KPC Carbapenemases Research Articles

Carbapenem-Resistant Enterobacteriaceae in Solid Organ Transplantation: Management Principles.

Carbapenem-resistant Enterobacteriaceae (CRE) have emerged as a worldwide problem. Given their degree of immunosuppression and the level of contact with the healthcare system, solid organ transplant (SOT) recipients are at a disproportionately higher risk of acquisition, colonization, and infection with CRE, and outcomes from infection tend to be worse compared to non-transplant patients. Therapeutic options are limited for CRE infections although several newer agents have recently been approved for use. How well these agents perform in the setting of immunosuppression and SOT is unclear. We sought to review the epidemiology of CRE in SOT and the management principles. CRE infections are becoming an increasing problem in SOT, and donor-derived infections present a challenge in the peri-transplant period. Newer treatments for CRE are emerging that are less toxic and potentially more effective than prior CRE-active agents, but supportive clinical data are limited. Newer beta-lactamase inhibitors have good activity against KPC carbapenemases, but they lack activity against metallo-beta-lactamases (e.g., NDM). Promising data is emerging with newer agents that have activity against most carbapenemases, but, again, clinical data is needed. Combination therapy in addition to optimal pharmacokinetic and pharmacodynamics may go some way to improve outcomes against these difficult-to-treat organisms. Other novel therapies that prevent the emergence of resistance (oral beta-lactamase inhibitors) and eradication of resistant Gram-negative colonization (fecal microbiota transplant) may eventually become part of a bundle approach to reduce CRE infections in the future. As in non-transplant patients, CRE infections in the transplant setting are challenging to treat and prevent. Infection prevention and control remains crucial to prevent widespread dissemination, and unique challenges exist with donor-derived CRE and how best to manage recipients in the peri-transplant period. Newer treatments are now in early-phase clinical studies, and in vitro activity data are supportive for several agents providing hope for improved outcomes with these typically difficult-to-treat and highly morbid infections in transplant recipients.

Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period

IntroductionThe rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. MethodsA retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). ResultsThe predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of β-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p<0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 β-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. ConclusionThis study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen.

Evaluation of a rapid immunochromatographic test for detection of KPC in clinical isolates of Enterobacteriaceae and Pseudomonas species

The KPC K-SeT immunochromatographic test (Coris BioConcept®, Gembloux, Belgium) has been widely used for detection of KPC in Enterobacteriaceae with reported sensitivities and specificities of 100%. However, to our knowledge, there are no reports of its use in KPC-positive Pseudomonas species. We evaluated the KPC K-SeT test in 36 clinical isolates of Enterobacteriaceae (21 KPC-positive and 15 KPC-negative) and 20 Pseudomonas species (5 KPC-positive and 15 KPC-negative) using conventional PCR for carbapenemase genes as the reference method. The KPC K-SeT test detected 25 out of 26 KPC-positive isolates (96.1%). The undetected isolate was 1 P. aeruginosa bearing the mutation D179Y in the omega loop region of KPC-2 carbapenemase. This mutation was already reported in Enterobacteriaceae as conferring resistance to ceftazidime-avibactam. To our knowledge, this is the first report of evaluation of KPC K-SeT test in KPC-positive P. aeruginosa isolates.

Evaluating the Efficacies of Carbapenem/β-Lactamase Inhibitors Against Carbapenem-Resistant Gram-Negative Bacteria in vitro and in vivo.

