Abstract
A Drigalski agar-based culture medium containing an ertapenem, cloxacillin, and zinc sulfate (Supercarba medium) was tested for screening carbapenemase-producing members of the family Enterobacteriaceae. OXA-48 (n = 44), NDM (n = 25), VIM or IMP (n = 27), and KPC producers (n = 18) were detected with a low detection limit. Its overall sensitivity (95.6%) was higher than those of the currently available ChromID ESBL (bioMérieux) and CHROMagar KPC (CHROMagar) screening media. The Supercarba medium provides a significant improvement for detection of the most common types of carbapenemase producers.
TEXT
A variety of carbapenemases are increasingly reported in members of the family Enterobacteriaceae worldwide. Carbapenemase producers are becoming a source of therapeutic failures in both hospital- and community-acquired infections. The detection of infected patients and carriers with multidrug-resistant isolates is therefore becoming a major issue, and it is a major health issue to prevent the spread of these isolates. The clinically significant carbapenemases in Enterobacteriaceae belong to several Ambler classes of β-lactamases that differ by chemical structures and biochemical properties (1). They are mostly of the Ambler class A (KPC) that hydrolyze all β-lactams, of the zinc-dependent Ambler class B (NDM, VIM, and IMP) that hydrolyze all β-lactams except aztreonam, and of the Ambler class D (OXA-48-like) that hydrolyze carbapenems and weakly hydrolyze (or do not hydrolyze) broad-spectrum cephalosporins (2, 5, 6, 8, 13, 15–17, 20–22). The level of resistance to carbapenems provided by those carbapenemase producers may vary significantly, making their detection difficult when based only on high-level carbapenem resistance (3, 4, 11, 12). A medium initially designed to screen for extended-spectrum β-lactamase (ESBL) producers that contains cefpodoxime (ChromID ESBL; bioMérieux, La Balme-les-Grottes, France) and a carbapenem-containing medium (CHROMagar KPC; CHROMagar Company, Paris, France) (11, 23, 24) were evaluated for screening carbapenemase producers. Both media contained chromogenic molecules that may contribute to the recognition of enterobacterial species. The ChromID ESBL medium has good sensitivity; its main disadvantage is its lack of detection of OXA-48-like producers that are susceptible to cefpodoxime in the absence of coproduction of an ESBL (3). In addition, this medium lacks specificity, since the widespread ESBL producers may be coselected on that medium. The CHROMagar KPC medium detects carbapenemase producers only if they are resistant to high levels of carbapenems. Therefore, its main disadvantage remains its lack of sensitivity, since it does not detect carbapenemase producers with a low level of resistance to carbapenems (3, 16). This is the case for many KPC-, IMP-, VIM-, NDM-, and OXA-48-producing Escherichia coli and Klebsiella pneumoniae.
Taking into account the current importance of detecting carbapenemase producers with accuracy, we have designed a novel screening medium called Supercarba medium. The rationale for the design of this medium was that it should be able to detect carbapenemase producers with low-level resistance to carbapenems and be as selective as possible by inhibiting the growth of carbapenem-resistant but non-carbapenemase-producing isolates.
Different concentrations of several carbapenem molecules were tested, and finally, ertapenem was added to Drigalski agar medium at a concentration of 0.25 μg/ml. ZnSO4 (70 μg/ml) was added to improve expression of metallo-β-lactamases (MBLs) by MBL producers (12). Cloxacillin (250 μg/ml), which is a cephalosporinase (AmpC-type β-lactamase) inhibitor, was used to prevent growth of isolates expressing high levels of cephalosporinases, such as Enterobacter cloacae, Enterobacter aerogenes, Morganella morgannii, and Serratia marcescens. These isolates are clinically significant sources of carbapenem resistance associated with an outer membrane permeability defect (9, 14).
A total of 114 carbapenemase-producing isolates belonging to various enterobacterial species of worldwide origin were included in the study, all having a β-lactamase content characterized at the molecular level (Table 1). The strains were as follows: KPC producers (n = 18), VIM producers (n = 12), IMP producers (n = 15), NDM-1 producers (n = 25), together with OXA-48- (n = 41) and OXA-181 producers (n = 3). Seventy-five of those isolates coexpressed an ESBL (Table 1). Strains that did not express any carbapenemase were used as controls, consisting of isolates showing reduced susceptibility to ertapenem due to an overexpressed AmpC (n = 10), or to an ESBL (n = 12), and/or porin deficiency. Wild-type ertapenem-susceptible isolates, restricted-spectrum β-lactamase producers, ESBL producers, and high-level AmpC producers were also included as controls (n = 40) (Table 1). Using an inoculum of ∼2 × 107 CFU/ml (range, 1.5 × 107 to 3.5 × 108 CFU/ml), serial 10-fold dilutions of the isolates were made in normal saline, and 100-μl portions were plated onto the Supercarba medium and compared to results obtained using CHROMagar KPC and ChromID ESBL media. Viable bacteria were counted after 24 h of culture at 37°C. The sensitivity and specificity cutoff values were set at 1 × 103 CFU/ml, i.e., a limit value of 1 × 103 CFU/ml and above was considered “not efficiently detected.”
