The difficult-to-control spread of carbapenemase producers among Enterobacteriaceae worldwide

The spread of carbapenemase producers in Enterobacteriaceae has now been identified worldwide. Three main carbapenemases have been reported; they belong to three classes of β-lactamases, which are KPC, NDM, and OXA-48. The main reservoirs of KPC are Klebsiella pneumoniae in the USA, Israel, Greece, and Italy, those of NDM are K. pneumoniae and Escherichia coli in the Indian subcontinent, and those of OXA-48 are K. pneumoniae and Escherichia coli in North Africa and Turkey. KPC producers have been mostly identified among nosocomial isolates, whereas NDM and OXA-48 producers are both nosocomial and community-acquired pathogens. Control of their spread is still possible in hospital settings, and relies on the use of rapid diagnostic techniques and the strict implemention of hygiene measures.     

Rapid evolution and spread of carbapenemases among Enterobacteriaceae in Europe

Plasmid-acquired carbapenemases in Enterobacteriaceae, which were first discovered in Europe in the 1990s, are now increasingly being identified at an alarming rate. Although their hydrolysis spectrum may vary, they hydrolyse most β-lactams, including carbapenems. They are mostly of the KPC, VIM, NDM and OXA-48 types. Their prevalence in Europe as reported in 2011 varies significantly from high (Greece and Italy) to low (Nordic countries). The types of carbapenemase vary among countries, partially depending on the cultural/population exchange relationship between the European countries and the possible reservoirs of each carbapenemase. Carbapenemase producers are mainly identified among Klebsiella pneumoniae and Escherichia coli, and still mostly in hospital settings and rarely in the community. Although important nosocomial outbreaks with carbapenemase-producing Enterobacteriaceae have been extensively reported, many new cases are still related to importation from a foreign country. Rapid identification of colonized or infected patients and screening of carriers is possible, and will probably be effective for prevention of a scenario of endemicity, as now reported for extended-spectrum β-lactamase (mainly CTX-M) producers in all European countries.     

Carbapénèmases de type KPC : quel enjeu en microbiologie clinique ?

Emergence and dissemination of carbapenem resistance in the world represent a significant threat for management of hospital-acquired infections. From the early 2000s, Enterobacteriaceae that produce Klebsiella pneumoniae carbapenemases (KPC) have initially been reported from the USA and now worldwide, becoming the most important carbapenemase. These KPC producing-bacteria are mostly involved in nosocomial and systemic infections. They are mostly Enterobacteriaceae and more rarely Pseudomonas aeruginosa. KPC β-lactamases confer decreased susceptibility or resistance to virtually all β-lactams. Therefore, carbapenems (imipenem, meropenem, ertapenem) may become inefficient for treating enterobacterial infections with KPC-producing bacteria, which are in addition resistant to many other non β-lactam molecules, leaving only few available therapeutic options. Detection of KPC-producing bacteria may be difficult based on routine antibiotic susceptibility testing. Several phenotypic tests have been proposed, but until now, only molecular methods are reliable techniques for their identification. It is therefore critical to implement efficient infection control measures to detect patients who are colonized or infected with these pathogens in order to limit their spread.     

Evaluation of a DNA microarray (Check-MDR CT102) for rapid detection of TEM, SHV, and CTX-M extended-spectrum β-lactamases and of KPC, OXA-48, VIM, IMP, and NDM-1 carbapenemases

The Check-MDR CT102 microarray, aimed at identifying bacteria producing extended-spectrum β-lactamase (ESBL) (SHV, TEM, and CTX-M) and carbapenemase (KPC, OXA-48, VIM, IMP, and NDM-1), was evaluated on a total of 144 Gram-negative strains expressing various β-lactamases. The sensitivity and specificity were 100% for most tested genes, suggesting that this assay allows accurate identification of common ESBL and carbapenemase producers from bacterial cultures.     

