Review of phenotypic assays for detection of extended-spectrum β-lactamases and carbapenemases: a microbiology laboratory bench guide

Abstract

Background: Infections caused by gram-negative antibiotic-resistant bacteria continue to increase. Despite recommendations by the Clinical Laboratory Standards Institute (CLSI) and the European Committee on Antimicrobial Susceptibility Testing (EUCAST) with regards to detection of antibiotic degrading enzymes secreted by these bacteria, the true prevalence of extended-spectrum β-lactamase (ESBL) and carbapenemase producers remains a difficult task to resolve. Describing of previously designed phenotypic detection assays for ESBLs and carbapenemases in a single document avails a summary that allows for multiple testing which increases the sensitivity and specificity of detection.

Methods and aims: This review, therefore, defined and classified ESBLs and carbapenemases, and also briefly described how the several previously designed phenotypic detection assays for the same should be performed.

Conclusion: Extended-spectrum β-lactamase and carbapenemase detection assays, once performed correctly, can precisely discriminate between bacteria producing these enzymes and those with other mechanisms of resistance to β-lactam antibiotics.

Keywords: Extended-spectrum β-lactamases; carbapenemases; phenotypic detection.

Fig. 1

DDS. Photograph showing extension and broadening of inhibitory zones of the third generation cephalosporins towards the amoxicillin-clavulanic acid disc. Reprinted from the European Journal of Clinical Microbiology and Infectious Diseases (vol. 8, Issue 6, pp 527–529), by Legrand, P., et al., 1989. Copyright 1989 by the European Journal of Clinical Microbiology and Infectious Diseases.

Fig. 2

MDDS. The organism shows an enhanced zone of inhibition between CAZ, CTX, cefepime, aztreonam, and amoxicillin-clavulanic acid (centre disc), indicating ESBL production. Reprinted from the British Journal of Biomedical Science (vol. 63.2 (2006): 51–54), by Kader, A. A., et al., 2006. Copyright 2006 by the British Journal of Biomedical Science.

Fig. 3

MDDS. The organism shows resistance to CTX, CAZ, aztreonam and an enhanced zone of inhibition between cefepime (top disc) and amoxicillin-clavulanic acid (centre disc), indicating ESBL production and presumptive AmpC β-lactamase production. Reprinted from the British Journal of Biomedical Science (vol. 63.2 (2006): 51–54), by Kader, A. A., et al., 2006. Copyright 2006 by the British Journal of Biomedical Science.

Fig. 4

The circular three-dimensional inoculation (indicated by the arrow) intersected the inhibition zone margins to produce major distortions (i.e., positive three-dimensional test results) that indicated enzymatic inactivation of piperacillin (A), cefamandole (D), cefoperazone (E), CTX (F), and ceftriaxone (G). Distortions did not occur (i.e., negative three-dimensional test results) in tests with aztreonam (B), imipenem (C), CAZ (H), or cefoxitin (I). The outer circle is the plastic rim on the bottom of the petri dish.

Fig. 5

The three-dimensional inoculation (arrow) resulted in minor distortions that indicated antibiotic inactivation (i.e., positive three-dimensional test results) at the intersection with the zone margins of cefamandole (D), cefoperazone (E), CTX (F), and ceftriaxone (G). Distortions did not occur in tests with aztreonam (B), imipenem (C), or cefoxitin (I). The inhibition zones were too small to interpret in the direct three-dimensional test in tests with piperacillin (A) and CAZ (H).

Fig. 6

Major zone distortions that indicated antibiotic inactivation (i.e., positive three dimensional test results) occurred in tests with piperacillin (A), cefuroxime (B), cefamandole (D), cefoperazone (E), CTX (F), ceftriaxone (G), and cefoxitin (I) but not in tests with aztreonam (C) or CAZ (H). Note the growth of small colonies in the cefoperazone (E) and CTX (F) zones in the vicinity of the three-dimensional inoculation site. The small colonies in this part of the zone also indicated enzymatic drug inactivation (i.e., positive three-dimensional test results). Reprinted from the Journal of Antimicrobial Agents and Chemotherapy (vol. 36, (9), pp.1877–1882), by Thomson, K.S. and Sanders, C.C., 1992. Copyright 1992 by the Journal of Antimicrobial Agents and Chemotherapy.

Fig. 7

Flow chart for detection of AmpC beta-lactamase production in Enterobacteriaceae. Superscript letters: a, this category includes Enterobacteriaceae spp. with no known chromosomal AmpC production plus E. coli; b, differences of zone diameters indicated as “inconclusive” means there were no visible inhibition zones around both cefoxitin discs with or without cloxacillin (pampC, plasmidic AmpC beta-lactamase); c, this category refers to mutations in the AmpC promoter region of E. coli that result in the overexpression of AmpC. Adapted from the Journal of Clinical Microbiology (vol. 49.8 (2011): 2798–2803), by Polsfuss, Silke, et al., 2011. Copyright 2011 by the Journal of Clinical Microbiology.

