Phenotypic screening of carbapenemases and associated β-lactamases in carbapenem-resistant Enterobacteriaceae

Abstract

Dissemination of carbapenem resistance among Enterobacteriaceae poses a considerable threat to public health. Carbapenemase gene detection by molecular methods is the gold standard but is available in only a few laboratories. The aim of this study was to test phenotypic methods for the detection of metallo-β-lactamase (MBL)- or Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae and associated mechanisms of β-lactam resistance against a panel of 30 genotypically characterized carbapenem-resistant Enterobacteriaceae : 9 MBL, 7 KPC, 6 OXA-48, and 8 extended-spectrum β-lactamase (ESBL) or AmpC β-lactamases associated with decreased permeability. We used carbapenemase inhibitor-impregnated agar to test for carbapenem-resistant strains. Differences in the inhibition zone sizes of the meropenem, imipenem, ertapenem, and doripenem disks were measured between control and inhibitor (EDTA or phenylboronic acid [PBA] with or without cloxacillin)-impregnated Mueller-Hinton agar with a cutoff of 10 mm. All 9 MBL- and 7 KPC-producing Enterobacteriaceae were identified from the differences in zone size in the presence and absence of specific inhibitors, regardless of the carbapenem MICs and including isolates with low-level resistance to carbapenems. We also detected their associated β-lactam resistance mechanisms (11 ESBL-type and 5 class A β-lactamase 2b). No differences in zone size were observed for OXA-48-producing strains or other carbapenem resistance mechanisms such as ESBL and decreased permeability. We propose a new strategy to detect carbapenemases (MBL- and KPC-type) and associated mechanisms of β-lactam resistance (ESBL or class A β-lactamase 2b) by the use of inhibitor-impregnated agar. A rapid phenotypic detection of resistance mechanisms is important for epidemiological purposes and for limiting the spread of resistant strains by implementing specific infection control measures.

Fig 1

Representative phenotypes of resistance. (Aa to Ad) The VIM-4- and SHV-5-producing K. pneumoniae strain (strain 10) is shown in a, b, c, and d. (a) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar (MHA) as a reference. (b) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. The difference in zone size in the presence and absence of EDTA was >10 mm for ETP, MEM, DORI, and IPM, suggesting MBL production. Synergy between amoxicillin-clavulanic acid or ticarcillin-clavulanic acid and broad-spectrum cephalosporins (FEP and CTX) suggests ESBL production. (c) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. No difference in zone size was observed in the presence and absence of PBA. (d) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. (Be to Bh) The NDM-1- and OXA-1-producing E. coli strain (strain 9) is shown in e, f, g, and h. (e) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (f) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting MBL production. (g) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. No difference in zone size was observed. (h) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. (Ci to Cl) The KPC-2-, CTX-M-15-, and TEM-1-producing K. pneumoniae strain (strain 25) is shown in i, j, k, and l. (i) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (j) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, indicating no MBL production. (k) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting class A carbapenemase production or a combination of plasmid-mediated AmpC production and porin loss. Synergy between amoxicillin-clavulanic acid or ticarcillin-clavulanic acid and broad-spectrum cephalosporins (FEP, CTX, and CAZ) suggested ESBL production. (l) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. One can conclude that the E. coli strain produces both a class A carbapenemase and an ESBL. (Dm to Dp) The E. cloacae strain exhibiting AmpC hyperproduction and decreased permeability (strain 23) is shown in m, n, o, and p. (m) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (n) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM, indicating the absence of MBL. (o) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting the production of a class A carbapenemase or a combination of AmpC hyperproduction and decreased permeability. (p) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml). Differences in the zone diameter were observed between native MHA and MHA-cloxacillin. Given the similar inhibition zones obtained with cloxacillin and PBA, carbapenemase production can be ruled out. (Eq to Et) The OXA-48- and CTX-M-9-producing E. coli strain (strain 27) is shown in q, r, s, and t. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, PBA, or cloxacillin. (Fu to Fx) The K. pneumoniae strain exhibiting CTX-M-15, OXA-1, TEM-1, and decreased permeability (strain 20) is shown in u, v, w, and x. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, PBA, or cloxacillin.