Modified CLSI Extended-Spectrum β-Lactamase (ESBL) Confirmatory Test for Phenotypic Detection of ESBLs among Enterobacteriaceae Producing Various β-Lactamases

The worldwide dissemination of Enterobacteriaceae producing AmpC β-lactamases and carbapenemases makes difficult the phenotypic detection of extended-spectrum β-lactamases (ESBLs), as they may be masked by these additional enzymes. A modification of the CLSI ESBL confirmatory test was developed and evaluated in a comparative study for its ability to successfully detect ESBLs among Enterobacteriaceae producing various carbapenemases (Klebsiella pneumoniae carbapenemase [KPC], VIM, NDM, and OXA-48) and plasmidic or derepressed AmpCs. The modified CLSI ESBL confirmatory test was performed with cefotaxime and ceftazidime disks with and without clavulanate, on which both boronic acid (BA) and EDTA were dispensed. A total of 162 genotypically confirmed ESBL-positive Enterobacteriaceae isolates (83 carbapenemase/ESBL producers, 25 AmpC/ESBL producers, and 54 ESBL-only producers) were examined. For comparison, 139 genotypically confirmed ESBL-negative Enterobacteriaceae isolates (94 of them possessed carbapenemases and 20 possessed AmpCs) were also tested. The standard CLSI ESBL confirmatory test was positive for 106 of the 162 ESBL producers (sensitivity, 65.4%) and showed false-positive results for 4 of the 139 non-ESBL producers (specificity, 97.1%). The modified CLSI ESBL confirmatory test detected 158 of 162 ESBL producers (sensitivity, 97.5%) and showed no false-positive results for non-ESBL producers (specificity, 100%). The findings of the study demonstrate that the modified CLSI ESBL confirmatory test using antibiotic disks containing both BA and EDTA accurately detects ESBLs in Enterobacteriaceae regardless of the coexistence of additional β-lactam resistance mechanisms.     

NDM-1, OXA-48 and OXA-181 carbapenemase-producing Enterobacteriaceae in Sultanate of Oman

Twenty-two carbapenem-resistant enterobacterial isolates were recovered from patients hospitalized between October 2010 and March 2011 at the Royal Hospital of Muscat, Sultanate of Oman. Eleven NDM-1, five OXA-48 and one NDM-1 plus OXA-181 producers of diverse ST types were recovered from clinical samples. All carbapenemase genes were located on self-conjugative plasmids and were nearly always associated with other resistance determinants, including extended-spectrum β-lactamases and the ArmA methylase encoding resistance to aminoglycosides. This work highlights the dissemination of NDM-1 and OXA-48-type producers in the Middle East.     

Faecal carriage of oxyiminocephalosporin-resistant Enterobacteriaceae among paediatric units in different hospitals in the south of France

The aim of this study was to determine the presence of oxyiminocephalosporin-resistant (OCR) Gram-negative bacilli and extended-spectrum β-lactamase (ESBL)-producing isolates in stool specimens obtained from paediatric patients hospitalised for acute diarrhoea. We conducted a prospective, multicentre study over a period of 6 months in seven hospitals in the south of France. Samplings were carried out from infants admitted for acute diarrhoea with no previous antibiotic treatment in the last week. Bacteria in stool specimens were screened for the presence of OCR Gram-negative bacilli on Drigalski agar supplemented with ceftazidime and ESBL CHROMagar® media, and confirmed by the Rosco tablets test. Genetic detection was performed by the Check MDR® microarray and by polymerase chain reaction (PCR) and sequencing with bacterial DNA extracted from isolates. The presence of OCR enterobacteria was markedly high (177/1,118 patients, 15.2 %), with an important community origin (66.1 %). The majority of multidrug-resistant (MDR) bacteria were Enterobacter cloacae (106, 59.9 %) and Escherichia coli (61, 34.5 %). The prevalence of ESBL and CTX-M producers represented 5.2 and 4.3 % of the isolates, respectively. The main proportion of these ESBL carriers was found in children less than 1 year of age (53.4 %). One carbapenemase (IMP-1) was detected. The study revealed the wide dissemination of MDR bacteria in infants attending hospitals in the south of France during a non-outbreak situation, in particular, the spread of cefotaximase and the detection of a carbapenemase. This worrisome situation must reinforce the use of hygiene procedures and appropriate antibiotics to control the emergence and spread of OCR organisms.     