BackgroundCarbapenem-resistant Gram-negative bacteria are a major clinical concern as they cause virtually untreatable infections since carbapenems are among the last-resort antimicrobial agents. β-Lactamases implicated in carbapenem resistance include KPC, NDM, and OXA-type carbapenemases. Antimicrobial combination therapy is the current treatment approach against carbapenem resistance in order to limit the excessive use of colistin; however, its advantages over monotherapy remain debatable. An alternative treatment strategy would be the use of carbapenem/β-lactamase inhibitor (βLI) combinations. In this study, we assessed the in vitro and in vivo phenotypic and molecular efficacies of three βLIs when combined with different carbapenems against carbapenem-resistant Gram-negative clinical isolates. The chosen βLIs were (1) Avibactam, against OXA-type carbapenemases, (2) calcium-EDTA, against NDM-1, and (3) Relebactam, against KPC-2.MethodsSix Acinetobacter baumannii clinical isolates were screened for blaOXA-23-like, blaOXA-24/40, blaOXA-51-like, blaOXA-58, and blaOXA-143-like, and eight Enterobacteriaceae clinical isolates were screened for blaOXA-48, blaNDM-1, and blaKPC-2. The minimal inhibitory concentrations of Imipenem (IPM), Ertapenem (ETP), and Meropenem (MEM) with corresponding βLIs for each isolate were determined. The efficacy of the most suitable in vitro treatment option against each of blaOXA-48, blaNDM-1, and blaKPC-2 was assessed via survival studies in a BALB/c murine infection model. Finally, RT-qPCR was performed to assess the molecular response of the genes of resistance to the carbapenem/βLI combinations used under both in vitro and in vivo settings.ResultsCombining MEM, IPM, and ETP with the corresponding βLIs restored the isolates’ susceptibilities to those antimicrobial agents in 66.7%, 57.1%, and 30.8% of the samples, respectively. Survival studies in mice revealed 100% survival rates when MEM was combined with either Avibactam or Relebactam against blaOXA-48 and blaKPC-2, respectively. RT-qPCR demonstrated the consistent overexpression of blaOXA-48 upon treatment, without hindering Avibactam’s activity, while blaNDM-1 and blaKPC-2 experienced variable expression levels upon treatment under in vitro and in vivo settings despite their effective phenotypic results.ConclusionNew carbapenem/βLI combinations may be viable alternatives to antimicrobial combination therapy as they displayed high efficacy in vitro and in vivo. Meropenem/Avibactam and Meropenem/Relebactam should be tested on larger sample sizes with different carbapenemases before progressing further in its preclinical development.

Antimicrobial and KPC/AmpC inhibitory activity of functionalized benzosiloxaboroles

A series of 22 benzosiloxaboroles, silicon analogues of strong antimicrobial agents - benzoxaboroles, have been synthesized and tested against β-lactamases KPC- and pAmpC-producing strains of Gram-negative rods. Comprehensive structural-property relationship studies supported by molecular modelling as well as biological studies reveal that 6-B(OH)2-substituted derivative 27 strongly inhibits the activity of cephalosporinases (chromosomally encoded AmpC and plasmid encoded CMY-2) and KPC carbapenemases. It also shows strong ability to inhibit growth of the strains producing KPC-3 when combined with meropenem. In addition, halogen-substituted (mono-, di- or tetra-) benzosiloxaboroles demonstrate high antifungal activity (MIC 1.56–6.25 mg/L) against C. tropicalis, C. guilliermondii and S. cerevisiae. The highest activity against pathogenic yeasts (C. albicans, C. krusei and C. parapsilosis - MICs 12.5 mg/L) and against Gram-positive cocci (S. aureus and E. faecalis - 6.25 mg/L and 25 mg/L respectively) was displayed by 6,7-dichloro-substituted benzosiloxaborole. The studied systems exhibit low cytotoxity toward human lung fibroblasts.

Whole genome assembly and functional portrait of hypervirulent extensively drug-resistant NDM-1 and KPC-2 co-producing Klebsiella pneumoniae of capsular serotype K2 and ST86.

To characterize an emergent carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) strain, NUHL30457, which co-produces NDM-1 and KPC-2 carbapenemases. We performed WGS analysis on a clinical carbapenemase-producing hypervirulent K. pneumoniae (CP-hvKP) strain NUHL30457. Sequence data were analysed using comparative genomics and phylogenetics. WGS was used to perform MLST, capsular genotyping and identification of virulence and antimicrobial resistance genes. The virulence of NUHL30457 was analysed by serum killing assay, neutrophil phagocytosis and mouse lethality assay. The NUHL30457 strain was carbapenem resistant and belonged to ST86 and serotype K2. A significant increase in resistance to serum killing and antiphagocytosis was found in the NUHL30457 strain compared with the reference strain. The murine lethality assay showed an LD50 of 2.5 × 102 cfu for the NUHL30457 strain, indicating hypervirulence. WGS revealed that NUHL30457 has a single 5.3 Mb chromosome (57.53% G + C content) and four plasmids in the range 49.2-215.7 kb. The incompatibility group (Inc)N plasmid p30457-4 carried the blaNDM-1 and qnrS1 genes. The IncFII(K) plasmid p30457-3 also carried an array of resistance elements, including blaCTX-M-65, blaTEM-1 and blaKPC-2. The IncHI1/IncFIB plasmid p30457-1, which carried virulence genes, was identical to a pLVPK plasmid reported previously. To the best of our knowledge, this is the first report to isolate an ST86 hvKP strain that co-produces NDM-1 and KPC-2 carbapenemase. Further investigation is required to reinforce our understanding of the epidemiology and virulence mechanisms of this clinically significant CP-hvKP.

