Serine Carbapenemases Research Articles

Frequency of BKC-1-Producing Klebsiella Species Isolates.

BKC-1 is a new class A serine carbapenemase that was recently identified in Klebsiella pneumoniae clinical isolates. The principal objective of this study was to evaluate the frequency of blaBKC-1 by testing a collection of Klebsiella isolates. Only 2 of 635 Klebsiella isolates (0.3%) carried blaBKC-1 The two BKC-1-producing isolates belonged to clonal complex 442 and possessed identical pulsed-field gel electrophoresis patterns. The blaBKC-1 gene was inserted into a 10-kb plasmid that was identical to the previously reported plasmid, p60136. The BKC-producing K. pneumoniae isolates presented also possessed other mechanisms for beta-lactam resistance, such as genes encoding extended-spectrum beta-lactamases and mutations in the genes ompK35 and ompK36, encoding the major porins.

Activity of Meropenem Combined with RPX7009, a Novel β-Lactamase Inhibitor, against Gram-Negative Clinical Isolates in New York City

Multidrug-resistant Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae are endemic to hospitals in New York City and other regions. RPX7009 is a novel β-lactamase inhibitor with activity against serine carbapenemases. We tested the activity of meropenem plus RPX7009 against 4,500 recent Gram-negative clinical isolates from 11 New York City hospitals. The meropenem-RPX7009 combination was found to have excellent in vitro activity against Escherichia coli, K. pneumoniae, and Enterobacter spp., including multidrug-resistant (MDR) KPC-producing strains. Overall, 131/133 (98.5%) KPC-producing Enterobacteriaceae strains were inhibited by meropenem (≤1 μg/ml) plus RPX7009 (8 μg/ml). In a limited number of strains, the combination appeared to have reduced activity against KPC-producing K. pneumoniae isolates with diminished ompK35 and ompK36 expression. The addition of RPX7009 did not affect the activity of meropenem against Acinetobacter baumannii and Pseudomonas aeruginosa. The meropenem-RPX7009 combination shows promise as a novel agent against KPC-producing Enterobacteriaceae and deserves further study. Other approaches will be needed to address multidrug-resistant A. baumannii and P. aeruginosa, which typically possess different mechanisms of carbapenem resistance.

Discovery of a Cyclic Boronic Acid β-Lactamase Inhibitor (RPX7009) with Utility vs Class A Serine Carbapenemases.

The increasing dissemination of carbapenemases in Gram-negative bacteria has threatened the clinical usefulness of the β-lactam class of antimicrobials. A program was initiated to discover a new series of serine β-lactamase inhibitors containing a boronic acid pharmacophore, with the goal of finding a potent inhibitor of serine carbapenemase enzymes that are currently compromising the utility of the carbapenem class of antibacterials. Potential lead structures were screened in silico by modeling into the active sites of key serine β-lactamases. Promising candidate molecules were synthesized and evaluated in biochemical and whole-cell assays. Inhibitors were identified with potent inhibition of serine carbapenemases, particularly the Klebsiella pneumoniae carbapenemase (KPC), with no inhibition of mammalian serine proteases. Studies in vitro and in vivo show that RPX7009 (9f) is a broad-spectrum inhibitor, notably restoring the activity of carbapenems against KPC-producing strains. Combined with a carbapenem, 9f is a promising product for the treatment of multidrug resistant Gram-negative bacteria.

Chromogenic agar medium for rapid detection of extended-spectrum β-lactamases and Klebsiella pneumoniae carbapenemases producing bacteria from human immunodeficiency virus patients.