Acquired carbapenemases in Gram-negative bacterial pathogens: detection and surveillance issues

Acquired carbapenemases are emerging resistance determinants in Gram-negative pathogens, including Enterobacteriaceae, Pseudomonas aeruginosa and other Gram-negative non-fermenters. A consistent number of acquired carbapenemases have been identified during the past few years, belonging to either molecular class B (metallo-β-lactamases) or molecular classes A and D (serine carbapenemases), and genes encoding these enzymes are associated with mobile genetic elements that allow their rapid dissemination in the clinical setting. Therefore, detection and surveillance of carbapenemase-producing organisms have become matters of major importance for the selection of appropriate therapeutic schemes and the implementation of infection control measures. As carbapenemase production cannot be simply inferred from the resistance profile, criteria must be established for which isolates should be suspected and screened for carbapenemase production, and for which tests (phenotypic and/or genotypic) should be adopted for confirmation of the resistance mechanism. Moreover, strategies should be devised for surveillance of carbapenemase producers in order to enable the implementation of effective surveillance programmes. The above issues are addressed in this article, as a follow-up to an expert meeting on acquired carbapenemases that was recently organized by the ESCMID Study Group for Antibiotic Resistance Surveillance.     

Controlling False-Positive Results Obtained with the Hodge and Masuda Assays for Detection of Class A Carbapenemase in Species of Enterobacteriaceae by Incorporating Boronic Acid

The modified Hodge method (MHT) has been recommended by the CLSI for confirmation of suspected class A carbapenemase production in species of Enterobacteriaceae. This test and the Masuda method (MAS) have advantages over traditional phenotypic methods in that they directly analyze carbapenemase activity. In order to identify the potential interferences of these tests, we designed a panel composed of diverse bacterial genera with distinct carbapenem susceptibility patterns (42 carbapenemase producers and 48 nonproducers). About 25% of results among carbapenemase nonproducers, mainly strains harboring CTX-M and AmpC hyperproducers, were observed to be false positive. Subsequently, we developed an optimized approach for more-accurate detection of suspicious isolates of carbapenemase by addition of boronic acid (BA) derivatives (reversible inhibitor of class A carbapenemases and AmpC cephalosporinases) and oxacillin (inhibitor of AmpCs enzymes). The use of the modified BA- and oxacillin-based MHT and MAS resulted in high sensitivity (>90%) and specificity (100%) for class A carbapenemase detection. By use of these methodologies, isolates producing KPCs and GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of ß-lactamases (extended-spectrum β-lactamases [ESBLs], AmpC β-lactamases, metallo-β-lactamases [MBLs], etc.). These methods will provide the fast and useful information needed for targeting of antimicrobial therapy and appropriate infection control.Class A carbapenemases (Klebsiella pneumoniae carbapenemases [KPCs], Sme, NMC-A, IMI, and some allelic variants of GES/IBC) have become more prevalent within the Enterobacteriaceae family (23, 25). Early recognition of carbapenemase producers has become mandatory, as clinical failure associated with these enzymes has been described (8). Therefore, different strategies are needed for their detection in any attempt aimed for their control and eradication. An accurate identification of class A carbapenemases will therefore rely on the availability of specific, sensitive, and simple assays able to differentiate carbapenemase producers from nonproducers. The modified Hodge test (MHT) (5) and similar tests, such as the Masuda assay (MAS) (15), directly analyze the carbapenemase activity in unbroken cells and enzyme crude extracts, respectively. These tests preformed better than routine phenotypic methods for detection of carbapenemase-producing bacteria, especially when combined mechanisms were present. Additionally, a practical and fast approach that efficiently disrupts bacterial cells has become available to the routine clinical microbiological laboratory, making the MAS even easier to perform (12). Thus, given these features, these methods started to gain more adherents among microbiologists. Moreover, the CLSI has issued recommendations for phenotypic screening of carbapenemase producers among species of Enterobacteriaceae and included the MHT as a confirmatory assay (5). Thus, MICs of ertapenem (ETP), meropenem (MEM), and imipenem (IPM) of 2, 2 to 4, and 2 to 4 μg/ml, respectively (or an ETP- or MEM-induced zone of inhibition of ≤21 mm in diameter in the disk diffusion assay), may indicate isolates with carbapenemase production, and this phenotype should be confirmed by the MHT (5). However, we have reported false-positive rates of up to 25% for MHT results (carbapenemase-like patterns) among carbapenemase nonproducers (as defined by spectrophotometric assays and molecular methods) (21). These unexpected outcomes were observed among CTX-M-producing bacteria, with uniformly decreased susceptibility to ETP or AmpC hyperproducers. Recent reports have also questioned the specificity (SP) of the MHT, suggesting that AmpC hyperproducers and several members of the CTX-M ESBL family (mostly CTX-M-2 but also CTX-M-15 and CTX-M-59) were the main cause of the unexpected performance (1, 4, 18).Cloxacillin, oxacillin (OXA), and boronic acid (BA) have long been known as reversible inhibitors of class C enzymes (2, 3, 26). More recently, it has been shown that an additional property of the BA derivatives, but not of OXA, is the ability to inhibit KPC enzymes (6, 21, 24) and the remaining members of the class A carbapenemase family (GES, Sme, IMI, and NMC-A) (21). Using a panel composed of diverse bacterial genera with distinct carbapenem susceptibility patterns, we designed strategies to improve the efficiency of the MHT and MAS for phenotypic confirmation of carbapenemase producers among species of Enterobacteriaceae by addition of BA and OXA as inhibitors. The proposed procedures, designated the boronic acid-based modified Hodge test (BA-MHT) plus the oxacillin-based modified Hodge test (OXA-MHT) and the boronic acid-based Masuda assay (BA-MAS) plus the oxacillin-based Masuda assay (OXA-MAS) may be suitable as confirmatory tests, as they displayed high sensitivity (SN) and specificity (SP) for detection of class A carbapenemase producers, in addition to being relatively simple and inexpensive.     