Table 1.
MICs and limits of detection of Supercarba, ChromID ESBL, and CHROMagar KPC mediaa
| Strain | β-Lactamase contentb | MIC (μg/ml) of antibioticc |
Lowest detection limit (CFU/ml) for the following mediumd: |
||||
|---|---|---|---|---|---|---|---|
| IPM | ETP | MEM | Supercarba | ChromID ESBL | CHROMagar KPC | ||
| Ambler class A carbapenemase (KPC)-producing strains | |||||||
| K. pneumoniae 2303 | KPC-2 + SHV-11 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae LIE | KPC-2 + TEM-1 + OXA-9 | >32 | >32 | >32 | 5 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae GES | KPC-2 + TEM-1 + SHV-11 | 6 | 12 | 1.5 | 1 × 101 | 1 × 101 | 1 × 103 |
| K. pneumoniae 588 | KPC-2 + TEM-1 + SHV-11 + OXA-9 | 24 | 32 | 16 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae YC | KPC-2 + TEM-1 + SHV-11 + SHV-12 + OXA-9 | 4 | 24 | 2 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae A28006 | KPC-2 + TEM-1 + CTX-M-2 + SHV-11 | 16 | 24 | 32 | 2 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae A33504 | KPC-2 + TEM-1 + SHV-11 + CTX-M-2 + OXA-9 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae MUS | KPC-2 + TEM-1 + SHV-11 + SHV-12 | 0.75 | 4 | 1.5 | 1 × 101 | 1 × 101 | 1 × 103 |
| K. pneumoniae KAM | KPC-3 + TEM-1 + SHV-11 | 8 | 12 | 2 | 1 × 101 | 1 × 101 | 5 × 103 |
| E. coli PSP | KPC-2 + TEM-1 + OXA-1 | 0.5 | 0.5 | 0.5 | 1 × 102 | 1 × 101 | 1 × 104 |
| E. coli DIN | KPC-2 + TEM-1 + OXA-1 | 1 | >32 | 0.5 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli COL | KPC-2 + TEM-1 + CTX-M-9 | 4 | 4 | 2 | 1 × 101 | 1 × 101 | 1 × 103 |
| E. coli LIL | KPC-2 + TEM-1 + OXA-9 | 2 | 1.5 | 1 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. cloacae HMG | KPC-2 + TEM-1 | 24 | >32 | 16 | 1 × 102 | 1 × 101 | 1 × 101 |
| E. cloacae CFVL | KPC-2 + TEM-3 | 4 | 2 | 1 | 1 × 101 | 1 × 101 | 5 × 105 |
| E. cloacae HPTU | KPC-2 + TEM-1 + SHV-11 | 2 | 4 | 1.5 | 1 × 101 | 1 × 101 | 1 × 101 |
| S. marcescens D6403 | KPC-2 + TEM-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| S. marcescens C7052 | KPC-2 + TEM-1 + SHV-12 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| Ambler class B carbapenemase-producing strains | |||||||
| K. pneumoniae OMA419 | NDM-1 + OXA-1 | 1.5 | 6 | 2 | 1 × 101 | 1 × 101 | 1 × 102 |
| K. pneumoniae KI2 | NDM-1 + CTX-M-15 + OXA-1 | 1 | 8 | 4 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae UK | NDM-1 + CTX-M-15 + CMY-4 + OXA-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 6642 GEN | NDM-1 + CTX-M-15 + OXA-1 + OXA-10 | 1 | 16 | 3 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 6759 GEN | NDM-1 + CTX-M-15 + CMY-16 + OXA-1 + OXA-9 + OXA-10 | 12 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae OMA601 | NDM-1 + CTX-M-15 + OXA-1 + OXA-9 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 7AFR | NDM-1 + TEM-1 + CTX-M-15 + CMY-6 + OXA-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae OM2 | NDM-1 + TEM-1+ CTX-M-3 + SHV-11 + OXA-1 | 0.75 | 8 | 1.5 | 1 × 101 | 1 × 101 | 3 × 104 |
