and CSF samples from patients in the various wards. A total of 245 consecutive, non-repetitive, clinical samples were obtained and tested for phenotypic ESBL production using the Double Disc Synergy Test using cefotaxime, ceftazidime, cefotaxime-clavulanic acid and ceftazidime-clavulanic acid.ResultsResults show that 47 % of the 245 samples had Enterobacteriaceae isolates. Of these isolates 62 % were ESBL producers while 38 % were of non-ESBL phenotype. E. coli was the most isolated organism (53.9 %), followed by K. pneumoniae (28.7 %). Majority of Enterobacteriaceae organisms were isolated from urine samples, followed by pus samples and of these 64.9 % and 47.4 % were ESBL-producers respectively. Klebsiella pneumoniae had the highest percentage of ESBL producers (72.7 %). There was a higher percentage of isolates showing resistance to ceftazidime (73 %) compared to cefotaxime (57.5 %). All Enterobacter cloacae isolates showed resistance to ceftazidime. There were no statistically significant association between phenotype (ESBL/non-ESBL) and patients’ age or gender or Enterobacteriaceae spp.ConclusionsThis study reveals a high prevalence of ESBL producing organisms in Mulago Hospital and high levels of resistance to third generation cephalosporins. In addition to undertaking appropriate infection control measures, there is urgent need for formulation of an antibiotic policy in Uganda to prevent spread of these organisms. This also calls for continuous monitoring and reporting of the presence of such organisms in order to ensure rational and judicious use of antibiotics by clinicians.