Sir, Bacterial infections in the human immunodeficiency virus (HIV) patients are mostly caused by the pathogenic bacteria with a high level of antibiotic resistance.[1] Extended-spectrum beta(β)-lactamases (ESBLs) are plasmid-encoded enzymes and confer resistance to penicillins, cephalosporins, and aztreonam.[2] Klebsiella pneumoniae carbapenemases (KPC) belong to the family of serine carbapenemases and are usually found in Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E.coli). KPC hydrolyze β-lactam antibiotics and thereby, reducing their activity and misidentification of KPC-producing bacteria is common with standard susceptibility testing.[3] The aim of this study is to identify the ESBLs and KPC-producing gram-negative bacteria from HIV patients using HiCrome agar medium. HiCrome ESBL Agar Base (HiMedia, India) is a chromogenic medium used for the selective isolation of ESBL-producing bacteria on the basis of colour colonies. HiCrome ESBL supplement, containing antibiotics such as ceftazidime, cefotaxime, ceftriaxone, aztreonam, and flucanazole, is used to inhibit other contaminating microorganisms and non-ESBL-producing bacteria. HiCrome KPC Agar (HiMedia, India) is used in the identification of KPC-producing bacteria and the supplement contains antibiotics that inhibit the growth of yeast, gram-positive organisms, and gram-negative organisms that do not produce carbapenemases. The HIV patients were identified by standard combination rapid tests as per National acquired immune deficiency syndrome Control Organisation (NACO) guidelines (NACO, 2007).[4] The median cluster of differentiation 4 (CD4) cell count of the HIV patients was 142 cells/mm3. Among the 173 bacterial isolates from HIV patients, 126 were from urine, 27 from pus, 16 from sputum, 2 from blood, 1 from fine needle aspiration cytology, and 1 from vaginal swab. Using ESBL and HiCrome KPC agar media, a total of 108 (P ≤ 0.001) and 132 (P ≤ 0.001) bacterial isolates were found to be positive for ESBLs and KPC production, respectively. Out of 108 ESBLs-producing strains, 64 were E. coli, 11 K. pneumoniae, 18 Klebsiella oxytoca (K. oxytoca), 10 Pseudomonas aeruginosa (P. aeruginosa), and 5 Proteus mirabilis (P. mirabilis). Of the 132 KPC-producing isolates, 73 were E. coli, 17 K. pneumoniae, 15 K. oxytoca, 18 P. aeruginosa, 5 P. mirabilis, 3 Proteus vulgaris, and 1 Acinetobacter baumannii. A number of 59 isolates showed positive for both ESBLs and KPC production. ESBLs production was compared by the combination disc method (CDM) using cefotaxime and ceftazidime alone and in combination with clavulanic acid and KPC production by the modified Hodge test (MHT) using meropenem disc.[5] It was found that only 88 (P ≤ 0.001) isolates showed positive for ESBLs production using CDM and 110 (P ≤ 0.001) for KPC production using MHT. Comparison of results of the above methods in this study thus revealed that HiCrome agar is more sensitive than CDM in ESBLs production, as well as MHT in KPC production. Ongut et al. (2014)[6] from Turkey reported that among 237 bacterial isolates from various clinical samples from non-HIV patients, 143 showed positive for ESBLs production using Brilliance ESBL agar (Oxoid; Thermo Fisher Scientific, UK) and among 143 ESBL-positive isolates, 76 were E.coli and 67 K. pneumoniae. Samra et al. (2008)[7] reported a new CHROMagar KPC medium for rapid and direct detection of carbapenem-resistant K. pneumoniae from clinical samples. They found that among 122 swab cultures, 43 K. pneumonaiae isolates showed positive for KPC production. This is the first report of the detection of ESBLs and KPC-producing gram-negative bacteria from HIV patients using chromogenic agar medium in India. Identification of ESBLs and KPC production among gram-negative bacteria using HiCrome agar is cost-effective and in addition, has the advantage of rapid identification within 24 h with higher sensitivity. The limitations of this method include the occurrence of false-positive results in case of multiple β-lactamases producing bacteria and that a few other carbapenemases producing bacteria may show positive results as well. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

In Memoriam: John P. Quinn, MD

In Memoriam: John P. Quinn, MD

Utility of a novel multiplex TaqMan PCR assay for metallo-β-lactamase genes plus other TaqMan assays in detecting genes encoding serine carbapenemases and clinically significant extended-spectrum β-lactamases