| K. pneumoniae OM4 | NDM-1 + TEM-1 + CTX-M-15 + SHV-12 + OXA-9 | 4 | >32 | 16 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae OM8 | NDM-1 + TEM-1 + CTX-M-15 + SHV-11 + OXA-1 | 2 | >32 | 4 | 2 × 101 | 1 × 101 | 1 × 102 |
| K. pneumoniae OM13 | NDM-1+ TEM-1 + CTX-M-15 + SHV-28 + OXA-1 + OXA-9 | 3 | 4 | 2 | 1 × 101 | 1 × 101 | 3 × 104 |
| K. pneumoniae OM15 | NDM-1 + CTX-M-15 + SHV-130 + OXA-1 | 1.5 | 12 | 3 | 1 × 101 | 1 × 101 | 3 × 105 |
| K. pneumoniae OM16 | NDM-1 + CTX-M-15 + OXA-1 + OXA-181 | 8 | >32 | 16 | 3 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae OM19 | NDM-1 + CTX-M-15 + SHV-12 + OXA-1 | 4 | 24 | 8 | 1 × 101 | 1 × 101 | 4 × 102 |
| K. pneumoniae KIE | NDM-1 + SHV-38 + CMY-16 + OXA-10 | 0.75 | 2 | 1 | 1 × 101 | 1 × 101 | 1 × 104 |
| E. coli GUE | NDM-1 + TEM-1 + OXA-1 | 3 | 3 | 2 | 1 × 101 | 1 × 101 | 1 × 105 |
| E. coli AUS | NDM-1 + TEM-1 + CTX-M-15 | 6 | 32 | 16 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli IR5 | NDM-1 + TEM-1 + CTX-M-15 | 16 | >32 | 16 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli GEN | NDM-1 + TEM-1 + CMY-30 + OXA-1 | 8 | >32 | 12 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli RIC | NDM-1 + CMY-16 + OXA-1 + OXA-10 | 1 | 3 | 1 | 1 × 101 | 1 × 101 | 1 × 105 |
| E. coli ALL | NDM-1 + TEM-1 + CTX-M-15 + OXA-1 + OXA-2 | 4 | >32 | 8 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli OM20 | NDM-1+ TEM-1 + CTX-M-15 | 2 | >32 | 8 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. cloacae IR38 | NDM-1 + CTX-M-15 | 2 | 16 | 2 | 1 × 101 | 3 × 102 | 4 × 104 |
| P. stuartii PS1 | NDM-1 + CMY-6 + OXA-1 | 12 | 0.38 | 1.5 | 1 × 107 | 1 × 103 | 1 × 107 |
| C. freundii STE | NDM-1 + TEM-1 + CTX-M-15 + VIM-4 + OXA-1 + OXA-9 + OXA-10 + OXA-181 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 0404024 | VIM-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 0511135 | VIM-1 + SHV-12 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae 0404020 | VIM-1 + SHV-5 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae ENN | VIM-1 + SHV-5 | 0.5 | 1.5 | 0.38 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae MAD | VIM-1 + CTX-M-3 | 1 | 0.5 | 1 | 1 × 101 | 3 × 101 | 2 × 104 |
| E. coli DIH | VIM-19 | 8 | 16 | 4 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli 0404018 | VIM-1 + CMY-6 | 3 | 1.5 | 1 | 5 × 101 | 1 × 101 | >1 × 108 |
| E. coli 1008077 | VIM-1 + TEM-1 + CTX-M-15 | >32 | 4 | 4 | 1 × 101 | 1 × 101 | >1 × 108 |
| E. coli MAD | VIM-1 + CTX-M-3 | 1.5 | 0.38 | 0.5 | 1 × 105 | 1 × 101 | 2 × 105 |
| E. cloacae KAR | VIM-1 + SHV-70 | 1 | 0.38 | 0.5 | 1 × 106 | 1 × 101 | >1 × 108 |
| E. cloacae 1008029 | VIM-1 + CTX-M-3 | >32 | >32 | >32 | 2 × 101 | 1 × 101 | 1 × 101 |
| S. marcescens 1008091 | VIM-1 + CTX-M-15 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae TUR | IMP-1 | 1 | 2 | 8 | 1 × 106 | 2 × 101 | 1 × 101 |
| K. pneumoniae 0709121 | IMP-1 | 1.5 | 3 | 1 | 1 × 101 | 1 × 101 | 1 × 103 |
| K. pneumoniae 0709124 | IMP-1 + TEM-15 | 8 | 3 | 2 | 1 × 101 | 1 × 101 | 1 × 104 |
