Emerging carbapenemases in Gram-negative aerobes

Carbapenemases may be defined as β -lactamases that significantly hydrolyze at least imipenem or/and meropenem. Carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. Class A, clavulanic acid-inhibited carbapenemases are rare. They are either chromosomally encoded (NMC-A, Sme-1 to Sme-3, IMI-1) in Enterobacter cloacae and Serratia marcescens , or plasmid encoded, such as KPC-1 in Klebsiella pneumoniae and GES-2 in Pseudomonas aeruginosa , the latter being a point-mutant of the clavulanic acid-inhibited extended-spectrum β -lactamase GES-1. The class B enzymes are the most clinically significant carbapenemases. They are metalloenzymes of the IMP or VIM series. They have been reported worldwide but mostly from South East Asia and Europe. Metalloenzymes, whose genes are plasmid and integron located, hydrolyze virtually all β -lactams except aztreonam. Finally, the class D carbapenemases are increasingly reported in Acinetobacter baumannii but compromise imipenem and meropenem susceptibility only marginally. The sources of the acquired carbapenemase genes remain unknown, as does the relative importance of the spread of epidemic strains as opposed to the spread of plasmid- or integron-borne genes. Because most of these carbapenemases confer only reduced susceptibility to carbapenems in Enterobacteriaceae, they may remain underestimated as a consequence of the lack of their detection.     

Occurrence and characterization of carbapenemase-producing Enterobacteriaceae: report from the SENTRY Antimicrobial Surveillance Program (2000-2004)

Emergence and dissemination of Enterobacteriaceae isolates harboring carbapenemases in various geographic regions represents a significant threat to the management of nosocomial infections. Enterobacteriaceae isolates from the SENTRY Antimicrobial Surveillance Program (2000-2004) demonstrating decreased susceptibility to imipenem and meropenem (minimum inhibitory concentration [MIC], > or =2 mg/L) were evaluated for the production of metallo-beta-lactamases and serine carbapenemases using disk approximation and polymerase chain reaction (PCR) tests. Carbapenemase-producing strains were epidemiologically typed by automated riboprinting and pulsed-field gel electrophoresis (PFGE) to establish clonality. Among 37,557 Enterobacteriaceae (5 genus groups) evaluated, 119 (0.32%) had increased carbapenem MIC values, and a carbapenemase was identified in 51 (42.9%) of these strains. KPC-2 and KPC-3 were the most frequently occurring carbapenemases (24 isolates, 20.2%) in the United States and were detected in Klebsiella spp, Citrobacter spp., Enterobacter spp., and Serratia marcescens strains isolated in New York, Arkansas, and Virginia. SME-2-producing S. marcescens were isolated in the New York City area, Texas, and Ohio, while NMC-A was found in one E. cloacae strain from New York. In contrast, metallo-beta-lactamases were prevalent in Europe. IMP-1-producing E. cloacae (11 isolates) were detected in Turkey, while VIM-1-producing strains were found in Italy (Enterobacter spp.) and Greece (Klebsiella pneumoniae). Clonal dissemination of carbapenemase-producing strains was observed in several medical centers on both continents. The occurrence of carbapenemases in various Enterobacteriaceae remains rare but appears to be spreading geographically (not in Latin America), mainly with metallo-beta-lactamases being found in Mediterranean Europe and KPC enzymes in the New York City area.     

A disc diffusion assay for detection of class A,B and OXA-48 carbapenemases in Enterobacteriaceae using phenyl boronic acid,dipicolinic acid and temocillin