Combined disc methods for the detection of KPC- and/or VIM-positive Klebsiella pneumoniae: improving reliability for the double carbapenemase producers

Klebsiella pneumoniae strains co-producing klebsiella pneumoniae carbapenemase (KPC) and verona integron-encoded metallo-beta-lactamase (VIM) are frequently isolated in Greece and have also occurred in other European countries. Conventional combined disc tests exhibit low sensitivity against these emerging pathogens. We have evaluated modifications of the KPC/Metallo-β-Lactamase Confirmation kit (ROSCO) exhibiting high diagnostic value against KPC, VIM and KPC + VIM producers. The key changes were the inclusion of additional combined tablets containing meropenem plus two inhibitors (dipicolinic acid (1000 μg per tablet) for metallo-β-lactamases and a boronic acid derivative for KPCs) and the replacement of aminophenylboronic acid by phenylboronic acid (400 μg per tablet).     

CarbAcineto NP Test for Rapid Detection of Carbapenemase-Producing Acinetobacter spp.

Multidrug-resistant Acinetobacter baumannii isolates, particularly those that produce carbapenemases, are increasingly reported worldwide. The biochemically based Carba NP test, extensively validated for the detection of carbapenemase producers among Enterobacteriaceae and Pseudomonas spp., has been modified to detect carbapenemase production in Acinetobacter spp. A collection of 151 carbapenemase-producing and 69 non-carbapenemase-producing Acinetobacter spp. were tested using the Carba NP test and a modified Carba NP protocol (the CarbAcineto NP test) in this study. The CarbAcineto NP test requires modified lysis conditions and an increased bacterial inoculum compared to those of the original Carba NP test. The Carba NP test detects metallo-β-lactamase producers but failed to detect the production of other carbapenemase types among Acinetobacter spp. In contrast, the newly designed CarbAcineto NP test, which is rapid and reproducible, detects all types of carbapenemases with a sensitivity of 94.7% and a specificity of 100%. This cost-effective technique offers a reliable and affordable technique for identifying carbapenemase production in Acinetobacter spp., which is a marker of multidrug resistance in those species. Its use will facilitate the recognition of these carbapenemases and prevent their spread.     

Bloodstream infections caused by IMP-8-producing Enterobacteriaceae isolates: the need for clinical laboratory detection of metallo-β-lactamases?