| K. pneumoniae 0709125 | IMP-1 + TEM-1 + SHV-12 | 1.5 | 4 | 2 | 1 × 101 | 1 × 101 | 1 × 103 |
| K. pneumoniae 0709127 | IMP-1 + TEM-1 | 0.5 | 4 | 1 | 1 × 101 | 1 × 101 | 1 × 104 |
| K. pneumoniae TWA | IMP-8 | 1 | 1 | 0.5 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae TAW | IMP-8 + SHV-12 | 0.5 | 0.5 | 0.5 | 4 × 102 | 1 × 101 | >1 × 108 |
| E. coli JAP | IMP-1 | 0.5 | 3 | 0.5 | 1 × 104 | 1 × 101 | 2 × 105 |
| E. coli TWA | IMP-8 + SHV-12 | 6 | 8 | 3 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli 1108013 | IMP-1 + TEM-1 | 0.5 | 4 | 1 | 1 × 101 | 1 × 101 | 1 × 106 |
| E. cloacae TWA | IMP-8 | 1.5 | 1 | 1 | 1 × 101 | 1 × 101 | 1 × 102 |
| E. cloacae TAW | IMP-8 + SHV-12 | 0.75 | 0.5 | 0.5 | 1 × 102 | 1 × 101 | >1 × 108 |
| E. cloacae 1008079 | IMP-1 | 8 | >32 | >32 | 1 × 101 | 1 × 102 | 1 × 107 |
| E. cloacae 1008187 | IMP-1 + CTX-M-15 | 8 | >32 | 4 | 1 × 101 | 1 × 101 | 1 × 104 |
| S. marcescens 0911033 | IMP-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| Ambler class D carbapenemase-producing strains | |||||||
| K. pneumoniae BIC | OXA-48 | 0.5 | 2 | 0.5 | 1 × 101 | >1 × 108 | 5 × 106 |
| K. pneumoniae BEL | OXA-48 | 1 | 4 | 1 | 1 × 101 | >1 × 108 | 1 × 106 |
| K. pneumoniae RAM | OXA-48 | 1 | 4 | 1 | 1 × 101 | >1 × 108 | 1 × 105 |
| K. pneumoniae LIB | OXA-48 | 16 | 16 | 16 | 1 × 101 | >1 × 108 | 5 × 104 |
| K. pneumoniae BOU | OXA-48 | 0.38 | 0.5 | 0.25 | 1 × 101 | >1 × 108 | 1 × 108 |
| K. pneumoniae SCO | OXA-48 | 0.5 | 0.75 | 0.25 | 1 × 101 | >1 × 108 | >1 × 108 |
| K. pneumoniae LOU | OXA-48 | 4 | 16 | 0.5 | 1 × 101 | >1 × 108 | >1 × 108 |
| K. pneumoniae TIK | OXA-48 | 0.75 | 2 | 0.38 | 1 × 101 | >1 × 108 | >1 × 108 |
| K. pneumoniae OM14 | OXA-48 + TEM-1 | 0.5 | 1 | 0.38 | 1 × 101 | >1 × 108 | 5 × 107 |
| K. pneumoniae CHA | OXA-48 + TEM-1 | 0.38 | 1 | 0.5 | 1 × 101 | >1 × 108 | >1 × 108 |
| K. pneumoniae EGY | OXA-48 + CTX-M-15 | 2 | 3 | 2 | 1 × 101 | 2 × 101 | 1 × 105 |
| K. pneumoniae ROU | OXA-48 + CTX-M-15 | 0.5 | 1.5 | 0.25 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae BEY | OXA-48 + TEM-1 + CTX-M-15 | 0.38 | 0.38 | 0.38 | 5 × 102 | 1 × 101 | 1 × 108 |
| K. pneumoniae DAL | OXA-48 + TEM-1 + CTX-M-15 | 0.38 | 2 | 0.38 | 1 × 101 | 1 × 101 | 4 × 105 |
| K. pneumoniae BAJ | OXA-48+ TEM-1 + CTX-M-15 + SHV-28 | 0.5 | 1.5 | 0.38 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae BEN | OXA-48 + TEM-1 + CTX-M-15 + SHV-28 | 0.38 | 1 | 0.25 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae DUW | OXA-48 + TEM-1 + CTX-M-15 + SHV-28 | 32 | 32 | 32 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae SIC | OXA-48 + CTX-M-15 + SHV-28 | 0.25 | 1 | 0.25 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae AEL | OXA-48 + CTX-M-15 + SHV-28 + OXA-1 | 0.5 | 6 | 0.38 | 1 × 101 | 1 × 101 | 5 × 102 |