Class A and B carbapenemases in Enterobacteriaceae may be detected using carbapenemase inhibition tests with boronic acid derivatives (BA) and dipicolinic acid (DPA)/EDTA, respectively. However, for OXA-48 (like) carbapenemases, no specific inhibitor is available. Because OXA-48 confers high-level temocillin resistance, a disc diffusion assay using temocillin as well as BA and DPA inhibition tests was evaluated for detection of class A, B and OXA-48 carbapenemases. The test collection included 128 well-characterized non-repeat Enterobacteriaceae isolates suspected of carbapenemase production; that is, with meropenem MICs ≥ 0.5 mg/L, including 99 carbapenemase producers (36 KPC, one GES, 31 MBL, four KPC plus VIM, 25 OXA-48, two OXA-162), and 29 ESBL and/or AmpC-producing isolates. PCR and sequencing of beta-lactamase genes was used as a reference test. Phenotypic carbapenemase detection was performed with discs (Rosco) containing meropenem (10 μg), temocillin (30 μg), meropenem + phenyl boronic acid (PBA), meropenem + DPA, meropenem + BA + DPA, and meropenem + cloxacillin (CL). Absence of synergy between meropenem and BA and/or DPA and a temocillin zone ≤10 mm was used to identify OXA-48. The sensitivity for identification of class A, B and OXA-48 carbapenemases was 95%, 90% and 100%, with 96–100% specificity. In non-Proteus species, the sensitivity for class B carbapenemase detection was 97%. All isolates without PBA or DPA synergy and a temocillin disc zone ≤10 mm were OXA-48 (like) positive. In conclusion, carbapenemase inhibition tests with PBA and DPA combined with a temocillin disc provide a reliable phenotypic confirmation method for class A, B and OXA-48 carbapenemases in Enterobacteriaceae.     

Prevalence of newer beta-lactamases in gram-negative clinical isolates collected in the United States from 2001 to 2002

Newer beta-lactamases such as extended-spectrum beta-lactamases (ESBLs), transferable AmpC beta-lactamases, and carbapenemases are associated with laboratory testing problems of false susceptibility that can lead to inappropriate therapy for infected patients. Because there appears to be a lack of awareness of these enzymes, a study was conducted during 2001 to 2002 in which 6,421 consecutive, nonduplicate clinical isolates of aerobically growing gram-negative bacilli from patients at 42 intensive care unit (ICU) and 21 non-ICU sites across the United States were tested on-site for antibiotic susceptibility. From these isolates, 746 screen-positive isolates (11.6%) were referred to a research facility and investigated to determine the prevalence of ESBLs in all gram-negative isolates, transferable AmpC beta-lactamases in Klebsiella pneumoniae, and carbapenemases in Enterobacteriaceae. The investigations involved phenotypic tests, isoelectric focusing, beta-lactamase inhibitor studies, spectrophotometric assays, induction assays, and molecular analyses. ESBLs were detected only in Enterobacteriaceae (4.9% of all Enterobacteriaceae) and were found in species other than those currently recommended for ESBL testing by the CLSI (formerly NCCLS). These isolates occurred at 74% of the ICU sites and 43% of the non-ICU sites. Transferable AmpC beta-lactamases were detected in 3.3% of K. pneumoniae isolates and at 16 of the 63 sites (25%) with no difference between ICU and non-ICU sites. Three sites submitted isolates that produced class A carbapenemases. No class B or D carbapenemases were detected. In conclusion, organisms producing ESBLs and transferable AmpC beta-lactamases were widespread. Clinical laboratories must be able to detect important beta-lactamases to ensure optimal patient care and infection control.     

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.     

Sensitive Screening Tests for Suspected Class A Carbapenemase Production in Species of Enterobacteriaceae