A retrospective study was conducted at a Taiwanese medical center to characterize bloodstream infections caused by IMP-8 metallo-β-lactamase (MBL)-producing Enterobacteriaceae isolates and to assess the need for laboratory detection of IMP producers. We analyzed 37 patients infected with IMP-8 producers (two Escherichia coli, nine Klebsiella pneumoniae, 25 Enterobacter cloacae, and one Citrobacter freundii) and 107 patients infected with non-IMP-8 producers (eight E. coli, 26 K. pneumoniae, 70 E. cloacae, and three C. freundii) that were interpreted as carbapenem-nonsusceptible based on the updated Clinical and Laboratory Standards Institute (CLSI) 2010 guidelines. Only 18 (48.6 %) of the IMP-8 producers were regarded as potential carbapenemase producers based on the CLSI 2012 guidelines. The production of extended-spectrum β-lactamases (ESBLs) was more common in the MBL group (73.0 %) than in the non-MBL group (41.1 %). There were no significant differences in carbapenem susceptibilities, clinical characteristics, carbapenem use for empirical and definitive treatment, and mortality rates between the two groups. Eighteen IMP-8 producers could be deemed as resistant to all carbapenems [minimum inhibitory concentration (MIC) of any carbapenem ≥2 μg/mL]; patients with these isolates had a lower, but non-significant, 28-day mortality rate (27.8 %) than patients infected with non-MBL producers having similar carbapenem MICs (39.0 %) (p?=?0.41). A multivariate analysis revealed severity of acute illness as the single independent variable associated with both 7-day and 28-day mortality rates (p?<?0.01) for infections caused by Enterobacteriaceae with decreased carbapenem susceptibilities. Our findings suggest that the clinical detection of IMP-producing Enterobacteriaceae is not required even when the “old” CLSI criteria are used.     

Rapid Detection of β-Lactamase-Hydrolyzing Extended-Spectrum Cephalosporins in Enterobacteriaceae by Use of the New Chromogenic βLacta Test

The chromogenic βLacta test developed for the rapid detection of β-lactamase-hydrolyzing extended-spectrum cephalosporins in Enterobacteriaceae revealed good performance with extended-spectrum β-lactamase (ESBL) producers (97.5% true-positive results). However, false-negative results occurred with chromosomal AmpC hyperproducers and plasmid AmpC producers, whereas uninterpretable results were mostly due to VIM-1 carbapenemase producers and possibly low levels of expressed ESBLs.     

Use of Faropenem as an Indicator of Carbapenemase Activity in the Enterobacteriaceae

The aim of this study was to determine the ability of a disc susceptibility test using faropenem (10 μg) to predict carbapenemase activity in Enterobacteriaceae. A collection of 166 isolates of carbapenemase-producing Enterobacteriaceae (CPE) and 82 isolates of Enterobacteriaceae that produced other β-lactamases was compiled from diverse sources. Disc susceptibility testing was performed using the CLSI/EUCAST methodology with discs of faropenem (10 μg), temocillin (30 μg), and four carbapenems (each 10 μg). A further prospective evaluation of the faropenem disc susceptibility test was performed using 205 consecutive isolates referred to a United Kingdom reference laboratory in parallel with molecular methods for carbapenemase detection. Of 166 isolates of CPE, 99% showed growth up to the edge of a 10-μg faropenem disc compared with only 6% of other β-lactamase producers (sensitivity, 99%; specificity, 94%). A “double zone” around 10-μg faropenem discs was frequently associated with OXA-48 producers. Of the carbapenems, the most useful agent was imipenem, where a zone diameter of ≤23 mm as a predictor of carbapenemase activity had a sensitivity of 99% and a specificity of 85%. The presence of no zone of inhibition around a 30-μg temocillin disc was a consistent feature of strains producing OXA-48 carbapenemase. For 205 isolates of Enterobacteriaceae referred to a United Kingdom reference laboratory, growth up to a 10-μg faropenem disc correctly identified 84 of 86 carbapenemase producers (98% sensitivity), with a specificity of 87%. Disc susceptibility testing using faropenem (10 μg) is a simple, convenient, and highly predictive screening test for carbapenemase-producing Enterobacteriaceae.     

How to detect NDM-1 producers

Enterobacterial isolates expressing the carbapenemase NDM-1 are emerging worldwide. Twenty-seven NDM-1-positive isolates of worldwide origin were included in this study to identify these strains as not only pathogens but also colonizers of normal flora for infection control screening. Although susceptibility to carbapenems varied, a combined test (IMP/IMP + EDTA), the Etest MBL, and automated susceptibility testing by Vitek2 (bioMérieux) identified those NDM-1 producers as verified by PCR using specific primers. Screening for carriers of NDM-1 producers may be based on media such as the ChromID ESBL culture medium routinely used to screen for extended-spectrum β-lactamase producers, which gives excellent detection levels with low limits of detection ranging from 8 × 10(0) to 5 × 10(2) CFU/ml. The CHROMagar KPC culture medium had higher limits of detection (1 × 10(1) to 5 × 10(5) CFU/ml) and may be proposed for the follow-up of outbreaks of infections with NDM-1 producers. Colonies growing on these screening media can be verified as NDM-1 producers with molecular methods as described herein.     