Prompt detection of infections caused by Enterobacteriaceae that produce therapeutically important β-lactamases [metallo-β-lactamases (MBLs), serine carbapenemases, acquired AmpC and CTX-M extended-spectrum β-lactamases (ESBLs)] is crucial for infection prevention and control and surveillance purposes, and, more contentiously, also for effective patient management. A novel TaqMan PCR assay was developed to detect genes encoding IMP, VIM, NDM, SPM, SIM and GIM MBLs. Published PCR assays for acquired genes encoding CTX-M ESBLs and AmpC β-lactamases were updated and adapted to the TaqMan format, respectively. A published TaqMan assay for serine carbapenemase genes was used. Assay specificity was tested using a panel of 59 isolates with known acquired genes from the four different β-lactamase groupings. The four TaqMan assays correctly identified the most clinically relevant acquired β-lactamase genes in the panel of 59 resistant Enterobacteriaceae, which included 3 VIM-, 7 NDM- and 12 IMP-producers. Consecutive, non-duplicate isolates of Enterobacteriaceae from 965 urinary and 343 blood cultures during 2010 were then screened for β-lactamase genes using these TaqMan assays. Amongst the urinary and blood culture isolates tested, 69 CTX-M-producers and 21 acquired AmpC β-lactamase-producers were identified; the CTX-M rate amongst blood culture isolates (9.3%) broadly reflects the UK national average. During the study period, one Klebsiella pneumoniae isolate producing an NDM carbapenemase was identified from a wound sample. The assays developed and/or used will enable the future surveillance and the rapid detection and appropriate early treatment of infections caused by Gram-negative bacteria producing clinically important β-lactamases, including carbapenemases.

Acinetobacter baumannii producing OXA-23 detected in the Czech Republic

BackgroundAcinetobacter baumannii is an opportunistic pathogen posing an increased risk to hospitalized persons, causing nosocomial pneumonias, urinary tract infections and postoperative infections.MethodsBetween 1 December 2011 and 30 September 2012, strains of Acinetobacter spp. were isolated from clinical samples obtained from hospitalized patients. Susceptibility to antibiotics was determined by the standard microdilution method and phenotypic testing was used to detect the presence of serine carbapenemases and metallo-beta-lactamases. The polymerase chain reaction was used to detect the genes encoding carbapenemases. Pulsed field gel electrophoresis was used to investigate the genetic relationship among the carbapenem resistant isolates of Acinetobacter baumannii.ResultsIn three strains of Acinetobacter baumannii enzyme OXA-23 was detected. This positive result was confirmed by restriction analysis and sequencing. The study reported an OXA-23-producing strains of Acinetobacter baumannii in the Czech Republic. All three strains isolated from Military Hospital patients had a completely identical restriction profile, indicating clonal spread of a strain carrying serine carbapenemase OXA-23 in this health care facility. Moreover this was the first time the strain was detected in the country in patients who had not stayed abroad.

Insights into β-Lactamases from Burkholderia Species, Two Phylogenetically Related yet Distinct Resistance Determinants

Burkholderia cepacia complex and Burkholderia pseudomallei are opportunistic human pathogens. Resistance to β-lactams among Burkholderia spp. is attributable to expression of β-lactamases (e.g. PenA in B. cepacia complex and PenI in B. pseudomallei). Phylogenetic comparisons reveal that PenA and PenI are highly related. However, the analyses presented here reveal that PenA is an inhibitor-resistant carbapenemase, most similar to KPC-2 (the most clinically significant serine carbapenemase), whereas PenI is an extended spectrum β-lactamase. PenA hydrolyzes β-lactams with k(cat) values ranging from 0.38 ± 0.04 to 460 ± 46 s(-1) and possesses high k(cat)/k(inact) values of 2000, 1500, and 75 for β-lactamase inhibitors. PenI demonstrates the highest kcat value for cefotaxime of 9.0 ± 0.9 s(-1). Crystal structure determination of PenA and PenI reveals important differences that aid in understanding their contrasting phenotypes. Changes in the positioning of conserved catalytic residues (e.g. Lys-73, Ser-130, and Tyr-105) as well as altered anchoring and decreased occupancy of the deacylation water explain the lower k(cat) values of PenI. The crystal structure of PenA with imipenem docked into the active site suggests why this carbapenem is hydrolyzed and the important role of Arg-220, which was functionally confirmed by mutagenesis and biochemical characterization. Conversely, the conformation of Tyr-105 hindered docking of imipenem into the active site of PenI. The structural and biochemical analyses of PenA and PenI provide key insights into the hydrolytic mechanisms of β-lactamases, which can lead to the rational design of novel agents against these pathogens.