Fig. 8

Organisms showing clear distortion in the zone of inhibition: (A) enhanced growth of the surface organism, E. coli ATCC 25922 near agar slits (arrows) containing extracts of AmpC producing E. coli (M563) and K. pneumoniae (M484) test isolates. The remaining slit contained a non-AmpC-producing E. coli isolate (M1601). Extract of AmpC-producing E. coli isolate M477 inhibited the growth of one surface organism, E. coli ATCC 25922 (B) (arrow), but did not inhibit the growth of the second surface organism, E. coli ATCC 11775 (C) (arrow). (D) Swarming growth (dark arrow) of unlysed cells in an extract of AmpCproducing P. mirabilis isolate M910 interfered with detection of growth of surface organism (white arrow) when Mueller-Hinton agar was used. (E) On MacConkey agar, growth of P. mirabilis was inhibited, and enhanced growth of the surface organism was easily seen (arrow). Reprinted from the Journal of Clinical Microbiology (vol. 38(5), pp.1791–1796), by Coudron, P.E., Moland, E.S. and Thomson, K.S., 2000. Copyright 2000 by the Journal of Clinical Microbiology.

Fig. 9

Positive AmpC test shown by indentation of the zone of inhibition around cephoxitin disc. Reprinted from the Journal of Clinical and Diagnostic Research (vol. 7, 2 (2013): 229–233), by Kaur, Jaspal et al., 2013. Copyright 2013 by the Journal of Clinical and Diagnostic Research.

Fig. 10

MHT. (1) K. pneumoniae ATCC BAA 1705, positive result (2) K. pneumoniae ATCC BAA 1706, negative result; and (3) a clinical isolate, positive result. Reprinted from the Iranian Journal of Microbiology (vol. 3, 4 (2011): 189–193), by Amjad, A et al., 2011. Copyright 2011 by the Iranian Journal of Microbiology.

Fig. 11

The phenotypic appearance of an MBL-producing gram-negative bacteria. (A) Combineddisk test, using two imipenem (10 g) disks, one with EDTA, showing an increase in zone inhibition of 4 mm around the disk with EDTA. (B) Double-disk synergy test, using an IPM (10 g) disk placed 20 mm (center to center) from a blank filter disk containing EDTA. (C) Aztreonam (30 g) disk with a 30-mm zone of inhibition. Reprinted from the Journal of Clinical Microbiology (vol. 44(9), pp.3139–3144), by Franklin, C., Liolios, L. and Peleg, A.Y., 2006. Copyright 2011 by the Journal of Clinical Microbiology.

Fig. 12

Inhibitory effects of 2-mercaptopropionic acid (2-MPA) on IMP-1 producers and non-IMP-1 producers. Three CAZ-resistant strains belonging to the gram-negative bacterial species P. aeruginosa, S. marcescens, and K. pneumoniae and producing IMP-1 metallo-β-lactamase or serine-β-lactamases (SHV-12 or AmpC) were tested. For each IMP-1 producer, a distinct growth inhibitory zone appeared between the KB disk containing CAZ and the filter disk containing 2-MPA (left column). No change is evident around the two KB disks containing CAZ with or without 2-MPA for each serine b-lactamase producer (right column).

Fig. 13

Appearance of growth-inhibitory zone in IMP-1-producing strains by use of CAZ and 2-mercaptopropionic acid (2-MPA). Various levels of growth inhibition were observed in the IMP-1-producing gram-negative bacterial species tested. Marked growth inhibitions were observed in Acinetobacter sp., Alcaligenes xylosoxidans, Enterobacter aerogenes, E. coli, Proteus vulgaris, and Pseudomonas putida, whereas weak and ambiguous growth inhibitions were observed in C. freundii and E. cloacae. Reprinted from the Journal of Microbiology (vol. 38.1 (2000): 40–43), by Arakawa, Yoshichika, et al., 2000. Copyright 2000 by the Journal of Microbiology.

Fig. 14

Representative results of the CLSI ESBL confirmatory test (A and C) and its modification using antibiotic disks containing BA (B and D) for ESBL PCR-positive (A and B) and ESBL PCR-negative (C and D) KPC-possessing isolates. Reprinted from the Journal of Clinical Microbiology (vol. 47.11 (2009): 3420–3426), by Tsakris, Athanassios, et al., 2009. Copyright 2009 by the Journal of Clinical Microbiology.

Fig. 15

Example of pinpoints within mCIM inhibition zone. During mCIM testing of some isolates, multiple small colonies could be observed growing throughout the zone of inhibition of the 10-g meropenem (MEM) disk; results are interpreted as positive when the zone of confluent growth inhibition measures 18 mm in diameter and as intermediate when the zone measures 19 mm in diameter.

Fig. 16

(A and B). Reading and interpretation of mCIM results. (A) A 22-mm zone of inhibition of growth of the carbapenem-susceptible indicator organism is present around the meropenem (MEM) disk incubated with the negative-control organism (N), while no zone of inhibition is present around the MEM disks incubated with the positive-control organism (P) or the test organism (T). (B) Closer examination of the zone around the MEM disk incubated with the negative-control organism reveals a narrow ring of growth abutting the disk, which represents carryover of the test organism from the tryptic soy broth; this growth is ignored when interpreting mCIM results. Reprinted from the Journal of Clinical Microbiology (vol. 55.8 (2017): 2321–2333), by Pierce, Virginia M., et al., 2009. Copyright 2017 by the Journal of Clinical Microbiology.