Simple disk-based method for detection of Klebsiella pneumoniae carbapenemase-type beta-lactamase by use of a boronic acid compound

A disk potentiation method using carbapenems as substrates and 3-aminophenyl boronic acid as an inhibitor was evaluated for the detection of Klebsiella pneumoniae carbapenemase (KPC)-type β-lactamases. When combined with nonsusceptibility to ertapenem, the method was easy to perform and reliably differentiated isolates producing KPC-type β-lactamases from those producing other types of β-lactamases.     

Evolution and epidemiology of extended-spectrum β-lactamases (ESBLs) and ESBL-producing microorganisms

The rapid and irrepressible increase in antimicrobial resistance of pathogenic bacteria that has been observed over the last two decades is widely accepted to be one of the major problems of human medicine today. Several aspects of this situation are especially worrying. There are resistance mechanisms that eliminate the use of last-choice antibiotics in the treatment of various kinds of infection. Many resistance mechanisms that emerge and spread in bacterial populations are those of wide activity spectra, which compromise all or a majority of drugs belonging to a given therapeutic group. Some mechanisms of great clinical importance require specific detection procedures, as they may not confer clear resistance in vitro on the basis of the interpretive criteria used in standard susceptibility testing. Finally, multiple mechanisms affecting the same and/or different groups of antimicrobials coexist and are even co-selected in more and more strains of pathogenic bacteria. The variety of β -lactamases with wide spectra of substrate specificity illustrates very well all the phenomena mentioned above. Being able to hydrolyze the majority of β -lactams that are currently in use, together they constitute the most important resistance mechanism of Gram-negative rods. Three major groups of these enzymes are usually distinguished, class C cephalosporinases (AmpC), extended-spectrum β -lactamases (ESBLs) and different types of β -lactamases with carbapenemase activity, of which the so-called class B metallo- β -lactamases (MBLs) are of the greatest concern. This review is focused on various aspects of the evolution and epidemiology of ESBLs; it does not cover the problems of ESBL detection and clinical relevance of infections caused by ESBL-producing organisms.     

Comparison of Three Biochemical Tests for Rapid Detection of Extended-Spectrum-β-Lactamase-Producing Enterobacteriaceae

Enterobacterial isolates producing clavulanic-inhibited extended-spectrum β-lactamases (ESBLs) are increasingly spreading in the community and are often responsible for nosocomial infections. Rapid biochemical tests have been developed recently for their detection. Three tests, namely, the Rapid ESBL NDP test, the β-Lacta test, and the Rapid ESBL Screen, have been evaluated with a collection of 108 well-characterized strains, including wild-type strains, strains producing ESBLs, overexpressed cephalosporinases, and carbapenemases. The ESBL NDP test and the Rapid ESBL Screen (a copy of the ESBL NDP test) are aimed at detecting ESBL producers, while the β-Lacta test is aimed at detecting not only ESBL producers but also cephalosporinase and carbapenemase producers. The sensitivity and specificity for detecting ESBL producers (n = 60) were 95% and 100% for the Rapid ESBL NDP test, 80% and 87% (after 30 min) and 92% and 83% (after 2 h) for the Rapid ESBL Screen, and 88% and 71% for the β-Lacta test, respectively. Varied and time-consuming detection (up to 2 h) of ESBLs by the Rapid ESBL Screen and concomitant and varied detection of producers of AmpC and several types of carbapenemases correspond to significant shortcomings of using the Rapid Screen ESBL and β-Lacta tests, respectively.     

Evaluation of Boronic Acid Disk Tests for Differentiating KPC-Possessing Klebsiella pneumoniae Isolates in the Clinical Laboratory

The worldwide increase in the occurrence and dissemination of KPC β-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 β-lactamases, metallo-β-lactamases, and plasmid-mediated AmpC β-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 μg 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 false-positive 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 β-lactamases are widespread.