Identification and Characterization of Carbapenem Hydrolysing β-lactamase – KPC among Enterobacteriaceae: A report from North India

Objective: The serine carbapenemase KPC (Klebsiella pneumoniae carbapenemase) has emerged as a beta-lactamase capable of inactivating carbapenem antibiotics. The emergence of carbapenem-resistant enterobacteria is therefore worrisome, since consequently the antimicrobial treatment options are very restricted. In the present study we have reported the presence of KPC ?-lactamase producing enterobacterial isolates from a tertiary referral hospital in north India. Methods: The isolates were subjected to phenotypic confirmatory test by boronic acid and clavulanic acid inhibition and presence of carbapenemase activity. They were further tested for PCR detection of blaKPC and the associated genetic component. Results: Three enterobacterial isolates were found to be phenotypically similar to that of KPC enzyme and genotypically showing positive results with primers specific for blaKPC gene were found to be located on integron. Conclusion: The findings indicate the need for continuous surveillance of this resistant determinant in this part of the world. DOI: http://dx.doi.org/10.3126/ajms.v3i2.5087 Asian Journal of Medical Sciences 3(2012) 11-15

OXA-181-producing Klebsiella pneumoniae establishing in Singapore

BackgroundCarbapenemase producing Enterobacteriaceae are becoming a major public health concern globally, however, relatively little is known about the molecular and clinical epidemiology of these organisms in many parts of the world.MethodsAs part of a laboratory surveillance programme, 96 carbapenem non-susceptible Enterobacteriaceae isolates from clinical samples from patients in seven hospitals were referred for investigation for carbapenemases. Using polymerase chain reaction (PCR) to screen for a collection of genes encoding carbapenemases, 33 of 96 (34.5%) isolates were confirmed as carbapenemase producers. NDM-1 producers were the most prevalent at 64% (21/33) whilst OXA-181 was the second most common carbapenemase constituting 24.5% (8/33) of the carbapenemase producing isolates. Seven of these eight OXA-181 positive isolates underwent further characterisation with screening for other transmissible antimicrobial resistance determinants using PCR. Clonal relatedness was explored using Multilocus sequence typing (MLST) and Pulsed Field Gel Electrophoresis (PFGE). Plasmid characterisation was performed including restriction analysis and transfer by conjugation or transformation.ResultsIn addition to the OXA-181 gene, all contained other transmissible resistance determinants including extended spectrum β-lactamases, oxacillinases or 16S rRNA methylase genes, but none contained metallo-β-lactamases or serine carbapenemases. All isolates had a multidrug resistant phenotype with two isolates being resistant to every antibiotic tested including colistin. Multilocus sequence typing confirmed five isolates belonged to ST17 and two to ST14, with those belonging to the same sequence type having identical PFGE profiles. The OXA-181 gene was typically carried on large plasmids which were mostly non-conjugative.ConclusionsOXA-181 carbapenemase appears to be an important and probably under-recognised cause of carbapenem resistance in Enterobacteriaceae in Singapore. Further coordinated research into clinical and molecular epidemiology of carbapenemases is urgently required in Singapore and throughout Asia.

Proliferation and significance of clinically relevant β‐lactamases

Inactivation of β-lactam antibiotics by β-lactamases in bacterial infections is associated with some of the most serious infectious disease issues that are currently encountered. The evolution of unique β-lactamases has resulted in more than 1,300 distinct enzymes that have been identified in natural clinical isolates. Of these enzymes, the most deleterious β-lactamases are the extended-spectrum β-lactamases, or ESBLs, that hydrolyze most penicillins and cephalosporins, and the carbapenemases that may inactivate all β-lactam classes of drugs. The most prominent ESBLs worldwide are the CTX-M-14 and CTX-M-15 enzymes. Among enzyme families, the TEM and OXA β-lactamases exhibit the greatest number of variants. The broad groups of carbapenemases are particularly treacherous, especially the KPC serine carbapenemases and the NDM family of metallo-β-lactamases, both of which appear in multidrug-resistant Gram-negative pathogens that are often resistant to most classes of antibiotics. Although new β-lactamase inhibitor combinations are being investigated as a means of controlling infections caused by these organisms, additional approaches are sorely needed.

