This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes, especially in regions where KPC- and MBL-possessing Enterobacteriaceae are highly prevalent.

The worldwide increase in the occurrence and dissemination of KPC ␤-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum ␤-lactamases, metallo-␤-lactamases, and plasmidmediated AmpC ␤-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 g of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave falsepositive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum ␤-lactamases are widespread.

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum ␤-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceae family. A total of 155 isolates of Klebsiella pneumoniae (n ‫؍‬ 141), Escherichia coli (n ‫؍‬ 6), Enterobacter aerogenes (n ‫؍‬ 6), and Klebsiella oxytoca (n ‫؍‬ 2) genotypically confirmed to be KPC producers were analyzed. As many as 118 isolates harbored ESBLs (103 harbored SHV-type ESBLs, 13 harbored CTX-M-type ESBLs, and 2 harbored both SHV-and CTX-M-type ESBLs); the remaining 37 isolates were genotypically negative for ESBL production. The CLSI ESBL confirmatory test was positive for 79 of the 118 ESBL producers (sensitivity, 66.9%), while all 37 non-ESBL producers were negative (specificity, 100%). When a >5-mm increase in the zone diameter of either the cefotaxime (CTX)-clavulanate (CA) or the ceftazidime (CAZ)-CA disks containing BA compared with the zone diameter of the CTX or CAZ disks containing BA was considered to be a positive result for ESBL production, the method detected all 118 ESBL producers (sensitivity, 100%) and showed no false-positive results for non-ESBL producers (specificity, 100%). Double-disk synergy tests, in which disks of CTX, CAZ, aztreonam, or cefepime in combination with BA were placed at distances of 20, 25, and 30 mm (center to center) from a disk containing amoxicillin (amoxicilline)-clavulanate-BA, were able to detect 116 (98.3%), 101 (85.6%), and 28 (23.7%) of the ESBL-positive isolates, respectively; no false-positive results for non-ESBL-producing isolates were detected. Our results demonstrate that the modified CLSI ESBL confirmatory test with antibiotic disks containing BA is the most accurate phenotypic method for the detection of ESBLs in Enterobacteriaceae producing KPC carbapenemases.

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Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 g/ml. Heteroresistant colonies had significantly elevated expression of the bla KPC gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.Since the beginning of the last decade, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) isolates have been increasingly detected in the United States and subsequently in several regions worldwide (3,4,13,17,21). KPC enzymes efficiently hydrolyze all ␤-lactam molecules (1, 22), conferring various levels of resistance to all ␤-lactam compounds, including carbapenems (13). However, KPC-producing K. pneumoniae may appear susceptible to carbapenems, mainly meropenem (2, 13), by reference CLSI agar dilution or broth microdilution methods as well as by automated systems (6,15,17). Characteristically, it has been reported that automated systems may identify as many as 87% of KPC-KP isolates to be susceptible to meropenem (13). The detection of the susceptibility level of KPC-KP isolates to carbapenems has been shown to be difficult due to the phenotypic heterogeneity that they commonly exhibit (3, 10, 13). For instance, in agar diffusion methods such as disk diffusion or Etest, the heterogeneous growth to carbapenems of KPC-KP results in the appearance of scattered colonies within the inhibition zones (9, 13).These issues raise the need for cautious evaluation of susceptibility testing in KPC-KP isolates that are recovered in clinical laboratories. In our clinical laboratories, several KPC-KP isolates that appear susceptible by automated susceptibility assays or reference dilution assays contain heterogeneous subpopulations (D. Sofianou and K. Themeli-Digalaki, personal communications). It has been also shown that among Greek KPC-KP isolates, meropenem tends to exhibit lower MICs than imipenem or ertapenem (17,20). In that respect, the aim of the present study was to characterize the heterogeneous mode of growth of apparently meropenem-susceptible KPC-KP clinical isolates by population analyses and bactericidal assays.Bacterial strains, susceptibility studies, and macrorestriction analysis. Six KPC-2-producing K. pneumoniae clinical isolates from our laboratory collection were randomly selected for the study among those that were meropenem susceptible by agar dilution (MIC Յ 4 g/ml) (5) but exhibited scattered heterogeneous colonies around carbapenem discs. The isolates were recovered from separate patients hospitalized in four hospitals located in different Greek regions. K. pneumoniae ATCC 13883, Pseudomonas aeruginosa ATCC 27853, ...