The detection of class A serine-carbapenemases among species of Enterobacteriaceae remains a challenging issue. Methods of identification for routine use in clinical microbiology laboratories have not been standardized to date. We developed a novel screening methodology suitable for countries with high basal levels of carbapenem resistance due to non-carbapenemase-mediated mechanisms and standardized several simple confirmatory methods that allow the recognition of bacteria producing class A carbapenemases, including KPC, Sme, IMI, NMC-A, and GES, by using boronic acid (BA) derivatives. A total of 28 genetically unrelated Enterobacteriaceae strains producing several class A carbapenemases were tested. Thirty-eight genetically unrelated negative controls were included. The isolates were tested against imipenem (IPM), meropenem (MEM), and ertapenem (ETP) by MIC and disk diffusion assays in order to select appropriate tools to screen for suspected carbapenemase production. It was possible to differentiate class A carbapenemase-producing bacteria from non-carbapenemase-producing bacteria by using solely the routine IPM susceptibility tests. The modified Hodge test was evaluated and found to be highly sensitive, although false-positive results were documented. Novel BA-based methods (a double-disk synergy test and combined-disk and MIC tests) using IPM, MEM, and ETP, in combination with 3-aminophenylboronic acid as an inhibitor, were designed as confirmatory tools. On the basis of the performance of these methods, a sensitive flow chart for suspicion and confirmation of class A carbapenemase production in species of Enterobacteriaceae was designed. By using this methodology, isolates producing KPC, GES, Sme, IMI, and NMC-A carbapenemases were successfully distinguished from those producing other classes of β-lactamases (extended-spectrum β-lactamases, AmpCs, and metallo-β-lactamases, etc). These methods will rapidly provide useful information needed for targeting antimicrobial therapy and appropriate infection control.Class A carbapenemases (KPCs, Sme, NMC-A, IMI, and some allelic variants of GES/IBC) have become more prevalent within the Enterobacteriaceae family (30, 33). Early recognition of producers of carbapenemases has become mandatory, as clinical failure associated with these enzymes has been described previously (12) and as recognition is crucial for controlling the spread of carbapenemase-producing bacteria. A uniform and standardized phenotypic tool for the detection of class A carbapenemases is still lacking. Recently, the CLSI issued recommendations for the phenotypic screening of carbapenemase producers among species of Enterobacteriaceae: MICs of ertapenem (ETP), meropenem (MEM), and imipenem (IPM) of 2, 2 to 4, and 2 to 4 μg/ml, respectively (or a zone of inhibition by ETP or MEM of ≤21 mm in diameter in the disk diffusion [DD] assay), may indicate isolates with carbapenemase production, and this phenotype should be confirmed by the Hodge method (5). In Argentina, large proportions of nosocomial Enterobacteriaceae strains meet the CLSI criteria for suspected carbapenemase production according to ETP and MEM DD assay results (about 14 and 5% of 6,700 tested strains, respectively, as reported by the WHONET-Argentina Network in 2007 (http://www.paho.org). Up to one-third of these isolates had a positive Hodge test result suggestive of carbapenemase production; still, almost all of them did not produce carbapenemases as determined by molecular methods (27). Therefore, in this work, we designed a panel composed of diverse bacterial genera with distinct carbapenem susceptibility patterns to identify potential problems in the current recommendations. In addition, we developed an optimized approach for the more accurate detection of isolates with possible carbapenemase production, suitable for countries in which strains have high baseline levels of resistance to carbapenems due to non-carbapenemase-mediated mechanisms, such as those resulting from the combination of extended-spectrum β-lactamases or AmpCs and porin mutations (2, 10, 15, 34).Boronic acids (BA) were reported in the early 1980s to be reversible inhibitors of class C enzymes (1). Unexpectedly, we discovered unusual inhibition of organisms possessing KPC by 3-aminophenylboronic acid (APB), which may be attributed solely to the presence of this carbapenemase (25). Afterwards, Doi et al. and, in a subsequent study with a larger series of strains, Tsakris et al. demonstrated the overwhelming effectiveness of APB for the detection of Klebsiella pneumoniae possessing KPC (7, 32). Given these promising results, we decided to explore the potential of APB methods as confirmatory tests for class A carbapenemase detection. To date, information on the efficiency of APB methods for KPC-possessing bacterial species different from K. pneumoniae is missing. Moreover, boronic methods for carbapenemase detection described in the literature so far have been limited to KPC enzymes and have not been evaluated for the remaining class A members. Thus, we examined the capabilities of BA-based methods to detect the entire class A carbapenemase family by using these methods to test a panel of well-characterized strains, including several bacterial hosts with KPC and also GES-, Sme-, IMI-, and NMC-A-producing bacteria.Thus, in this work we propose novel and accurate phenotypic methods, appropriate for clinical laboratories, to both screen for and confirm the presence of class A carbapenemases in species of Enterobacteriaceae.     

Nosocomial spread of Pseudomonas aeruginosa isolates expressing the metallo-beta-lactamase VIM-2 in a hematology unit of a French hospital

Dissemination of metallo-beta-lactamases (carbapenemases) was investigated retrospectively among ceftazidimeand imipenem-resistant Pseudomonas aeruginosa isolates in a hematology unit in Marseilles, France, from September, 1995, to March, 1999. Sixteen clinical isolates and 23 environmental strains were identified, with a same bla (VIM-2) gene that encoded a carbapenemase identified in Southern Europe and South Korea. Five different genotypes were identified among clinical and environmental P. aeruginosa isolates all harboring an approximately 45-kb plasmid with bla (VIM-2)-positive class 1 integrons varying in structures. This study identified a hidden reservoir of carbapenemase producers.     