| K. pneumoniae AMS | OXA-48 + TEM-1 + CTX-M-15 + OXA-1 | 0.5 | 2 | 0.38 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae ELK | OXA-48 + TEM-1 + CTX-M-15 + SHV-11 | 0.5 | 3 | 0.38 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae VER | OXA-48 + TEM-1 + CTX-M-15 + SHV-11 | 0.38 | 2 | 0.38 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae VSG | OXA-48 + TEM-1 + CTX-M-15 + OXA-1 | 0.75 | 3 | 0.75 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae HPA | OXA-48 + TEM-1 + CTX-M-15 + OXA-1 | 1.5 | >32 | 12 | 1 × 101 | 1 × 101 | >1 × 108 |
| K. pneumoniae OM11 | OXA-48 + TEM-1 + CTX-M-14 | 0.5 | 0.75 | 0.25 | 1 × 101 | 1 × 101 | 5 × 107 |
| K. pneumoniae DIA | OXA-48 + TEM-1b + CTX-M-15 + SHV-11 + OXA-1 | >32 | >32 | >32 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. coli ROB | OXA-48 | 0.5 | 0.75 | 0.25 | 2 × 101 | >1 × 108 | >1 × 108 |
| E. coli HAN | OXA-48 + CTX-M-15 | 3 | 16 | 1 | 5 × 101 | 1 × 101 | 3 × 104 |
| E. coli BOU | OXA-48 + CTX-M-15 | 0.5 | 0.75 | 0.125 | 2 × 101 | 1 × 101 | >1 × 108 |
| E. coli OM3 | OXA-48 + TEM-1 + CTX-M-15 | 0.5 | 1 | 0.38 | 1 × 101 | 1 × 101 | >1 × 108 |
| E. coli OM22 | OXA-48 + TEM-1 + CTX-M-15 | 0.5 | 1 | 0.25 | 1 × 101 | 1 × 101 | >1 × 108 |
| E. coli BER | OXA-48 + TEM-1 + CTX-M-15 | 0.38 | 1.5 | 0.19 | 5 × 101 | 1 × 101 | >1 × 108 |
| E. coli AME | OXA-48 + CTX-M-24 | 0.25 | 0.5 | 0.19 | 2 × 101 | 1 × 101 | >1 × 108 |
| E. coli ZAN | OXA-48 + TEM-1 + CTX-M-14 | 0.38 | 8 | 0.75 | 1 × 101 | 1 × 101 | >1 × 108 |
| E. coli BON | OXA-48 + TEM-1 + CTX-M-24 | 0.38 | 0.5 | 0.19 | 1 × 101 | 1 × 101 | >1 × 108 |
| E. coli BOK | OXA-48 + CTX-M-15 | 0.25 | 0.38 | 0.19 | 2 × 101 | 1 × 101 | >1 × 108 |
| E. cloacae TUR | OXA-48 + SHV-5 | 0.5 | 0.5 | 0.5 | 1 × 101 | 2 × 101 | 1 × 107 |
| E. cloacae 501 | OXA-48 + TEM-1 + CTX-M-15 | 1 | 16 | 1.5 | 1 × 101 | 1 × 101 | 1 × 101 |
| E. cloacae BEU | OXA-48 + TEM-1 + CTX-M-15 + SHV-12 | 0.5 | 8 | 0.5 | 1 × 101 | 1 × 101 | 1 × 104 |
| C. koseri ROU | OXA-48 | 0.38 | 2 | 0.38 | 1 × 101 | >1 × 108 | >1 × 108 |
| C. koseri VER | OXA-48 | 0.75 | 2 | 0.38 | 1 × 101 | >1 × 108 | >1 × 108 |
| K. pneumoniae HOL | OXA-181 + CTX-M-15 | 1 | 4 | 1 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae OMA | OXA-181 + CTXM-15 + OXA-1 | 0.5 | 2 | 0.5 | 1 × 101 | 1 × 101 | >1 × 108 |
| P. rettgeri RAP | OXA-181 + OXA-1 | 8 | 1 | 2 | 5 × 102 | 1 × 101 | 1 × 101 |
| Non-carbapenemase-producing strains | |||||||
| K. pneumoniae 7725 | SHV-1 | 0.19 | 0.006 | 0.032 | >1 × 108 | >1 × 108 | >1 × 108 |
| K. pneumoniae 0227 | SHV-1 | 0.19 | 0.008 | 0.016 | >1 × 108 | >1 × 108 | >1 × 108 |
| K. pneumoniae 648236e | SHV-2a | 0.25 | 2 | 0.38 | 1 × 102 | 1 × 101 | >1 × 108 |
| K. pneumoniae 1022 | SHV-2a + SHV-28 | 0.5 | 0.016 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| K. pneumoniae BERe | SHV-28 + TEM-1 | 1 | 4 | 1 | 1 × 102 | 1 × 103 | 1 × 103 |
| K. pneumoniae KPN | CTX-M-15 | 0.12 | 0.012 | 0.012 | 1 × 107 | 1 × 101 | >1 × 108 |
| K. pneumoniae 10112 | CTX-M-15 + TEM-1 + SHV-11 | 0.5 | 0.016 | 0.023 | 6 × 107 | 1 × 101 | >1 × 108 |
