Klebsiella Pneumoniae Carbapenemase Enzymes Research Articles

Detection of KPC enzyme by MALDI-TOF MS from bacteria impregnated in filter paper

The main mechanism that causes resistance to carbapenem, one of the most potent antibiotic available, in Enterobacterales bacterial isolates, is due to Klebsiella pneumoniae carbapenemase (KPC) production by the bacterium. KPC is spread worldwide, requiring laboratories to be capable of identifying this enzyme, however some methods can be expensive for small laboratories, especially in developing countries. Therefore, the development of methods with low cost of reagents for the detection of KPC enzyme is necessary. The objective of this study was to evaluate the detection of KPC enzyme by MALDI-TOF MS from inactivated bacteria impregnated in filter paper. A total of 129 Enterobacterales isolates were impregnated in filter paper, and after 7 days at room temperature, they were subjected to a protein extraction protocol and spectra acquisition, in triplicates, by MALDI-TOF MS. The spectra were evaluated and KPC was identified according to the presence of a peak of 28,712.62 ± 27.80 m/z. Considering the presence of the KPC peak in at least one spectrum of the triplicates, this method presented 60.8% sensitivity and 96.4% specificity. However, considering the presence of KPC peak in at least two spectra of the triplicate, a specificity of 100% was achieved. The detection of KPC enzyme from inactivated bacteria impregnated in filter paper can be used as a method to confirm the presence of KPC, which could be very significant for small laboratories with limited resources.

Ceftazidime-avibactam resistance in Klebsiella pneumoniae sequence type 37: a decade of persistence and concealed evolution.

The first reports of carbapenem-resistant Enterobacterales in our hospital date back to 2006. In that period, few ertapenem-resistant but meropenem-susceptible Klebsiella pneumoniae isolates belonging to sequence type (ST) 37 were retrieved from clinical samples. These strains produced the CTX-M-15 extended spectrum β-lactamase, OmpK35 was depleted due to a nonsense mutation, and a novel OmpK36 variant was identified. Yet, starting from 2010, Klebsiella pneumoniae carbapenemase (KPC)-producing ST512 isolates started prevailing and ST37 vanished from sight. Since 2018 the clinical use of the combination of ceftazidime-avibactam (CZA) has been introduced in clinical practice for the treatment of bacteria producing serine-β-lactamases, but KPC-producing, CZA-resistant K. pneumoniae are emerging. In 2021, four CZA-resistant ST37 isolates producing KPC variants were isolated from the same number of patients. blaKPC gene cloning in Escherichia coli was used to define the role of those KPC variants on CZA resistance, and whole genome sequencing was performed on these isolates and on three ST37 historical isolates from 2011. CZA resistance was due to mutations in the blaKPC genes carried on related pKpQIL-type plasmids, and three variants of the KPC enzyme have been identified in the four ST37 strains. The four ST37 isolates were closely related to each other and to the historical isolates, suggesting that ST37 survived without notice in our hospital for 10 years, waiting to re-emerge as a CZA-resistant K. pneumoniae clone. The ancestor of these contemporary isolates derives from ST37 wild-type porin strains, with no other mutations in chromosomal genes involved in conferring antibiotic resistance (parC, gyrA, ramR, mgrB, pmrB).

In vitro-obtained meropenem-vaborbactam resistance mechanisms among clinical Klebsiella pneumoniae carbapenemase-producing K. pneumoniae isolates