In vitro activity of imipenem-relebactam against non-MBL carbapenemase-producing Klebsiella pneumoniae isolated in Greek hospitals in 2015-2016.

Relebactam is a β-lactamase inhibitor of class A and class C β-lactamases, including carbapenemases. We evaluated the ability of relebactam to restore imipenem susceptibility against a collection of Klebsiella pneumoniae isolates from Greek hospitals. We tested 314 non-MBL carbapenemase-producing K. pneumoniae consecutive clinical strains isolated from unique patients at 18 hospitals in Greece, between November 2014 and December 2016. Susceptibility testing of imipenem, imipenem-relebactam, meropenem, doripenem, gentamicin, and colistin was performed using broth microdilution. Additionally, MICs of ceftazidime-avibactam, fosfomycin, and tigecycline were determined by MIC Test Strips. MICs were interpreted per EUCAST breakpoints. Imipenem-relebactam MICs were interpreted using the breakpoints proposed for imipenem. Carbapenemase genes were detected using PCR. Whole genome sequencing was performed for selected isolates. Imipenem-relebactam inhibited 98.0% of the KPC-producing isolates at ≤ 2mg/L (MIC50/90, 0.25/1mg/L) and was considerably more active than imipenem (MIC50/90, 32/> 64mg/L). Reduced activity of imipenem-relebactam was rarely detected (2%) and was associated with chromosomal factors (ompK35 disruption and/or mutated ompK36). Only ceftazidime-avibactam showed in vitro activity comparable to imipenem-relebactam (99.6% susceptible). Relebactam provided only weak potentiation of imipenem activity against K. pneumoniae with class D OXA-48-like enzymes. Relebactam exhibited strong potential for restoring the in vitro activity of imipenem against KPC-producing K. pneumoniae, lowering the imipenem MIC50 and MIC90 from 32 to 0.25mg/L, and from > 64 to 1mg/L, respectively. Production of KPC carbapenemase represents the main cause of carbapenem resistance among K. pneumoniae in Greek hospitals (66.5%), and this carbapenemase appears to be very well inhibited by relebactam.

Structure of the capsular polysaccharide of the KPC-2-producing Klebsiella pneumoniae strain KK207-2 and assignment of the glycosyltransferases functions.

Klebsiella pneumoniae strain KK207-2 was isolated in 2010 from a bloodstream infection of an inpatient at an Italian hospital. It was previously found to produce the KPC-2 carbapenemase and to belong to clade 1 of sequence type 258. Genotyping of the conserved wzi and wzc genes from strain KK207-2 yielded contrasting results: the wzc-based method assigned the cps207-2 to a new K-type, while the wzi-based method assigned it to the known K41 K-type. In order to resolve this contradiction, the capsular polysaccharide of K. pneumoniae KK207-2 was purified and its structure determined by using GLC-MS of appropriate carbohydrate derivatives, ESI-MS of both partial hydrolysis and Smith degradation derived oligosaccharides, and NMR spectroscopy of oligosaccharides, and the lithium degraded, native and de-O-acetylated polysaccharide. All the collected data demonstrated the following repeating unit for the K. pneumoniae KK207-2 capsular polysaccharide: The polysaccharide contains about 0.60 acetyl groups per repeating unit on C6 of the Gal residue. The reactions catalyzed by each glycosyltransferase in the cpsKK207-2 gene cluster were assigned on the basis of structural homology with other Klebsiella K antigens.

Plasmid-Mediated Transmission of KPC-2 Carbapenemase in Enterobacteriaceae in Critically Ill Patients.