| K. pneumoniae 1025 | CTX-M-14 + TEM-1 + SHV-11 | 0.12 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| K. pneumoniae MEKe | CTX-M-15 + SHV-11 | 1.5 | >32 | 6 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae SIMe | CTX-M-15 + TEM-1 + SHV-1 | 8 | >32 | 6 | 1 × 101 | 1 × 101 | 1 × 102 |
| K. pneumoniae SHMe | CTX-M-15 +TEM-1 + SHV-11 | 3 | >32 | 3 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae COOe | CTX-M-15 + SHV-28 | 8 | >32 | 4 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae FOSe | CTX-M-15 + TEM-1 + SHV-11 | 6 | >32 | >32 | 1 × 102 | 1 × 101 | 1 × 101 |
| K. pneumoniae BEDe | CTX-M-15 + TEM-1 + SHV-11 | 1.5 | >32 | 4 | 1 × 101 | 1 × 101 | 1 × 101 |
| K. pneumoniae SHIe | CTX-M-15 + TEM-1 + SHV-11 | 0.25 | 1 | 1 | 7 × 104 | 1 × 101 | >1 × 108 |
| K. pneumoniae LEGe | CTX-M-15 + TEM-1 + SHV-12 | 0.75 | >32 | 3 | 2 × 104 | 2 × 101 | 2 × 101 |
| K. pneumoniae ALEe | CTX-M-15 + SHV-1 | 1 | >32 | 4 | 1 × 105 | 1 × 101 | 1 × 101 |
| K. pneumoniae KDHf | DHA-2 | 0.12 | 0.5 | 0.12 | 1 × 102 | 1 × 101 | >1 × 108 |
| E. coli 6252 | None (wild type) | 0.12 | 0.004 | 0.008 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. coli 6367 | None (wild type) | 0.19 | 0.006 | 0.012 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. coli 1082 | TEM-1 | 0.19 | 0.019 | 0.016 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. coli 1034 | TEM-1 + SHV-38 | 0.19 | 0.006 | 0.016 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. coli 1048 | TEM-1 + SHV-2a | 0.19 | 0.012 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 1008 | CTX-M-1 + TEM-1 | 0.19 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 10122 | CTX-M-1 + TEM-1 | 0.19 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 1020 | CTX-M-1 + TEM-1 | 0.19 | 0.023 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 10121 | CTX-M-2 | 0.19 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 1023 | CTX-M-2 + TEM-1 | 0.12 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli E14 | CTX-M-14 | 0.12 | 0.012 | 0.012 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli FOR | CTX-M-15 | 0.12 | 0.012 | 0.012 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 1033 | CTX-M-15 | 0.19 | 0.012 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli EVB | VEB-1 | 0.12 | 0.012 | 0.012 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli 1092 | OXA-1 | 0.12 | 0.19 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli ECA | ACC-1 | 0.12 | 0.012 | 0.012 | >1 × 108 | 5 × 103 | >1 × 108 |
| E. coli SYD | CMY-2 | 0.12 | 0.012 | 0.012 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli MET | Chromosome-encoded extended-spectrum cephalosporinase | 0.12 | 0.012 | 0.012 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. coli MARf | Overexpressed AmpC | 16 | >32 | 2 | 1 × 102 | 1 × 101 | 1 × 101 |
| E. coli HB4e (OmpC−, OmpF−) | None | 0.12 | 1 | 0.25 | 1 × 101 | >1 × 108 | >1 × 108 |