A novel ß-lactam-β-lactamase inhibitor (BLBI), meropenem (MEM), combined with the boronate-based inhibitor vaborbactam (VAB), has recently been introduced for the treatment of infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacterales. The purpose of this study was to select for MEM-VAB resistance using a collection of eight KPC-producing K. pneumoniae clinical isolates, including three that produce KPC variants conferring ceftazidime-avibactam (CAZ-AVI) resistance, and subsequently decipher the corresponding resistance mechanisms. Mutants were selected in a stepwise process on agar plates containing different MEM-VAB concentrations. Susceptibility testing was performed by broth microdilution, and complementation assays were performed with wildtype ompK36. Whole genome sequencing was performed on mutants, and KPC copy number was assessed by quantitative polymerase chain reaction . Mutants were obtained from 6/8 tested isolates and reduced susceptibility to all tested β-lactams, and BLBIs, including CAZ-AVI, imipenem-relebactam, and aztreonam-AVI, were observed. No mutations were identified in the blaKPC. However, mutations in ompK36 were observed in four mutant lineages, and complementation with a wild-type ompK36 resulted in a reduction of minimal inhibitory concentrations to both MEM-VAB and other ß-lactams/BLBIs. blaKPC gene copy numbers were significantly increased in four mutant lineages. Whole genome sequencing identified genomic rearrangements in two lineages comprising mutations in the plasmid replicon encoding gene and duplication of the Tn4401 transposon bearing the blaKPC gene into a ColE-like, high copy number plasmid. In contrast to what is observed with KPC-producing mutants exhibiting resistance to CAZ-AVI, mainly corresponding to mutated KPC enzymes, here the MEM-VAB-resistant mutants showed permeability defects combined with increased KPC production, resulting from genomic rearrangement.

Omic insights into various ceftazidime-avibactam-resistant Klebsiella pneumoniae isolates from two southern Italian regions.

Ceftazidime-avibactam (CZA) is one of the best therapeutic options available for infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing bacteria. However, sporadic reports of CZA-resistant strains have been rapidly increasing in patients. Herein, we provide detailed case reports of the emergence of ceftazidime-avibactam resistance to identify their resistome and virulome using genomic molecular approaches. Sixteen isolates were collected from 13 patients at three hospitals in Catania and Catanzaro (Italy) between 2020-2021. Antimicrobial susceptibility was determined by broth microdiluition. The samples included in study were analyzed for resistome, virulome and Sequence Type (ST) using Whole Genome Sequencing (WGS). All strains were resistant to ceftazidime/avibactam, ciprofloxacin, extended-spectrum cephalosporins and aztreonam, 13/16 to meropenem, 8/16 to colistin and 7/16 to fosfomycin; 15/16 were susceptible to meropenem/vaborbactam; all strains were susceptible to cefiderocol. Molecular analysis showed circulation of three major clones: ST101, ST307 and ST512. In 10/16 strains, we found a bla KPC-3 gene; in 6/16 strains, four different bla KPC variants (bla KPC28-31-34-50) were detected. A plethora of other beta-lactam genes (bla SHV28-45-55-100-106-187-205-212, bla OXA1-9-48, bla TEM-181 and bla CTX-M-15) was observed; bla OXA-9 was found in ST307 and ST512, instead bla OXA48 in one out four ST101 strains. With regard to membrane permeability, ompK35 and ompK36 harbored frameshift mutations in 15/16 strains; analysis of ompK37 gene revealed that all strains harbored a non-functional protein and carry wild-type PBP3. There is an urgent need to characterize the mechanisms underlying carbapenem resistance and the intrinsic bacterial factors that facilitate the rapid emergence of resistance. Furthermore, it is becoming increasingly important to explore feasible methods for accurate detection of different KPC enzymes.

In vitro activity of imipenem/relebactam against Gram-negative clinical isolates in two Spanish tertiary hospitals.

ObjetiveThe aim of this study was to analyze the activity of the imipenem-relebactam combination (IMI/REL) against a collection of multidrug-resist Enterobacterales, Pseudomonas aeruginosa and Acinetobacter baumannii clinical isolates.Material and methodsThe study was conducted in two tertiary hospitals in Spain and included 192 clinical isolates of these 3 genera (139 resistant and 53 susceptible to IMI). The MICs for IMI with and without REL (at a fixed concentration of 4 mg/L) were determined by a standard broth microdilution method according to international recommendations.ResultsAll IMI-susceptible E. coli strains were also susceptible to IMI/REL. Enterobacterales resistant to IMI due to the production of carbapenemases, the MIC50 and MIC90 de-creased from 64/256 with IMI to 8/64 mg/L with IMI/REL. This high activity was principally detected among isolates with KPC enzymes. Enterobacterales with class B carbapenemases, P. aeruginosa carrying VIM carbapenemase and A. baumannii strains showed no changes on IMI MIC50 or MIC90 after adding REL. Among P. aeruginosa strains without carbapenemase the MIC for IMI/REL was reduced between 1 to 5 dilutions.ConclusionsIMI/REL showed high activity against the strains that carry Klebsiella pneumoniae carbapenemase (KPC) and against carbapenem-resistant P. aeruginosa unrelated to the VIM enzyme, mainly AmpC beta lactamase associated with impermeability. Against strains carrying oxacillinase 48 (OXA-48) associated with extended-spectrum beta-lactamase (ESBL), IMI/REL presented activity only slightly better than IMI and had no beneficial effect superior to IMI against A. baumannii.