Detection systems for carbapenemase gene identification should include the SME serine carbapenemase

Carbapenemase detection has become a major problem in hospitals that encounter outbreaks of infections caused by carbapenem-resistant Gram-negative bacteria. Rapid detection systems have been reported using multiplex PCR analyses and DNA microarray assays. Major carbapenemases that are detected by these systems include the KPC and OXA serine carbapenemases, and the IMP, VIM and NDM families of metallo-β-lactamases. However, increasing numbers of the SME serine carbapenemase are being reported from Serratia marcescens, especially from North and South America. These organisms differ from many of the other carbapenemase-producing pathogens in that they are generally susceptible to the expanded-spectrum cephalosporins ceftazidime and cefepime while retaining resistance to almost all other β-lactam antibiotics. Thus, multiplex PCR assays or DNA microarray testing of carbapenem-resistant S. marcescens isolates should include analyses for production of the SME carbapenemase. Confirmation of the presence of this enzyme may provide reassurance that oxyimino-cephalosporins can be considered for treatment of infections caused by these carbapenem-resistant pathogens.

Carbapenemase-ProducingKlebsiella pneumoniaein Hospital, Singapore, 2011

Carbapenemase-Producing<i>Klebsiella pneumoniae</i>in Hospital, Singapore, 2011

Rationale for the 2010 Revised Susceptibility Breakpoints for Cephalosporins, Aztreonam, and Carbapenems for Enterobacteriaceae.

β-Lactam antimicrobial agents are mainstays for treatment of infections due to many members of the Enterobacteriaceae. Since the introduction of these agents into clinical use in the 1970s and 1980s, there have been considerable changes in the potency of these drugs against clinical isolates of Enterobacteriaceae, largely due to the evolution and dissemination of extended spectrum β-lactamases (ESBLs) that hydrolyze penicillins, cephalosporins, and monobactams. The worldwide dissemination of these ESBLs played a major role in the first revision of susceptibility breakpoints (minimum inhibitory concentrations [MICs]) for these drugs by the Clinical Laboratory Standards Institute in June 2010. The increasing problem of β-lactamase-mediated resistance has recently extended to the carbapenem class of antimicrobial agents. The serine carbapenemase KPC (for Klebsiella pneumoniae carbapenemase) has been detected in Enterobacteriaceae from several major population regions in the United States, and is problematic in many institutions in Europe. More recently, a metallo β-lactamase (NDM-1) that hydrolyzes carbapenems and most β-lactams has spread rapidly from Asia to the United Kingdom and other regions in the world. The Centers for Disease Control and Prevention has issued alerts for both of these carbapenemases and recommendations for limiting its spread in hospitals [1, 2]. Carbapenemases of the KPC type produced a situation similar to that for ESBLs; strains producing KPC or ESBLs were often classified as susceptible using breakpoints established in the former era. Thus, new lower breakpoints for cephalosporins and carbapenems against Enterobacteriaceae were established to ensure optimal exposures with US Food and Drug Administration (FDA)-approved dosage regimens, and would also classify these isolates as nonsusceptible. Clinical, pharmacokinetic-pharmacodynamic (PKPD), and MIC distribution data were considered in revising the breakpoints [3, 4]. While highly desirable, clinical data were difficult to acquire. Critical information concerning MIC, concomitant drug treatment, site of infection, and cephalosporin dosage regimen was generally not available in studies of ESBL-producing isolates. Thus, other analyses were required. PK-PD analyses were conducted to determine whether usual FDA-approved dosage regimens of the Pediatric ID Consultant

Occurrence and phenotypic detection of class A carbapenemases among Escherichia coli and Klebsiella pneumoniae blood isolates at a tertiary care center