Carbapenem-resistant Enterobacteriaceae (CRE) cause health care-associated infections worldwide, and they are of severe concern due to limited treatment options. We report an outbreak of KPC-2-producing CRE that was caused by horizontal transmission of a promiscuous plasmid across different genera of bacteria and hospitals in Germany. Eleven isolates (8 Citrobacter freundii, 2 Klebsiella oxytoca, and 1 Escherichia coli) were obtained from seven critically ill patients during the six months of the outbreak in 2016. One patient developed a CRE infection while the other six patients were CRE-colonized. Three patients died in the course of the hospital stay. Six of the seven patients carried the same C. freundii clone; one K. oxytoca clone was found in two patients, and one patient carried E. coli and C. freundii. Molecular analysis confirmed the presence of a conjugative, blaKPC-2-carrying 70 kb-IncN plasmid in C. freundii and E. coli and an 80 kb-IncN plasmid in K. oxytoca. All transconjugants harbored either the 70 or 80 kb plasmid with blaKPC-2, embedded within transposon variant Tn4401g. Whole genome sequencing and downstream bioinformatics analyses of all plasmid sequences showed an almost perfect match when compared to a blaKPC-2-carrying plasmid of a large outbreak in another German hospital two years earlier. Differences in plasmid sizes and open reading frames point to the presence of inserted mobile genetic elements. There are few outbreak reports worldwide on the transmission of blaKPC-2-carrying plasmids across different bacterial genera. Our data suggest a regional and supraregional spread of blaKPC-2-carrying IncN-plasmids harboring the Tn4401g isoform in Germany.

Characterize carbapenem-resistant Klebsiella pneumoniae isolates for nosocomial pneumonia and their Gram-negative bacteria neighbors in the respiratory tract.

This study was conducted to perform an epidemiological survey of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates for nosocomial pneumonia (NP) and visit their Gram-negative bacteria neighbors (GNNs) from the respiratory tracts. Pulsed-field gel electrophoresis and multi locus sequence typing were performed to evaluate the clonal relationships of these isolates. Statistical methods were then used to understand their possible relationship between CRKP and their GNNs. Among the 63 CRKP isolates, 84.1% produced a KPC-2 carbapenemase gene, followed by blaNDM-1 and blaIMP-38 like. 13 different STs and 29 clones were identified. Less heterogeneous clone backgrounds were observed in 53 KPC-2K. pneumoniae isolates, and 6 STs have been found to contain KPC-2, of which the predominant ST11 harbored 47 KPC-2-producing K. pneumoniae isolates. Particularly, the two infrequent isolates co-possessing blaKPC-2 and blaIMP-38 like shared a different clonal relationship. 87 GNNs isolates of CRKP were established, the analysis present that different strains showed significant or no difference with CRKP upon antibiotics susceptibility. Conclusion, ST11 harboring KPC-2-producing K. pneumoniae isolates were dominant for NP, and both clonal spread and horizontal transfer contributed to the dissemination of CRKP. However, additional studies are needed to explore the biological relationship with their GNNs.

Virulence and resistance determinants of Klebsiella pneumoniae isolated from a Portuguese tertiary university hospital centre over a 31-year period

IntroductionThe rapid and complex evolution of bacterial resistance mechanisms in Klebsiella pneumoniae producing extended-spectrum β-lactamases and carbapenemases in Klebsiella pneumoniae is one of the most significant threats to public health. However, questions and controversies regarding the interactions between resistance and virulence in multidrug-resistant K. pneumoniae isolates remain unclear. MethodsA retrospective cohort study was performed with 100 K. pneumoniae isolates recovered from a tertiary care university hospital centre in Lisbon over a 31-year period. Resistance and virulence determinants were screened using molecular methods (PCR, M13-PCR and MLST). ResultsThe predominant virulence profile (fimH, mrkDv1, khe) was shared by all isolates, indicative of an important role of type 1 and 3 fimbrial adhesins and haemolysin, regardless of the type of β-lactamase produced. However, accumulation of virulence factors was identified in KPC-3-producers, with a higher frequency (p<0.05) of capsular serotype K2 and iucC aerobactin when compared with non-KPC-3 β-lactamases or carbapenemases. Additionally, 9 different virulence profiles were found, indicating that the KPC-3 carbapenemase producers seem to adapt successfully to the host environment and maintain virulence via several pathways. ConclusionThis study describes an overlapping of multidrug-resistance and virulence determinants in ST-14K2 KPC-3 K. pneumoniae clinical isolates that may impose an additional challenge in the treatment of infections caused by this pathogen.

Transcriptional Landscape of a bla KPC-2 Plasmid and Response to Imipenem Exposure in Escherichia coli TOP10.

