Phenotypic methods for detection of various β-lactamases in Gram-negative clinical isolates: Need of the hour

Abstract

Background: Many clinical laboratories have problems detecting various β-lactamases. Confusion exists about the importance of these resistance mechanisms, optimal test methods, and appropriate reporting conventions. It is more imperative to use various phenotypic methods for detection of various β-lactamases in routine microbiology laboratory on day-to-day basis to prevent antimicrobial resistance by evidence-based judicious use of antimicrobials. Aims: In view of the multidrug-resistant organisms being reported world over, we planned a cross-sectional prospective analytical study to determine resistance mechanism by various β-lactamases in Gramnegative clinical isolates using various phenotypic methods. Materials and Methods: All nonrepeat, nonenteric clinical isolates of Gram-negative bacilli, resistant to at least two third-generation cephalosporins, were first screened by Novel disc placement method, and isolates showing multiple mechanisms of resistance and reduced zone of inhibition for imipenem were further confirmed for AmpC and metallo β-lactamases. Statistical Analysis: All the data was managed and analyzed in Microsoft Excel. Results: Out of 807 isolates tested, as many as 795 (98.51%) revealed the presence of extended-spectrum β-lactamases (ESBLs). Only 10 isolates of Escherichia coli and 2 of Klebsiella pneumoniae did not show production of ESBL. A total of 450 (55.76%) isolates produced single enzyme,while 345 (42.75%) strains revealed multiple enzyme production simultaneously. Only ESBL production was seen in 315 (39.03%) strains, only AmpC in 75 (9.29%) and only MBL in 60 (7.44%) strains, while ESBL and AmpC together were seen in 219 (27.14%) and AmpC plus MBL in 92 (11.40%) strains. However, ESBL plus MBL were never observed together. All three enzymes were simultaneously detected in 34 (4.21%) strains. Conclusion: This innovative method of disc placement makes it easy, affordable, and reliable method for routine use by basic microbiology laboratories for detection of various β-lactamases, pending confirmation for AmpC and metallo β-lactamase by three-dimensional test and double disc potentiation test, respectively.

Antimicrobial Resistance Patterns and Molecular Characterization of <i>Klebsiella pneumoniae</i> in Clinical Isolates at Mbarara Regional Referral Hospital

“…The genes blaKPC, blaOXA-48 and blaVIM were amplified using primers (Table 5) A multiplex PCR was developed and used for simultaneous detection of bla OXA-48 and bla KPC genes, other PCRs were performed for bla VIM . PCRs were carried out by using the HotStart DNA polymerase master mix (QIAGEN) with [30][31][32][33][34][35] cycles at an annealing temperature of 52˚C for bla OXA-48 and bla kpc, and 50˚C for bla VIM. PCR products were analyzed by agarose gel electrophoresis.…”

“…10 μl of 0.5 MEDTA was added to one of the imipenem disc to get the desired concentration of 750 μg. After incubation at 37˚C overnight, increase of inhibition zone diameter of more ≥ 5 mm in the disc potentiated with the EDTA was interpreted as positive for metallo-β-lactamase production as described elsewhere (Figure 6)[30].…”