Resistance to carbapenems is a significant therapeutic threat. The increasing frequency of car bapenemase enzymes among Gram-negative bacilli makes their early detection and differentiation urgent. Carbapenemases belonging to Class A are most commonly produced by members of family Enterobacteriaceae and are inhibited to various degrees by clavulanic acid. The present study is aimed to determine the occurrence and phenotypic detection of Class A carbapenemases in Escherichia coli and Klebsiella pneumoniae blood isolates from septicemic patients. A total of 75 isolates of K. pneumoniae and 25 E. coli were screened for resistance to carbapenems by using meropenem and imipenem discs and meropenem E-test. Positive strains were then subjected to a modified Hodge test combined with carbapenemase inhibition tests to phenotypically detect and differentiate Class A serine carbapenemases from other classes of carbapenem hydrolyzing enzymes. The screening test showing the number of isolates resistant to meropenem and imipenem were 41 and 35 for K. pneumoniae and nine and four for E. coli, respectively. A total of 25 (33.3%) K. pneumoniae isolates and two (8.0%) E. coli isolates were classified as Class A carbapenemase producers. Multidrug resistance with coexistence of extended spectrum-beta-lactamases occurred in 44.4% isolates. However, all of the isolates were susceptible to colistin, polymyxin B, and tigecycline by disc diffusion test. We conclude from the present study that Class A carbapenemases appear to be the predominant cause of resistance to carbapenems in Enterobacteriaceae at our center and, thus, phenotypic detection based on simple methods should be employed routinely in clinical microbiology laboratories.

Molecular diversity in mechanisms of carbapenem resistance in paediatric Enterobacteriaceae

Development of carbapenem resistance in Enterobacteriaceae has impacted Clinical and Laboratory Standards Institute (CLSI) guidelines, infection control approaches and treatment strategies. The clinical, phenotypic and genotypic characteristics of carbapenem-resistant Enterobacteriaceae (CRE) infections at paediatric referral centres are not well described. CRE were identified through the clinical microbiology laboratory at Seattle Children's Hospital (Seattle, WA). Clinical data were retrieved from medical records. Resistance testing, polymerase chain reaction (PCR) for resistance determinants, and Escherichia coli transformation were carried out for each isolate. Multilocus sequence typing (MLST) and pulsed-field gel electrophoresis (PFGE) were used to characterise strain relatedness. PCR amplification and sequencing as well as sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) were used to investigate porin alterations. Six CRE isolates were identified between 2002 and 2010. Significant molecular diversity was documented in their mechanisms of resistance, including plasmid-mediated serine carbapenemase (KPC) and metallo-β-lactamase (IMP), chromosomally encoded β-lactamase (SME) and porin alterations with extended-spectrum β-lactamases. Patients had underlying health conditions and were from geographically diverse regions. In one case, PFGE of serial isolates documented the development of resistance in a previously susceptible strain. Molecular investigation of this strain identified insertion of the genetic mobile element insertion sequence IS Ecp1 in the ompK36 gene, conferring a functional porin alteration as demonstrated by SDS-PAGE. This is the first description of porin disruption by IS Ecp1 in a CTX-M-15-positive isolate. This is the largest report of paediatric CRE to date. This diverse description of demographic, phenotypic and molecular characteristics highlights the challenge of CRE infections in high-risk paediatric patients and that attention to emerging resistance mechanisms (including membrane alteration) at paediatric referral centres is essential.

Epidemiological Expansion, Structural Studies, and Clinical Challenges of New β-Lactamases from Gram-Negative Bacteria

β-Lactamase evolution presents to the infectious disease community a major challenge in the treatment of infections caused by multidrug-resistant gram-negative bacteria. Because over 1,000 of these naturally occurring β-lactamases exist, attempts to correlate structure and function have become daunting. Although new enzymes in the extended-spectrum β-lactamase (ESBL) families are frequently identified, the older CTX-M-14 and CTX-M-15 enzymes have become the most prevalent ESBLs in global surveillance. Carbapenemases with either serine-based or zinc-facilitated hydrolysis mechanisms are posing some of the most critical problems. Most geographical regions now report KPC serine carbapenemases and the metallo-β-lactamases VIM, IMP, and NDM-1, even though NDM-1 was only recently identified. The rapid emergence of these newer enzymes, with multiple β-lactamases appearing in a single organism, makes the design of new β-lactamase inactivators or β-lactamase-stable β-lactams all the more difficult. Combination therapy will likely be required to counteract the continuing evolution of these insidious enzymes in multidrug-resistant pathogens.