Biofilm Formation and Phenotypic Detection of ESBL, MBL, KPC and AmpC Enzymes and Their Coexistence in Klebsiella spp. Isolated at the National Reference Laboratory, Kathmandu, Nepal

Klebsiella spp. are associated with several nosocomial and opportunistic infections. Increasing antimicrobial resistance of Klebsiella species is aggravated by a number of intrinsic and extrinsic factors. The main aim of this study is to determine antimicrobial resistance due to production of β-lactamase enzymes, extended spectrum beta-lactamase (ESBL), metallo-beta-lactamase (MBL) and AmpC and Klebsiella pneumoniae carbapenemase (KPC) and biofilm formation in Klebsiella isolates. A total of 2197 non-duplicate specimens of urine, sputum and pus were obtained from the National Public Health Laboratory (NPHL), Kathmandu, Nepal, between February and August 2019. Klebsiella species were isolated, identified and screened for antimicrobial susceptibility testing with the disk diffusion method. Phenotypic detection of ESBL, MBL, KPC and AmpC production was observed and biofilm production was detected by the microtiter plate method. Out of a total of 2197 clinical specimens, bacterial growth was detected in 8% (175/2197) of the specimens. Of the total isolates, 86.3% (151/175) were Gram-negative bacteria and 37.7% (57/151) were Klebsiella spp. Of the total Klebsiella spp., 56% (32/57) were multi drug resistant (MDR), 16% (9/57) were ESBL, 26% (15/57) were MBL, 4% (2/57) were KPC (class A carbapenemase), 16% (9/57) were AmpC producers and 95% (54/57) were biofilm producers. Gentamicin was the most effective antibiotic, followed by cotrimoxazole, as 68% (39/57) and 47% (27/57) of the Klebsiella isolates were susceptible towards these drugs, respectively. The study results show evidence of β-lactamase production, high prevalence of MDR and biofilm producing Klebsiella species. Integrating the test parameters for phenotypic confirmation of ESBL, MBL, AmpC β lactamase and KPC in routine diagnostic procedures can help in the early detection and management of these resistant strains.

Klebsiella pneumoniae carbapenemase (KPC) producer resistant to ceftazidime–avibactam due to a deletion in the blaKPC3 gene

ObjectivesCarbapenemase-producing strains of Klebsiella pneumoniae (KPC) are a great health concern, and therapy with ceftazidime–avibactam represents a choice for the treatment of infections involving these strains. We report a strain resistant to ceftazidime–avibactam due to a deletion of six nucleotides in the blaKPC gene sequence. MethodsTwo strains, namely AMP920 and AMP2009, were isolated from the same patient a month apart. Antimicrobial susceptibility testing was performed both by broth microdilution and by Etest. Immunoenzymatic assay to detect carbapenemase was performed for both strains. The blaKPC gene of both strains was amplified by PCR and sequenced. Enzyme activity towards carbapenems was tested by the CarbaNP test and hydrolysis spectrophotometer assay. ResultsThe two isolates differed in antimicrobial susceptibility. AMP920 showed meropenem and imipenem resistance (MIC 32 and 32 mg/mL). A month later the carbapenem MIC decreased to 8 and 1 mg/mL respectively, while the ceftazidime–avibactam MIC increased from 1 to 16 mg/mL. Both isolates showed a positive immunoenzymatic test for the KPC enzyme, but only AMP920 showed a positive CarbaNP test hydrolysing imipenem. The BlaKPC gene was amplified in both strains. After sequencing, the two amplicons showed a KPC3 variant. The gene of the second isolate showed a deletion of six nucleotides at 498–503, resulting in a mutant variant with the deletion of glutamic acid and leucine residues at positions 167 and 168. ConclusionsWe detected a new deletion in the blaKPC gene of a clinical strain of K. pneumoniae which resulted in resistance to ceftazidime–avibactam. The amino acids deleted are in the Ω loop (amino acids 165–179) of the KPC enzyme, enhancing ceftazidime affinity and preventing avibactam binding.