Fig 1

Representative phenotypes of resistance. (Aa to Ad) The VIM-4- and SHV-5-producing K. pneumoniae strain (strain 10) is shown in a, b, c, and d. (a) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar (MHA) as a reference. (b) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. The difference in zone size in the presence and absence of EDTA was >10 mm for ETP, MEM, DORI, and IPM, suggesting MBL production. Synergy between amoxicillin-clavulanic acid or ticarcillin-clavulanic acid and broad-spectrum cephalosporins (FEP and CTX) suggests ESBL production. (c) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. No difference in zone size was observed in the presence and absence of PBA. (d) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. (Be to Bh) The NDM-1- and OXA-1-producing E. coli strain (strain 9) is shown in e, f, g, and h. (e) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (f) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting MBL production. (g) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. No difference in zone size was observed. (h) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. (Ci to Cl) The KPC-2-, CTX-M-15-, and TEM-1-producing K. pneumoniae strain (strain 25) is shown in i, j, k, and l. (i) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (j) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, indicating no MBL production. (k) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting class A carbapenemase production or a combination of plasmid-mediated AmpC production and porin loss. Synergy between amoxicillin-clavulanic acid or ticarcillin-clavulanic acid and broad-spectrum cephalosporins (FEP, CTX, and CAZ) suggested ESBL production. (l) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml) (AES Chemunex). No differences in zone size were observed. One can conclude that the E. coli strain produces both a class A carbapenemase and an ESBL. (Dm to Dp) The E. cloacae strain exhibiting AmpC hyperproduction and decreased permeability (strain 23) is shown in m, n, o, and p. (m) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar as a reference. (n) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 2 ml of EDTA 5 × 10⁻³ M. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM, indicating the absence of MBL. (o) Antimicrobial susceptibility testing was performed on Mueller-Hinton agar impregnated with 0.75 ml of 10 mg/ml PBA. The difference in zone size was >10 mm for ETP, MEM, DORI, and IPM, suggesting the production of a class A carbapenemase or a combination of AmpC hyperproduction and decreased permeability. (p) Antimicrobial susceptibility testing was performed on MHA-cloxacillin (250 μg/ml). Differences in the zone diameter were observed between native MHA and MHA-cloxacillin. Given the similar inhibition zones obtained with cloxacillin and PBA, carbapenemase production can be ruled out. (Eq to Et) The OXA-48- and CTX-M-9-producing E. coli strain (strain 27) is shown in q, r, s, and t. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, PBA, or cloxacillin. (Fu to Fx) The K. pneumoniae strain exhibiting CTX-M-15, OXA-1, TEM-1, and decreased permeability (strain 20) is shown in u, v, w, and x. No significant difference in zone size was observed with ETP, MEM, DORI, or IPM in the presence and absence of EDTA, PBA, or cloxacillin.

Fig 2

(A) The K. pneumoniae strain producing VIM-4 and SHV-5 (strain 10) is shown. Two lines of disks containing ATM (10 μg), AMC (10 μg), and CAZ (10 μg) were placed 20 mm apart on an MHA plate seeded with the test strain. Then, 5 μl of 0.5 M EDTA was added along the second line. Enhancement of the zone of inhibition in the area between the ATM and AMC disks and between the AMC and CAZ disks in the presence of EDTA is considered to suggest ESBL production in addition to MBL. (B) The K. pneumoniae strain producing KPC-2, CTX-M-15, and TEM-1 (strain 25) is shown. Three lines of disks containing ATM (10 μg), AMC (10 μg), and CAZ (10 μg) were placed 20 mm apart on an MHA plate seeded with the test strain. Then, 10 μl and 20 μl of phenylboronic acid (20 mg/ml) were added to lines 2 and 3, respectively. Enhancement of the zone of inhibition between the ATM and AMC disks and between the AMC and CAZ disks in the presence of PBA suggests ESBL production in addition to KPC or AmpC expression and decreased permeability. Cloxacillin-impregnated agar is necessary to draw firm conclusions, as it is otherwise impossible to tell whether the enhancement of the inhibition zone is due to the effect of boronic acid on an AmpC β-lactamase or on a KPC carbapenemase (PBA can inhibit both enzymes).

Fig 3

Strategy for identifying the type of carbapenemase.