The diffusion of KPC-2 carbapenemase is closely related to the spread of Klebsiella pneumoniae of the clonal-group 258 and linked to IncFIIK plasmids. Little is known about the biology of multi-drug resistant plasmids and the reasons of their successful dissemination. Using E. coli TOP10 strain harboring a multi-replicon IncFIIK-IncFIB blaKPC−2-gene carrying plasmid pBIC1a from K. pneumoniae ST-258 clinical isolate BIC-1, we aimed to identify basal gene expression and the effects of imipenem exposure using whole transcriptome approach by RNA sequencing (RNA-Seq). Independently of the antibiotic pressure, most of the plasmid-backbone genes were expressed at low levels. The most expressed pBIC1a genes were involved in antibiotic resistance (blaKPC−2, blaTEM and aph(3′)-I), in plasmid replication and conjugation, or associated to mobile elements. After antibiotic exposure, 34% of E. coli (pBIC1a) genome was differentially expressed. Induction of oxidative stress response was evidenced, with numerous upregulated genes of the SoxRS/OxyR oxydative stress regulons, the Fur regulon (for iron uptake machinery), and IscR regulon (for iron sulfur cluster synthesis). Nine genes carried by pBIC1a were up-regulated, including the murein DD-endopeptidase mepM and the copper resistance operon. Despite the presence of a carbapenemase, we observed a major impact on E. coli (pBIC1a) whole transcriptome after imipenem exposure, but no effect on the level of transcription of antimicrobial resistance genes. We describe adaptive responses of E. coli to imipenem-induced stress, and identified plasmid-encoded genes that could be involved in resistance to stressful environments.

In silico identification and experimental validation of hits active against KPC-2 β-lactamase.

Bacterial resistance has become a worldwide concern, particularly after the emergence of resistant strains overproducing carbapenemases. Among these, the KPC-2 carbapenemase represents a significant clinical challenge, being characterized by a broad substrate spectrum that includes aminothiazoleoxime and cephalosporins such as cefotaxime. Moreover, strains harboring KPC-type β-lactamases are often reported as resistant to available β-lactamase inhibitors (clavulanic acid, tazobactam and sulbactam). Therefore, the identification of novel non β-lactam KPC-2 inhibitors is strongly necessary to maintain treatment options. This study explored novel, non-covalent inhibitors active against KPC-2, as putative hit candidates. We performed a structure-based in silico screening of commercially available compounds for non-β-lactam KPC-2 inhibitors. Thirty-two commercially available high-scoring, fragment-like hits were selected for in vitro validation and their activity and mechanism of action vs the target was experimentally evaluated using recombinant KPC-2. N-(3-(1H-tetrazol-5-yl)phenyl)-3-fluorobenzamide (11a), in light of its ligand efficiency (LE = 0.28 kcal/mol/non-hydrogen atom) and chemistry, was selected as hit to be directed to chemical optimization to improve potency vs the enzyme and explore structural requirement for inhibition in KPC-2 binding site. Further, the compounds were evaluated against clinical strains overexpressing KPC-2 and the most promising compound reduced the MIC of the β-lactam antibiotic meropenem by four-fold.

700. Identification and Whole-Genome Sequencing (WGS) of Meropenem-Vaborbactam (MV) Resistant Klebsiella pneumoniae (MVRKP) Among Patients Without Prior Exposure to MV: Collateral Damage