High-performance method to detection of Klebsiella pneumoniae Carbapenemase in Enterobacterales by LC-MS/MS.

Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.

Identification of blaVIM-1 Gene in ST307 and ST661 Klebsiella pneumoniae Clones in Italy: Old Acquaintances for New Combinations.

In the past years, the Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae sequence type 258 (ST258) became an important worldwide spread nosocomial pathogen. Recent evidence shows that the global epidemiology is changing, with the rise of new lineages. In this study we report the microbiological and genomic features of two VIM-1-producing K. pneumoniae isolates belonging to the emerging ST307. Two extensively drug-resistant K. pneumoniae strains, collected between May and June 2017, were confirmed as blaVIM positive by GeneXpert system. The whole-genome sequencing revealed that both KpV_S_1 and KpV_S_2 isolates harbored blaVIM-1 and blaCTX-M-15 genes, besides qnrS1 and qnrB1, strB, mphA, tetR, and tetA determinants. KpV_S_1 and KpV_S_2 isolates belonged to ST661 and ST307, respectively. Both STs have been recently reported as responsible of outbreaks in several European countries. The detection of blaVIM-1 gene in nonpredominant K. pneumoniae clones in a hospital setting should alert on the changing of the epidemiological situation in Italy, usually endemic reservoir of KPC enzyme.

The Epidemiology, Evolution, and Treatment of KPC-Producing Organisms.

The purpose of this review is to investigate the evolution and epidemiology of Klebsiella pneumoniae carbapenemase (KPC)-producing organisms and the current and future treatment options for infections caused by KPC-producing isolates. The emergence of resistance in Enterobacteriaceae producing carbapenemases globally has increased the challenges in treating infections caused by these organisms. One of the prominent mechanisms of resistance is the production of KPC enzymes. Infections caused by organisms producing KPCs have limited treatment options and are associated with poor clinical outcomes. The rapid rise of KPC-producing organisms necessitated the use of drugs with pharmacokinetic and toxicity limitations, including polymyxins, tigecycline, fosfomycin, and aminoglycosides. The availability of new beta-lactamase inhibitor combinations that are effective against KPC-producing organisms represent an advance in safety and efficacy. Several agents are currently being studied that have activity against KPC-producing organisms and appear to represent promising additions to our armamentarium. KPC-producing organisms cause infections with high morbidity and mortality. Limited treatment options are available, though new therapies have been developed. Pipeline agents are likely to have a place in therapy for the treatment of infections caused by KPC-producing isolates.

Major role of pKpQIL-like plasmids in the early dissemination of KPC-type carbapenemases in the UK.

Klebsiella pneumoniae carbapenemase (KPC)-producing Enterobacteriaceae were first seen in the UK in 2003 and have been increasingly reported since 2010, largely owing to an ongoing outbreak in North-West England. We examined the role of clonal spread and plasmid transmission in their emergence. Isolates comprised KPC-positive K. pneumoniae ( n = 33), Escherichia coli ( n = 7) and Enterobacter spp. ( n = 4) referred to the national reference laboratory between 2008 and 2010 from 17 UK centres, including three in North-West England. Isolates were typed by MLST. Plasmids were transferred by electroporation and characterized by PCR or sequencing. PCR screening assays were developed to distinguish plasmid pKpQIL variants. The K. pneumoniae isolates included 10 STs, of which three belonged to clonal group (CG) 258. CG258 ( n = 19) isolates were detected in 13 centres but accounted for only 7/19 (36.8%) of those from North-West England. Most KPC-producers (37/44, 84.1%), including 16/19 CG258 K. pneumoniae , carried bla KPC on IncFII K2 plasmids. Sequencing of a subset of these plasmids ( n = 11) revealed similarities with published pKpQIL. One variant, pKpQIL-UK [identified in K. pneumoniae CG258 ( n = 5) and ST468 ( n = 1) isolates from distinct centres] had only a few nucleotide changes from classical pKpQIL, whereas pKpQIL-D1 ( n = 1) and pKpQIL-D2 ( n = 4), from isolates of various species in the North-West, harboured large variations, reflecting replacement of the partitioning and replication functions and potentially thereby facilitating spread. PCR revealed that 36/37 (97.3%) IncFII K2 -type plasmids in KPC-positive isolates had pKpQIL markers. pKpQIL-like plasmids played a major role in the early dissemination of KPC enzymes in the UK.