The sequences of seven class D β-lactamases isolated from carbapenem-resistant Acinetobacter baumannii from four continents

Carbapenem resistance associated with class D beta-lactamases is an increasing problem in Acinetobacter baumannii. Most enzymes of this class reported so far belong to two subgroups, 1 and 2; however, a novel class D carbapenemase (OXA-51) has been reported recently which shares 56% and < 63% amino-acid identity with subgroups 1 and 2, respectively, and which belongs to a third subgroup. This study describes a further seven novel subgroup 3 beta-lactamases in carbapenem-resistant A. baumannii isolates from four continents.     

Evaluation of a New Phenotypic OXA-48 Disk Test for Differentiation of OXA-48 Carbapenemase-Producing Enterobacteriaceae Clinical Isolates

The current phenotypic methods for detecting carbapenemase-producing Enterobacteriaceae (CPE) allow differentiation between class A and B carbapenemases, but they cannot confirm in a single test class D OXA-48 carbapenemase producers. In this study, we evaluated a new phenotypic test, the OXA-48 disk test, which is based on an imipenem disk and two blank disks adjacent to the imipenem disk, loaded with the tested strain and impregnated with EDTA and EDTA plus phenyl boronic acid (PBA), respectively. The evaluation of the OXA-48 disk test was performed with 81 genotypically confirmed OXA-48-type-producing Enterobacteriaceae isolates (41 extended-spectrum β-lactamase [ESBL] producers, 3 AmpC producers, and 37 non-ESBL, non-AmpC producers). To measure the specificity of the test, 173 genotypically confirmed OXA-48-negative Enterobacteriaceae isolates (57 Klebsiella pneumoniae carbapenemase [KPC] producers, 34 VIM producers, 23 KPC/VIM producers, 22 NDM producers, and 37 AmpC or ESBL producers and porin deficient) that were nonsusceptible to at least one carbapenem were chosen for testing. Using the imipenem disk and the distortion of the inhibition halo around both blank disks containing EDTA and EDTA/PBA, the test differentiated all but 3 of the 81 OXA-48 producers (sensitivity of 96.3%). The test was negative for OXA-48 production in all but 4 of the 173 carbapenem-nonsusceptible isolates producing other carbapenemases, AmpCs, or ESBLs (specificity of 97.7%). This evaluation shows that the OXA-48 disk test is an accurate phenotypic method for the direct differentiation of OXA-48-producing Enterobacteriaceae. Its use along with combined disk tests employing inhibitor-supplemented carbapenem disks might allow the differentiation of the currently known carbapenemase types in Enterobacteriaceae species and provide important infection control information.     

Carbapenemases‐producing Klebsiella pneumoniae in hospitals of two regions of Southern Italy

Carbapenem‐resistant Klebsiella pneumoniae infections are reported with increasing frequency elsewhere in the world, representing a worrying phenomenon for global health. In Italy, there are hotspot data on the diffusion and type of carbapenemase‐producing Enterobacteriaceae and K. pneumoniae in particular, with very few data coming from Apulia and Basilicata, two regions of Southern Italy. This study was aimed at characterizing by phenotypic and genotypic methods carbapenem‐resistant K. pneumoniae isolated from several Hospitals of Apulia and Basilicata, Southern Italy. Antibiotic susceptibility was also evaluated. The relatedness of carbapenemase‐producing K. pneumoniae strains was established by pulsed‐field gel electrophoresis (PFGE). Among the 150 K. pneumoniae carbapenemase producers, KPC‐3 genotype was the most predominant (95%), followed by VIM‐1 (5%). No other genotypes were found and no co‐presence of two carbapenemase genes was found. A full concordance between results obtained by both the phenotypic and the genotypic tests was observed. All strains were resistant to β‐lactam antibiotics including carbapenems, and among antibiotics tested, only tetracycline and gentamycin showed low percentage of resistance (18% and 15%, respectively). Resistance to colistin was detected in 17.3% of strains studied. The analysis of PFGE profiles of the carbapenemases‐positive strains shows that one group (B) of the five (A to E) main groups identified was the most prevalent and detected in almost all the hospitals considered, while the other groups were randomly distributed. Three different sequence types (ST 307, ST 258, and ST 512) were detected with the majority of isolates belonging to the ST 512. Our results demonstrated the wide diffusion of K. pneumoniae KPC‐3 in the area considered, the good concordance between phenotypic and genotypic tests. Gentamicin and colistin had a good activity against these strains.     