BackgroundMV is a newly approved β-lactam/β-lactamase inhibitor combination (BLIC) for the treatment of complicated urinary tract infections (cUTI). Vaborbactam is a cyclic boronic acid BLI that was mainly developed as a potent inhibitor of KPC carbapenemases and other Ambler class A&C enzymes. Vaborbactam is inactive against metallo-β-lactamases (MBL) and certain Class D enzymes (e.g. OXA-2 and OXA-48). We encountered a case of MV-resistant Klebsiella pneumoniae (MVRKP)and sought to explore the various mechanisms of MV resistance within KP.MethodsA 65-year-old nursing home resident with multiple prior hospitalizations and recent exposure to antibiotics (Timeline) developed sepsis secondary to carbapenem-resistant Klebsiella pneumoniae (CRKP) cUTI. WGS of the patient’s isolate was performed. This was followed by random screening for MV resistance and WGS of other isolates from a historical database.ResultsResults of WGS are seen in the table below. Sequencing of our patient’s isolate revealed strain ST258 with a premature stop in aa89 of OmpK35 as well as insertions at Gly134 and Asp135 (i.e., the GD repeat) of OmpK36. Furthermore, the KPC plasmid’s copy number was approximately five times higher than the chromosome. No mutations encoding efflux system AcrAB-TolC were found.ConclusionResistance to MV in KP was found in isolates that predate the drug’s availability. Notably, resistance occurred in the absence of MBLs and OXAs. The mechanism seems to involve outer membrane porin mutations in OmpK35 and/or OmpK36. WGS is a useful tool in identifying the mechanism of resistance especially for newer agents.Table: Characterization of MVRKP by WGS Typing Enzymes Efflux Outer Membrane Porin Variant Strain Date MV MIC (mg/mL) MLST β-Lactamase Multidrug Transporters and Regulators OmpK35 OmpK36 Notes 12-1-1216 ST258KPC-2 & SHV-160 Emr, mexk, oqxAB, smeD FS 121insG S to CZA and TGC 24-3-1216ST258KPC-2 & SHV-160 Emr, mexk, oqxAB, smeD FS 121insG S to CZA and TGC 32013 16 ST258 KPC-2 & SHV-160 Emr, mexk, oqxAB, smeD FS 121insG S to CZA and TGC 420134ST258KPC-2 & SHV-160 Emr, mexk, oqxAB, smeD FS 121insG S to CZA and TGC 52017 32 ST258KPC-2, SHV-11, SHV-12 N/A STOP aa89 134-135 GD ins CZA MIC 8 Disclosures All authors: No reported disclosures.

In vitro and in vivo bactericidal activity of ceftazidime-avibactam against Carbapenemase–producing Klebsiella pneumoniae

BackgroundIn recent years, the incidence of carbapenem-resistant Enterobacteriaceae (CRE) infections has increased rapidly. Since the CRE strain is usually resistant to most of antimicrobial agents, patients with this infection are often accompanied by a high mortality. Therefore, it instigates a severe challenge the clinical management of infection. In this study, we study the in vitro and in vivo bactericidal activity of ceftazidime-avibactam administrated either alone or in combination with aztreonam against KPC or NDM carbapenemase-producing Klebsiella pneumoniae, and explore a new clinical therapeutic regimen for infections induced by their resistant strains.MethodsThe microdilution broth method was performed to analyze the minimal inhibitory concentration (MIC). The time-kill curve assay of ceftazidime-avibactam at various concentrations was conducted in 16 strains of KPC-2 and 1 strain of OXA-232 carbapenemase–producing Klebsiella pneumoniae. The in vitro synergistic bactericidal effect of ceftazidime-avibactam combined with aztreonam was determined by checkerboard assay on 28 strains of NDM and 2 strains of NDM coupled with KPC carbapenemase–producing Klebsiella pneumoniae. According to calculating grade, the drugs with synergistic bactericidal effect were selected as an inhibitory concentration index. The in vitro bactericidal tests of ceftazidime-avibactam combined with aztreonam were implemented on 12 strains among them. Effect of ceftazidime-avibactam antibiotic against KPC carbapenemase–producing K. pneumoniae strain Y8 Infection was performed in the mouse model.ResultsThe time-kill assays revealed that ceftazidime-avibactam at various concentrations of 2MIC, 4MIC and 8MIC showed significant bactericidal efficiency to the resistant bacteria strains. However, in 28 strains of NDM and 2 strains of NDM coupled with KPC carbapenemase- producing Klebsiella pneumoniae, only 7 strains appeared the susceptibility to ceftazidime-avibactam treatment, MIC50 and MIC90 were 64 mg/L and 256 mg/L, respectively. Antimicrobial susceptibility testing of ceftazidime-avibactam combined with aztreonam disclosed the synergism of two drugs in 90% (27/30) strains, an additive efficiency in 3.3% (1/30) strains, and irrelevant effects in 6.6% (2/30) strains. No antagonism was found. The subsequent bactericidal tests also confirmed the results mentioned above. Therapeutic efficacy of Ceftazidime-Avibactam against K. pneumoniae strain Y8 infection in mouse indicated 70% of infection group mice died within 4 days, and all mice in this group died within 13 days. Bacterial load testing results showed that there was no significant difference in the amount of bacteria in the blood between the infected group and the treatment group. However, the spleen and liver of treatment group mice showed lower CFU counts, as compare with infected group, indicating that ceftazidime-avibactam has a significant effect on the bacteria and led to a certain therapeutic efficacy.ConclusionThis study indicated ceftazidime-avibactam therapy occupied significant bactericidal effects against KPC-2 and OXA-232 carbapenemase-producing Klebsiella pneumoniae. While combined with aztreonam, the stronger synergistic bactericidal effects against NDM carbapenemase-producing Klebsiella pneumoniae were achieved.