Global Dissemination of blaKPC into Bacterial Species beyond Klebsiella pneumoniae and In Vitro Susceptibility to Ceftazidime-Avibactam and Aztreonam-Avibactam.

The Klebsiella pneumoniae carbapenemase (KPC), first described in the United States in 1996, is now a widespread global problem in several Gram-negative species. A worldwide surveillance study collected Gram-negative pathogens from 202 global sites in 40 countries during 2012 to 2014 and determined susceptibility to β-lactams and other class agents by broth microdilution testing. Molecular mechanisms of β-lactam resistance among carbapenem-nonsusceptible Enterobacteriaceae and Pseudomonas aeruginosa were determined using PCR and sequencing. Genes encoding KPC enzymes were found in 586 isolates from 22 countries (76 medical centers), including countries in the Asia-Pacific region (32 isolates), Europe (264 isolates), Latin America (210 isolates), and the Middle East (19 isolates, Israel only) and the United States (61 isolates). The majority of isolates were K. pneumoniae (83.4%); however, KPC was detected in 13 additional species. KPC-2 (69.6%) was more common than KPC-3 (29.5%), with regional variation observed. A novel KPC variant, KPC-18 (KPC-3[V8I]), was identified during the study. Few antimicrobial agents tested remained effective in vitro against KPC-producing isolates, with ceftazidime-avibactam (MIC90, 4 μg/ml), aztreonam-avibactam (MIC90, 0.5 μg/ml), and tigecycline (MIC90, 2 μg/ml) retaining the greatest activity against Enterobacteriaceae cocarrying KPC and other β-lactamases, whereas colistin (MIC90, 2 μg/ml) demonstrated the greatest in vitro activity against KPC-positive P. aeruginosa This analysis of surveillance data demonstrated that KPC is widely disseminated. KPC was found in multiple species of Enterobacteriaceae and P. aeruginosa and has now become a global problem.

Differentiation of Klebsiella pneumoniae carbapenemase (KPC) variants by pyrosequencing

A fast and reliable protocol using the pyrosequencing technique was developed to identify 11 different types of the KPC enzyme. A total of 65 blaKPC positive bacterial isolates were tested and characterized. In the end, the pyrosequencing proved to be a powerful tool for epidemiological studies of KPC producer isolates.

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

Detection of the plasmid-mediated KPC-2 carbapenem-hydrolysing enzyme in three unusual species of the Enterobacteriaceae family in Israel