| E. aerogenes 1009 | TEM-24 | 0.19 | 0.12 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. aerogenes 1085 | TEM-24 | 0.12 | 0.19 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae 7746 | None (wild type) | 0.38 | 0.064 | 0.032 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. cloacae 7725 | None (wild type) | 0.19 | 0.008 | 0.012 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. cloacae 5434 | None (wild type) | 0.38 | 0.016 | 0.032 | >1 × 108 | >1 × 108 | >1 × 108 |
| E. cloacae 1012 | TEM-1 + SHV-12 | 0.19 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae 1072f | TEM-1 + OXA-1 | 0.38 | 0.5 | 0.064 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae CLO | CTX-M-15 | 0.12 | 0.12 | 0.12 | 1 × 107 | 1 × 101 | >1 × 108 |
| E. cloacae 10111f | TEM-1 + CTX-M-15 | 0.5 | 0.75 | 0.094 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae 1027 | TEM-1 + CTX-M-15 | 0.19 | 0.016 | 0.016 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae CVB | VEB-1 | 0.12 | 0.12 | 0.12 | 1 × 104 | 1 × 101 | >1 × 108 |
| E. cloacae 1019f | TEM-1 | 0.25 | 1 | 0.094 | >1 × 108 | 1 × 101 | >1 × 108 |
| E. cloacae ARFf | Overexpressed AmpC | 0.12 | 1 | 0.12 | 1 × 107 | 1 × 101 | >1 × 108 |
| E. cloacae BLAf | Overexpressed AmpC | 0.12 | 1 | 0.12 | 1 × 107 | 1 × 101 | >1 × 108 |
| E. cloacae CONf | Overexpressed AmpC | 0.25 | 4 | 0.25 | 1 × 107 | 1 × 101 | >1 × 108 |
| E. cloacae AZAf | Overexpressed AmpC | 0.12 | 1 | 0.12 | 1 × 107 | 1 × 106 | >1 × 108 |
| C. freundii 7767 | None (wild type) | 0.25 | 0.008 | 0.016 | >1 × 108 | >1 × 108 | >1 × 108 |
| C. freundii 10107 | TEM-1 + SHV-12 | 0.38 | 0.016 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| C. freundii 1003 | CTX-M-15 + TEM-1 | 0.38 | 0.016 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| C. freundii 10135 | CTX-M-15 | 0.38 | 0.016 | 0.023 | >1 × 108 | 1 × 101 | >1 × 108 |
| C. freundii MAUf | Overexpressed AmpC + TEM-3 | 1 | 8 | 1 | 1 × 105 | 1 × 101 | 1 × 105 |
| S. Typhimurium 1081 | CTX-M-1 | 0.25 | 0.19 | 0.032 | >1 × 108 | 1 × 101 | >1 × 108 |
| P. mirabilis 1031 | CTX-M-14 + TEM-1 + SHV-11 | 1.5 | 0.047 | 0.032 | >1 × 108 | 1 × 101 | >1 × 108 |
| P. mirabilis PMA | ACC-1 | 0.25 | 0.094 | 0.064 | >1 × 108 | >1 × 108 | >1 × 108 |
The MICs of imipenem, ertapenem, and meropenem and the detection limits of Supercarba medium for 176 carbapenemase- and/or ESBL/AmpC-producing enterobacterial isolates compared to the detection limits obtained with ChromID ESBL and CHROMagar KPC media are shown. The 176 enterobacterial isolates belong to the following species: Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae, Serratia marcescens, Providencia stuartii, Citrobacter freundii, Citrobacter koseri, Providencia rettgeri, Enterobacter aerogenes, Salmonella enterica serotype Typhimurium, and Proteus mirabilis.
β-Lactamase names shown in boldface type are carbapenemases.
Abbreviations: IMP, imipenem; ETP, ertapenem; MP, meropenem.
Underlined CFU counts are considered negative results (cutoff values set at ≥1 × 103 CFU/ml).
Reduced susceptibility to ertapenem due to porin deficiency.
Reduced susceptibility to ertapenem due to overexpressed AmpC.