Longitudinal survey of carbapenem resistance and resistance mechanisms in Enterobacteriaceae and non-fermenters from the USA in 2007–09

Antibiotic resistance is problematic in Enterobacteriaceae, Pseudomonas aeruginosa and Acinetobacter baumannii, and is often associated with serious infections. Carbapenems are often one of the few remaining therapeutic options, so it is important to monitor carbapenem activity against these pathogens and to identify resistance mechanisms. Carbapenem susceptibilities were determined for 14 359 Enterobacteriaceae, 3614 P. aeruginosa and 994 A. baumannii from the USA (2007-09). Klebsiella pneumoniae with doripenem MICs ≥2 mg/L (n = 88), and P. aeruginosa (n = 452), A. baumannii (n = 349) and other enterics (n = 13) with doripenem MICs ≥4 mg/L were screened for carbapenem resistance mechanisms. Doripenem/meropenem and imipenem susceptibilities for Enterobacteriaceae were >99% and 89%, respectively. Doripenem susceptibility (2007-09) for P. aeruginosa was 87.4%-84.1%; comparable to meropenem and higher than imipenem. For A. baumannii, doripenem susceptibility (2007-09) was 63%-58.2%; lower than imipenem and meropenem. Resistant K. pneumoniae had KPC and lacked porins OmpK35/OmpK36. In 2009, 3.4% of all K. pneumoniae possessed KPC. Five other enterics and one P. aeruginosa possessed KPC. Resistance mechanisms in P. aeruginosa were loss of porin OprD (90%), efflux (55%) and elevated AmpC activity (25%). Acquired carbapenemases OXA-23/-24 were present in 48% of resistant A. baumannii. VIM metallo-β-lactamases were present in three P. aeruginosa and one A. baumannii isolates. Doripenem and meropenem were more active than imipenem against Enterobacteriaceae and P. aeruginosa from the USA. Carbapenem resistance mechanisms included serine carbapenemases, elevated AmpC activity, efflux and porin deficiencies occurring mostly in P. aeruginosa. Metallo-β-lactamases were found in <0.1% of isolates.

Therapeutic Options for Infections with Enterobacteriaceae Producing Carbapenem-Hydrolyzing Enzymes

Enterobacteriaceae that produce serine carbapenemases or metallo-β-lactamases, such as KPC, OXA-48, VIM or NDM, respectively, are spreading mostly as nosocomial pathogens worldwide. Such strains are typically resistant to most if not all available antimicrobials. Specific relevant clinical data are scarce to guide the determination of the most appropriate treatment options. Data on antimicrobial susceptibility, resistance development, synergy, pharmacokinetic and pharmacodynamic parameters of the candidate regimens, as well as the experience from the treatment of infections with nonfermenting Gram-negative pathogens, can aid in this regard. Colistin and tigecycline are most likely to be active in vitro against Enterobacteriaceae producing carbapenem-hydrolyzing β-lactamases, but resistance development is of concern. Individual members of the aminoglycoside class can also be active in vitro, while carbapenems or aztreonam (specifically for metallo-β-lactamase producers) can have low minimum inhibitory concentrations. Current data do not reliably support the use of these agents as monotherapy for systemic infections. Several expanded-spectrum cephalosporins, such as ceftazidime, may be active against OXA-48 type producers. Fosfomycin might be useful as a last-resort option as part of combination regimens. Combination antimicrobial therapy with agents exhibiting synergy might also be of benefit, until novel effective agents could become clinically available.

Real-time TaqMan PCR for rapid detection of genes encoding five types of non-metallo- (class A and D) carbapenemases in Enterobacteriaceae

A real-time TaqMan multiplex polymerase chain reaction (PCR) assay was developed to detect genes encoding five types of serine carbapenemases (GES, IMI/NMC, KPC, OXA-48 and SME). The assay was validated using control strains known to produce each of these types of enzyme and was then further assessed by ‘blindly’ testing 59 previously characterised clinical isolates, including 19 with serine (KPC or OXA-48) carbapenemases, 22 with metallo- (IMP, VIM or NDM) carbapenemases, and 18 with carbapenem resistance contingent upon extended-spectrum β-lactamase (ESBL) or AmpC production combined with porin loss. The assay detected and correctly assigned the serine carbapenemases in all five positive control strains and in 19 clinical isolates. No false-positive results were seen for isolates with metallo-enzymes or for those that lacked a carbapenemase. The five serine carbapenemase genotypes could also be distinguished by melt-curve analysis or the molecular size of the amplicons.