Biological assay for the detection of metallo-beta-lactamases in Bacteroides fragilis.

A biological assay was developed for the detection of carbapenemases, particularly metallo-beta-lactamases, in Bacteroides fragilis. The isolates tested possessed the gene (cfiA) responsible for metallo-beta-lactamase production, and showed reduced susceptibility to imipenem. Carbapenemase activity was investigated spectrophotometrically and by a biological assay in which sonicates of bacterial cells were mixed with imipenem in wells cut into Isosensitest agar inoculated with an Escherichia coli indicator organism. After incubation, zones of inhibition were measured. Reductions in zone size compared to a beta-lactamase-negative control, indicating carbapenemase production, were observed with all strains that exhibited hydrolysis of imipenem when measured spectrophotometrically, and with one isolate in which activity was not detected by spectrophotometry. Inclusion of EDTA in the well mixtures abolished the reduction in zone size, indicating the presence of metallo-beta-lactamase. This simple method can detect weak carbapenemase activity that may be overlooked by spectrophotometry.     

A possible alternative to the error prone modified Hodge test to correctly identify the carbapenemase producing Gram-negative bacteria

Context: The modified Hodge test (MHT) is widely used as a screening test for the detection of carbapenemases in Gram-negative bacteria. This test has several pitfalls in terms of validity and interpretation. Also the test has a very low sensitivity in detecting the New Delhi metallo-β-lactamase (NDM). Considering the degree of dissemination of the NDM and the growing pandemic of carbapenem resistance, a more accurate alternative test is needed at the earliest. Aims: The study intends to compare the performance of the MHT with the commercially available Neo-Sensitabs - Carbapenemases/Metallo-β-Lactamase (MBL) Confirmative Identification pack to find out whether the latter could be an efficient alternative to the former. Settings and Design: A total of 105 isolates of Klebsiella pneumoniae resistant to imipenem and meropenem, collected prospectively over a period of 2 years were included in the study. Subjects and Methods: The study isolates were tested with the MHT, the Neo-Sensitabs - Carbapenemases/MBL Confirmative Identification pack and polymerase chain reaction (PCR) for detecting the bla_NDM-1 gene. Results: Among the 105 isolates, the MHT identified 100 isolates as carbapenemase producers. In the five isolates negative for the MHT, four were found to produce MBLs by the Neo-Sensitabs. The Neo-Sensitabs did not have any false negatives when compared against the PCR. Conclusions: The MHT can give false negative results, which lead to failure in detecting the carbapenemase producers. Also considering the other pitfalls of the MHT, the Neo-Sensitabs - Carbapenemases/MBL Confirmative Identification pack could be a more efficient alternative for detection of carbapenemase production in Gram-negative bacteria.     

Evaluation of the New NucliSENS EasyQ KPC test for rapid detection of Klebsiella pneumoniae carbapenemase genes (blaKPC)

KPC-type carbapenemases are emerging in Klebsiella pneumoniae and other Gram-negative pathogens worldwide. Rapid and sensitive detection of these resistance determinants has become relevant to clinical management and infection control. We evaluated the bioMérieux EasyQ real-time PCR assay for bla(KPC) detection with 300 members of the Enterobacteriaceae, including 29 control strains producing known carbapenemases and 271 nonreplicate clinical isolates. The EasyQ assay correctly detected all of the 111 isolates harboring bla(KPC) genes, with no false positives, and results were available within 2 h.     

