Twenty-eight (28) strains of ceftazidime-resistant Klebsiella pneumoniae were isolated from blood cultures of in-patients from University Hospital, Kuala Lumpur between March 1995 and August 1996. Three methods were used to detect the production of ESBL enzymes by these strains. These three methods include the double-disc synergy test (DDST), inhibitor-potentiated disc-diffusion test (IPDD) and the E-test ESBL method. All strains could be identified as ESBL producers using the DDST method by a minimum of two beta-lactams and these included either a combination of ceftazidime and ceftriaxone with clavulanate respectively or cefotaxime and aztreonam with clavulanate respectively. Similarly using a combination of either cefotaxime and ceftriaxone with clavulanate or ceftriaxone and aztreonam with clavulanate respectively would have detected all strains as ESBL producers. The IPDD method could also detect for ESBL activity based on combinations of beta-lactam antibiotics with clavulanate respectively. All combinations of beta-lactam antibiotics could detect for ESBL activity in all the strains except a combination of either ceftazidime and aztreonam or cefotaxime and ceftriaxone with clavulanate respectively. The E-Test method using ceftazidime alone and in combination with clavulanate was found to be the most effective method in the presumptive identification of ESBL activity in all the strains.
Klebsiella pneumoniae are opportunistic bacteria found in the gut. In recent years they have been associated with nosocomial infections. The increased incidence of multiple drug-resistant K. pneumoniae makes it necessary to find new alternatives to treat the disease. In this study, phage UPM2146 was isolated from a polluted lake which can lyse its host K. pneumoniae ATCC BAA-2146. Observation from TEM shows that UPM2146 belongs to Caudoviriales (Order) based on morphological appearance. Whole genome analysis of UPM2146 showed that its genome comprises 160,795 bp encoding for 214 putative open reading frames (ORFs). Phylogenetic analysis revealed that the phage belongs to Ackermannviridae (Family) under the Caudoviriales. UPM2146 produces clear plaques with high titers of 1010 PFU/ml. The phage has an adsorption period of 4 min, latent period of 20 min, rise period of 5 min, and releases approximately 20 PFU/ bacteria at Multiplicity of Infection (MOI) of 0.001. UPM2146 has a narrow host-range and can lyse 5 out of 22 K. pneumoniae isolates (22.72%) based on spot test and efficiency of plating (EOP). The zebrafish larvae model was used to test the efficacy of UPM2146 in lysing its host. Based on colony forming unit counts, UPM2146 was able to completely lyse its host at 10 hours onwards. Moreover, we show that the phage is safe to be used in the treatment against K. pneumoniae infections in the zebrafish model.
Antimicrobial resistance is a major threat to human health, hence there is an urgent need to discover antibacterial molecule(s). Previously, we hypothesized that microbial gut flora of animals are a potential source of antibacterial molecules. Among various animals, Cuora amboinensis (turtle) represents an important reptile species living in diverse ecological environments and feed on organic waste and terrestrial organisms and have been used in folk medicine. The purpose of this study was to mine turtle's gut bacteria for potential antibacterial molecule(s). Several bacteria were isolated from the turtle gut and their conditioned media were prepared. Conditioned media showed potent antibacterial activity against several Gram-positive (Bacillus cereus, Streptococcus pyogenes and methicillin-resistant Staphylococcus aureus) and Gram-negative (neuropathogenic Escherichia coli K1, Serratia marcescens, Pseudomonas aeruginosa, Salmonella enterica and Klebsiella pneumoniae) pathogenic bacteria. Conditioned media-mediated bactericidal activity was heat-resistant when treated at 95°C for 10 min. By measuring Lactate dehydrogenase release, the results showed that conditioned media had no effect on human cell viability. Tandem Mass Spectrometric analysis revealed the presence of various secondary metabolites, i.e., a series of known as well as novel N-acyl-homoserine lactones, several homologues of 4-hydroxy-2-alkylquinolines, and rhamnolipids, which are the signature metabolites of Pseudomonas species. These findings are significant and provide the basis for rational development of therapeutic interventions against bacterial infections.