Evaluation of the EntericBio CPE assay for the detection of carbapenemase-producing organisms.

Carbapenemase-producing organisms (CPOs) represent an increasing threat in healthcare facilities and detection of these organisms in the diagnostic laboratory can be challenging. The EntericBio CPE (EBCPE) real-time PCR assay (Serosep Ltd) was evaluated for the detection of NDM, KPC, OXA-48-like, VIM, IMP and GES carbapenemase genes from a panel of 145 multidrug-resistant organisms (29 NDM, 35 OXA-48, 21 VIM, 4 OXA-23, 3 KPC, 5 NDM+OXA-48, 3 GES-5, 1 OXA-23+NDM, 1 IMI, 2 IMP-1 and 41 multidrug-resistant carbapenemase-negative isolates). The EBCPE assay performed well, with 100 % sensitivity and specificity for the detection of all genotypes included in the assay. Turnaround time and laboratory workflow were improved compared to culture-based assays. Users must remain aware of the limitations of molecular assays for CPO detection to ensure implementation of the most suitable CPO diagnostic pathways.

Selection of mutants with resistance or diminished susceptibility to ceftazidime/avibactam from ESBL- and AmpC-producing Enterobacteriaceae.

Difficult Gram-negative infections are increasingly treated with new β-lactamase inhibitor combinations, e.g. ceftazidime/avibactam. Disturbingly, mutations in KPC carbapenemases can confer ceftazidime/avibactam resistance, which is sometimes selected during therapy. We explored whether this risk extended to AmpC and ESBL enzymes. Mutants were selected by plating AmpC-derepressed strains, ESBL producers and ceftazidime-susceptible controls on agar containing ceftazidime + avibactam (1 or 4 mg/L). MICs were determined by CLSI agar dilution; WGS was by Illumina methodology. Using 2× MIC of ceftazidime + 1 mg/L avibactam, mutants were selected from all strain types at frequencies of 10-7-10-9. Rates diminished to <10-9 with 4 mg/L avibactam or higher MIC multiples, except with AmpC-derepressed Enterobacteriaceae. Characterized mutants (n = 10; MICs 4-64 mg/L) of AmpC-derepressed strains had modifications in ampC, variously giving Arg168Pro/His, Gly176Arg/Asp, Asn366Tyr or small deletions around positions 309-314. Mutants of ESBL producers (n = 19; MICs 0.5-16 mg/L) mostly had changes affecting permeability, efflux or β-lactamase quantity; only one had an altered β-lactamase, with an Asp182Tyr substitution in CTX-M-15, raising the ceftazidime/avibactam MIC, but abrogating other cephalosporin resistance. Mutants of ceftazidime-susceptible strains were not sequenced, but phenotypes suggested altered drug accumulation or, for Enterobacter cloacae only, AmpC derepression. In further experiments, avibactam reduced, but did not abolish, selection of AmpC-derepressed Enterobacteriaceae by ceftazidime. Most mutants of AmpC-derepressed Enterobacteriaceae had structural mutations in ampC; those of ESBL producers mostly had genetic modifications outside β-lactamase genes, commonly affecting uptake, efflux, or β-lactamase quantity. The clinical significance of these observations remains to be determined.

Rapid detection of NDM, KPC and OXA-48 carbapenemases directly from positive blood cultures using a new multiplex immunochromatographic assay.

Bloodstream infections caused by carbapenemase-producing Enterobacteriaceae (CPE) are associated with treatment failure and increased mortality. Detection of CPE from blood cultures (BC) by standard methods takes 16–72 hours, which can delay the initiation of appropriate antimicrobial therapy and compromise patient outcome. In the present study, we developed and evaluated a new method for the rapid detection of carbapenemases directly from positive BC using a new multiplex immunochromatographic test (ICT). The new ICT was assessed using 170 molecularly characterized Enterobacteriaceae clinical isolates including 126 CPE (OXA-48-like (N = 79), KPC (N = 18) and NDM (N = 29)). After spiking with bacteria and incubation in a BC system, blood from positive BC bottles was hemolyzed, bacteria concentrated by centrifugation and lysed. The lysate was transferred to the RESIST-3 O.K.N. ICT (Coris BioConcept, Gembloux, Belgium), which detects OXA-48-like, KPC and NDM carbapenemases. The final results of the ICT were read when they became positive, at the latest after 15 min. All CPE isolates (126/126) were correctly detected with the new protocol (100% sensitivity, 100% specificity). There was perfect concordance between ICT results and molecular characterization. Total time to result was 20–45 min.Conclusions: This proof-of-principle study demonstrates that with the newly developed method, OXA-48-like, KPC and NDM carbapenemases can be reliably detected directly from positive BC bottles. The new method is more rapid than other currently available assays and can be performed in any routine microbiology laboratory. This can help to rapidly identify patients with CPE BSI and optimize the management of patients with these difficult-to-treat infections. Further studies are needed to assess the performance of the ICT in routine diagnostics.