Sir, Carbapenem-resistant Enterobacteriaceae (CRE) pose a major threat to public health worldwide. One of the most frequently encountered mechanisms is the Ambler class A Klebsiella pneumoniae carbapenemase (KPC)-type enzyme. Since the initial report of a KPC-producing K. pneumoniae in 2001, these organisms have spread worldwide, mainly due to the spread of a single successful clone, sequence type 258 (ST258). In addition, KPC enzymes have been detected in multiple genera and species of the Enterobacteriaceae family, including Salmonella spp., Klebsiella oxytoca, Enterobacter spp., Citrobacter freundii, Escherichia coli, Proteus mirabilis and Serratia marcescens. The blaKPC gene is typically located on the Tn4401 transposon, which may be carried on various plasmids. Transfer of mobile genetic elements may serve as a vector for inter-species transmission and thus poses a serious challenge for infection control measures. In this report, we describe three unusual Enterobacteriaceae species carrying the blaKPC-2 gene: Leclercia adecarboxylata, Kluyvera cryocrescens and Kluyvera ascorbata. The molecular and microbiological characteristics of these isolates are discussed. All strains in this research were isolated from rectal surveillance cultures of patients from three medical centres: Laniado Medical Center (LMC), Tel Aviv Sourasky Medical Center (TASMC) and Rambam Medical Center (RMC). Table 1 summarizes the molecular and microbiological characteristics of the isolates. Bacterial identification was performed with the VITEK-2 system using GN-ID and GN09 cards (bioMerieux, Marcy l’Etoile, France) and confirmed by 16S rDNA sequencing. We identified the isolates as K. cryocrescens (two isolates), K. ascorbata (one isolate) and L. adecarboxylata (one isolate). All isolates carried the blaKPC-2 gene. 5 Plasmid DNAs purified from the four isolates were transformed into E. coli DH10B. Transformants were selected on lysogeny broth (LB) agar supplemented with 100 mg/L ampicillin and confirmed by PCR. Susceptibility to ertapenem, imipenem and meropenem was tested by agar dilution and interpreted based on the 2012 CLSI criteria. All isolates were resistant to ertapenem, imipenem and meropenem. In the transformants, the MICs of ertapenem, imipenem and meropenem were 2–8, 4–8 and 2–4 mg/L, respectively. Determination of plasmid sizes was done as previously described, revealing various sizes of the blaKPC-2-harbouring plasmids. The plasmid incompatibility group (Inc) was identified as IncN. The blaKPC-2 gene was located on the Tn4401 transposon and was typed by sequencing as Tn4401c. The presence of the Tn4401c variant in these isolates is in contrast with isolates belonging to the Israeli epidemic clone, K. pneumoniae ST258, which harbours the Tn4401a variant on the pKpQIL plasmid, which belongs to the IncFII incompatibility group. It was shown previously that isolates harbouring Tn4401c had a 10-fold reduction in their blaKPC expression. This also correlated with lower MICs of carbapenems. The identical plasmid incompatibility group and Tn4401 type in all isolates suggests horizontal inter-species transmission of the Tn4401c element between different plasmids of the same type, as recently described by Mathers et al. Kluyvera spp. and L. adecarboxylata are present ubiquitously in the environment in water and soil and were also described as part of the normal gastrointestinal flora in humans. Human infections with these bacteria are rare. Several cases of L. adecarboxylata infections in humans have been reported, especially in immunocompromised patients. In such patients, L. adecarboxylata was reported to cause bacteraemia, sepsis, peritonitis, cellulitis, endocarditis and cholecystitis. Reported human infections caused by Kluyvera spp. include bacteraemia, soft tissue infections, intra-abdominal abscesses and urinary tract infections. Recently, Ribeiro et al. reported on the first isolate of a carbapenem-resistant Kluyvera georgiana possessing the blaKPC-2 gene. Although clinical infections were absent in all of these cases, the spread of blaKPC-carrying plasmids into unusual Enterobacteriaceae species is of concern from an infection control perspective. These bacteria, though commonly regarded as avirulent, could serve as a reservoir for the spread of resistance genes between different species, leading to dissemination of resistant bacteria between patients and in the environment.

Inhibitor-based methods for the detection of KPC carbapenemase-producing Enterobacteriaceae in clinical practice by using boronic acid compounds

Enterobacteriaceae clinical strains that produce the class A carbapenem-hydrolysing enzyme KPC (Klebsiella pneumoniae carbapenemase) are increasingly reported worldwide, and are already endemic in North and South America, China, Israel and Greece. The accurate detection of KPC enzymes is of utmost importance for containing the global spread of KPC producers. Currently, the detection of putative carbapenemase production is based on an initial phenotypic screen for carbapenem resistance followed by the modified Hodge test (MHT) as a confirmatory test. However, the MHT is often difficult to interpret, is not specific for carbapenemase activity due to KPC and there are reports of false-positive results with CTX-M-positive or AmpC-hyperproducing Enterobacteriaceae. Boronic acid compounds are serine-type beta-lactamase inhibitors that were employed originally for the detection of class C plasmidic AmpCs in Enterobacteriaceae. Recently, they have also been evaluated for the differentiation of KPC-producing Enterobacteriaceae. In that respect, combined-disc tests using carbapenems with and without phenylboronic acid (PBA) have been proposed as the most accurate phenotypic tests for detecting KPC production. When these disc tests are extended to include carbapenem discs with EDTA or both PBA and EDTA on the same plate, the production of metallo-beta-lactamase (MBL) or both KPC and MBL, respectively, can also be accurately detected. Phenotypic tests based on the inhibitory activity of boronic acid compounds are very easy to perform and interpret, and may be applied from the first day of isolation of the suspected resistant Enterobacteriaceae. We think that they could effectively replace MHT for the convenient and early detection of KPC carbapenemases in regions where these enzymes are common.