A simple test for the detection of KPC and metallo-β-lactamase carbapenemase-producing Pseudomonas aeruginosa isolates with the use of meropenem disks supplemented with aminophenylboronic acid,dipicolinic acid and cloxacillin

We evaluated the ability of the combination disk test (CDT) and the Modified Hodge Test (MHT) to discriminate between various carbapenemase-producing Pseudomonas aeruginosa isolates (KPC, n = 36; metallo-β-lactamase (MBL), n = 38) and carbapenemase non-producers (n = 75). For the CDT, the optimal inhibitor concentrations and cut-off values were: 600 µg of 3-aminophenylboronic acid (APB) per disk (an increment of ≥4 mm), 1000 µg of dipicolinic acid (DPA) per disk (an increment of ≥5 mm) and 3000 µg of cloxacillin per disk (an increment of ≥3 mm). APB had excellent sensitivity (97%) and specificity (97%) for the detection of KPC enzymes. DPA detected MBL enzymes with a sensitivity and specificity of 97% and 81%, respectively. The MHT resulted in a low sensitivity (78%) and specificity (57%). The CDT could be very useful in daily practice to provide fast and reliable detection of KPC and MBL carbapenemases among P. aeruginosa isolates.     

Rapid emergence of blaCTX-M among Enterobacteriaceae in U.S. Medical Centers: molecular evaluation from the MYSTIC Program (2007)

A total of 220 gram-negative isolates showing distinct beta-lactam resistance profiles recovered in U.S. medical centers during the MYSTIC Program 2007 were evaluated to determine the presence of selected beta-lactamase genes. CTX-M-encoding genes, considered rare in the United States, were detected in 38.8% (28/70; three species) of the extended spectrum beta-lactamase-positive isolates and were observed in 80.0% of the participating hospitals. CTX-M-14 and -15 were found in multiple institutions (eight and nine medical centers, respectively), and CTX-M-3 was detected in only one isolate. The OXA-2 and -10 were identified in nine Enterobacteriaceae strains, and plasmid-mediated AmpC enzymes CMY-2 and FOX-5 were identified in six and four isolates, respectively, displaying negative clavulanate inhibition. Genes encoding OXA-23 and -24 were detected in 30.0% (15/50) of carbapenem-resistant Acinetobacter spp. strains. Retrospective sampling showed that these OXA enzymes were present since 2004 in the MYSTIC Program isolates. The KPC serine carbapenemases were observed in the majority of the carbapenem-resistant Enterobacteriaceae (usually Klebsiella pneumoniae), confirming an epidemic problem in the New York City area. The association of beta-lactamase production and transferable quinolone resistance genes (qnr; 6.7%) in Enterobacteriaceae strains was higher than previously reported. This study illustrates the emergence and rapid dissemination of some beta-lactamases, such as CTX-M, broad-spectrum oxacillinases, and serine carbapenemases, that compromised the treatment of gram-negative infections in numerous U.S. hospitals participating in the MYSTIC Program in 2007.     

Evaluation of the NucliSENS EasyQ KPC Assay for Detection of Klebsiella pneumoniae Carbapenemase-Producing Enterobacteriaceae

The NucliSENS EasyQ KPC assay (bioMérieux SA, Marcy l''Etoile, France) was compared with a routinely used phenotypic method for detection of Enterobacteriaceae producing Klebsiella pneumoniae carbapenemase (KPC)-type carbapenemases, using 806 stool samples and rectal swabs. Compared with the phenotypic method, the EasyQ KPC assay had a sensitivity and specificity of 93.3% and 99.0%, respectively, in this setting, with diverse KPC producers not limited to ST258 Klebsiella pneumoniae.     

Carbapenem resistance in Acinetobacter baumannii: mechanisms and epidemiology

The increasing trend of carbapenem resistance in Acinetobacter baumannii worldwide is a concern since it limits drastically the range of therapeutic alternatives. Metallo-beta-lactamases (VIM, IMP, SIM) have been reported worldwide, especially in Asia and western Europe, and confer resistance to all beta-lactams except aztreonam. The most widespread beta-lactamases with carbapenemase activity in A. baumannii are carbapenem-hydrolysing class D beta-lactamases (CHDLs) that are mostly specific for this species. These enzymes belong to three unrelated groups of clavulanic acid-resistant beta-lactamases, represented by OXA-23, OXA-24 and OXA-58, that can be either plasmid- or chromosomally-encoded. A. baumannii also possesses an intrinsic carbapenem-hydrolysing oxacillinase, the expression of which may vary, that may play a role in carbapenem resistance. In addition to beta-lactamases, carbapenem resistance in A. baumannii may also result from porin or penicillin-binding protein modifications. Several porins, including the 33-kDa CarO protein, that constitute a pore channel for influx of carbapenems, might be involved in carbapenem resistance.     

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.