Klebsiella grimontii, a New Species Acquired Carbapenem Resistance.

Klebsiella grimontii is a newly identified species closely related to Klebsiella oxytoca, but carbapenem resistance was not identified in the species before. We found a carbapenem-resistant K. oxytoca-like clinical strain, WCHKG020121. The strain was subjected to whole genome sequencing using Illumina HiSeq X10. The precise species identification was established based on average nucleotide identity (ANI) and in silico DNA–DNA hybridization (isDDH) between strain WCHKG020121 and type strains of Klebsiella species. Antimicrobial resistance genes were identified from the genome sequence. The sequence of the blaKPC-2-carrying plasmid was completed using PCR and Sanger sequencing. Conjugation experiments were performed to obtain the plasmid carrying blaKPC-2. All K. grimontii genomes were retrieved from GenBank and were analyzed for antimicrobial resistance genes. Strain WCHKG020121 was resistant to imipenem and meropenem (MIC for both, 32 μg/ml) but was susceptible to colistin (1 μg/ml). Strain WCHKG020121 was initially identified as K. oxytoca using Vitek II but it actually belongs to K. grimontii as it had a 98.81% ANI and 83.4% isDDH value with K. grimontii type strain. Strain WCHKG020121 had blaKPC-2; by contrast, none of other K. grimontii genomes carry any known carbapenemase genes. blaKPC-2 was carried by a 95,734-bp plasmid, designated pKPC2_020121, which contained two different FII(Y) replicons. pKPC2_020121 was closest (93% coverage, 99% identity) to blaKPC-2-carrying plasmids from Enterobacter hormaechei recovered in 2014 at the same hospital. pKPC2_020121 was not self-transmissible, which could be explained by the absence of a conjugation essential gene, traY. In conclusion, we reported the first K. grimontii strain that produced the KPC carbapenemase. Carbapenem resistant K. grimontii may represent a new threat.

Genomic analysis of the first isolate of KPC-2-producing Klebsiella pneumoniae from Uruguay.

Since KPC-2-producing Klebsiella pneumoniae are associated with successful dissemination of a major clone, defined as sequence type 258 (ST258), the aim of this study was to perform whole-genome sequencing (WGS) of the first colistin-resistant K. pneumoniae strain (Kpn666) carrying blaKPC-2 identified in Uruguay in 2011 in order to identify genomic and phylogenetic traits. WGS of strain Kpn666 isolated from an asymptomatic urinary tract infection was performed using Illumina MiSeq, and de novo assembly was performed using SPADES v.3.11. Contigs were re-ordered using the ST258 reference genome NJST258_1 (GenBank CP006923) and were oriented with the MAUVE Contig Mover. Twenty complete genomes of K. pneumoniae identified as ST258 using the Pasteur MLST site were downloaded from GenBank (May 2017). A maximum-likelihood tree was created using MEGA7 based on core single nucleotide polymorphisms (SNPs) from whole-genome alignment obtained with SNP sites (https://github.com/sanger-pathogens/snp-sites). WGS analysis revealed a genome of 5448179bp (5232 CDS, 108 RNAs). Phylogenetic analysis identified that Kpn666 belonged to clade I lineage of ST258. Further studies also identified IncR, IncFIB(K) and IncFII(K) plasmid replicons and 11 transferable associated antimicrobial resistance genes (ARGs) comprising four drug classes. The mgrB gene involved in colistin resistance was shown to be disrupted by insertion of an IS5-like element. The first isolate of KPC-2-producing K. pneumoniae detected in Uruguay was sequenced and the results confirm the ability of this bacterium to capture several ARGs. The KPC-2 carbapenemase in Uruguay is likely to have been introduced by the high-risk clone ST258.