The lowest limit of detection of OXA-48, OXA-181, NDM-1, and KPC producers ranged from 1 × 101 to 1 × 102 CFU/ml (Table 1). A single NDM producer (NDM-1-producing Providencia stuartii isolate [19]) was not efficiently detected on the Supercarba medium (detection limit of 1 × 107 CFU/ml) (Table 1). Its lack of detection might be explained by its low MIC value of ertapenem (0.38 μg/ml) and a likely weak expression of the blaNDM-1 gene, related to chromosomal insertion of the blaNDM-1 gene. As expected, OXA-181-producing K. pneumoniae was also detected well with the Supercarba medium. The lowest limit of detection of VIM and IMP producers ranged from 1 × 101 to 1 × 106 CFU/ml (Table 1). Although the addition of zinc sulfate significantly decreased the detection limits for VIM and IMP producers, a few VIM and IMP producers were not efficiently detected on this medium (detection limit of ≥1 × 103 CFU/ml). As expected, growth of isolates that do not express any carbapenemase (i.e., AmpC and/or ESBL producers) were inhibited by the Supercarba medium (with a detection limit much higher than 1 × 103 CFU/ml). In particular, the addition of cloxacillin prevented growth of the isolates expressing cephalosporinases (Table 1). As previously shown, a porin defect resulting in a decreased outer membrane permeability leads to a reduced susceptibility to ertapenem of E. coli and K. pneumoniae (7, 9, 10). In this study, among the 19 non-ertapenem-susceptible isolates with MIC values of ertapenem of >0.25 μg/ml (1 Citrobacter freundii isolate, 2 E. coli isolates, 4 Enterobacter cloacae isolates, and 12 K. pneumoniae isolates) and for which a porin defect was involved in ertapenem resistance, 58% (n = 11) were detected by selection on the Supercarba medium (lower detection limit of ≤102 CFU/ml) (Table 1). The addition of zinc sulfate and cloxacillin was useful for prevention of growth of many non-carbapenemase-producing carbapenem-resistant isolates (up to 42%; n = 8). Noticeably, non-carbapenemase-producing Acinetobacter baumannii and Pseudomonas aeruginosa grew on the Supercarba medium (data not shown). Similar growth results of nonenterobacterial Gram-negative rods were obtained using the ChromID ESBL and CHROMagar KPC media (data not shown). These three media are suitable only for selection of members of the Enterobacteriaceae.
A comparison of the results obtained with the ChromID ESBL and CHROMagar KPC media with those obtained with the Supercarba medium showed that the latter screening medium is more efficient in detecting carbapenemase-producing isolates (Tables 1 and 2). Indeed, the sensitivity of the Supercarba medium was 95.6%, which was higher than the sensitivity of the ChromID ESBL (87.7%) medium and of the CHROMagar KPC (40.3%) medium. Moreover, the sensitivities of the Supercarba medium determined for each class of carbapenemase producers was higher (100%, 90%, and 100% for classes A, B, and D, respectively) than those obtained for the two other screening media (Table 2). The specificity of the Supercarba medium was also high (82.2%). A further improvement of the Supercarba medium would be the addition of chromogenic molecules that would permit recognition of species.
Table 2.
Sensitivity and specificity of Supercarba, ChromID ESBL, and CHROMagar KPC media
| Sensitivity or specificity | Value for sensitivity (%) or specificity (%) on the following medium: |
||
|---|---|---|---|
| Supercarba | ChromID ESBL | CHROMagar KPC | |
| Sensitivity | 95.6 | 87.7 | 40.3 |
| Specificity | 82.2 | 24.2 | 85.5 |
| Sensitivity for Ambler class of carbapenemasea | |||
| Class A | 100 | 100 | 66.7 |
| Class B | 90 | 98 | 55.8 |
| Class D | 100 | 70 | 13.6 |
Sensitivity was determined for each Ambler class of carbapenemase: class A carbapenemases are of the KPC type, class B carbapenemases are of the VIM, IMP, and NDM types, whereas class D carbapenemases are of the OXA-48 type.
To assess the storage ability of the Supercarba medium, E. cloacae ARF that overexpressed AmpC was subcultured daily onto Drigalski agar plates from a single batch of Supercarba medium stored at 4°C. Growth of this isolate was consistently inhibited on the Supercarba agar during a 7-day period.
We propose here the very first screening medium that may detect not only KPC and MBL producers but also OXA-48 producers. This medium represents a significant improvement compared to the available screening media to detect carbapenemase producers, and particularly for detection of OXA-48 producers that do not coexpress any ESBL. Taking into account the fact that Supercarba medium contains ertapenem at a low concentration, using this medium may detect carbapenemase producers with low-level resistance to carbapenems, which is a situation frequently observed for OXA-48 producers. In addition, this medium is useful for selecting specifically carbapenemase producers in stools that also contain a large amount of ESBL producers and inhibiting the growth of ESBL producers. This property is particularly relevant, since high rates of ESBL carriage are now reported worldwide (18).
Finally, a further improvement of the Supercarba medium would be the addition of chromogenic molecules for identification of enterobacterial species.
ACKNOWLEDGMENT
This work was funded by a grant from the INSERM (UMR914), Paris, France.
Footnotes
Published ahead of print 22 February 2012
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