Emergence of clonally related Klebsiella pneumoniae isolates of sequence type 258 producing KPC-2 carbapenemase in Belgium

Sir, The Klebsiella pneumoniae carbapenemase (KPC) is a class A b-lactamase that confers resistance to virtually all b-lactams including carbapenems. Initially reported in the USA in a Klebsiella isolate in 1996, KPC-producing K. pneumoniae have been responsible for large nosocomial outbreaks in the USA, Israel and Greece where these isolates are now endemic in hospitals. The rapid dissemination of KPC enzymes among and across different enterobacterial species is likely to be due to the localization of blaKPC genes on transferable broad host range plasmids and their association with a transposon, but is also linked with a disseminated international clone of KPC-producing K. pneumoniae isolates of sequence type (ST) 258. Here we report on the first three cases of KPC-2-producing ST258 K. pneumoniae that were detected in Belgium, subsequent to the transfer of patients from three Greek hospitals. In 2009, a patient with high-grade bladder neoplasm with pelvic metastases was transferred from a hospital in Greece to St-Pierre University Hospital in Brussels. Six weeks later, a patient with severe trauma was transferred from another hospital in the same region of Greece to the intensive care unit (ICU) of the same Brussels hospital. Finally, a few days later, the third polytrauma patient was transferred from the ICU of a hospital in Rhodes to the ICU of Erasme University Hospital in Brussels. Upon admission, a urine culture for the first patient and rectal swabs from the two other patients revealed the presence of multidrug-resistant K. pneumoniae (isolates Hk1, Hk2 and Rh1, respectively) with reduced susceptibility to carbapenems. No secondary local transmission occurred in the two hospitals following the rapid implementation of strict infection control measures. MICs produced using the Vitek2 system (N046 AST cards) and Etest (AB BIODISK, Solna, Sweden) were interpreted according to clinical breakpoints from the European Committee for Antimicrobial Susceptibility Testing (EUCAST). The three multidrug-resistant isolates showed high-level resistance to all penicillins (including temocillin) and to all expanded-spectrum cephalosporins except cefepime to which they all remained borderline susceptible (MICs of 8–16 mg/L) (Table 1). Interestingly, the three isolates appeared to be of intermediate susceptibility to meropenem using the Vitek2 system (MIC of 4–8 mg/L) while they were resistant to meropenem by Etest (MIC of 32 mg/L). All the isolates remained susceptible to gentamicin but were of intermediate

Carbapenemases in Enterobacteriaceae: Activity, Epidemiology, and Laboratory Detection

Carbapenemases are β-lactamases that confer resistance to the carbapenems (e.g., imipenem, meropenem, ertapenem, and doripenem). Most often these enzymes confer resistance to the other β-lactam agents as well, including extended-spectrum cephalosporins. The enzymes are usually found in bacterial isolates that are already resistant to nearly all other antimicrobial agents, and treatment options for infections caused by them are significantly limited. This important mechanism of resistance is emerging in the United States, and the most common enzyme is the Klebsiella pneumoniae carbapenemase (KPC). The KPC enzyme is found in many different genera of Enterobacteriaceae, but most commonly in K. pneumoniae. Currently, KPC-producing isolates are commonly isolated in the northeastern part of the U.S., but reports of isolates from other locations are increasing. Prevention and control of this emerging resistance is complicated by the occurrence of KPC-positive isolates producing low-level carbapenem resistance that is hard to detect in the clinical microbiology laboratory. The most sensitive methods for detecting carbapenemase-producing isolates are to look for elevated but susceptible carbapenem susceptibility results. In addition to discussing KPC, this article will review the epidemiology of other carbapenemases and their potentials for dissemination in the U.S.