scholarly journals Novel Carbapenem-Hydrolyzing β-Lactamase, KPC-1, from a Carbapenem-Resistant Strain of Klebsiella pneumoniae

2001 ◽  
Vol 45 (4) ◽  
pp. 1151-1161 ◽  
Author(s):  
Hesna Yigit ◽  
Anne Marie Queenan ◽  
Gregory J. Anderson ◽  
Antonio Domenech-Sanchez ◽  
James W. Biddle ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Clavulanic Acid ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Kinetic Studies ◽  
The Novel ◽  
E Coli ◽  
Carbapenem Resistant ◽  
Extended Spectrum ◽  
High Level

ABSTRACT A Klebsiella pneumoniae isolate showing moderate to high-level imipenem and meropenem resistance was investigated. The MICs of both drugs were 16 μg/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. The strain was also resistant to extended-spectrum cephalosporins and aztreonam. Isoelectric focusing studies demonstrated three β-lactamases, with pIs of 7.2 (SHV-29), 6.7 (KPC-1), and 5.4 (TEM-1). The presence of bla SHV andbla TEM genes was confirmed by specific PCRs and DNA sequence analysis. Transformation and conjugation studies withEscherichia coli showed that the β-lactamase with a pI of 6.7, KPC-1 (K. pneumoniae carbapenemase-1), was encoded on an approximately 50-kb nonconjugative plasmid. The gene,bla KPC-1, was cloned in E. coli and shown to confer resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of the novel carbapenem-hydrolyzing β-lactamase, KPC-1, showed 45% identity to the pI 9.7 carbapenem-hydrolyzing β-lactamase, Sme-1, fromSerratia marcescens S6. Hydrolysis studies showed that purified KPC-1 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and monobactams. KPC-1 had the highest affinity for meropenem. The kinetic studies also revealed that clavulanic acid and tazobactam inhibited KPC-1. An examination of the outer membrane proteins of the parent K. pneumoniae strain demonstrated that the strain does not express detectable levels of OmpK35 and OmpK37, although OmpK36 is present. We concluded that carbapenem resistance in K. pneumoniae strain 1534 is mainly due to production of a novel Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase, KPC-1, although alterations in porin expression may also play a role.

scholarly journals Effects of Klebsiella pneumoniae Carbapenemase Subtypes, Extended-Spectrum β-Lactamases, and Porin Mutations on theIn VitroActivity of Ceftazidime-Avibactam against Carbapenem-Resistant K. pneumoniae

2015 ◽  
Vol 59 (9) ◽  
pp. 5793-5797 ◽  
Author(s):  
Ryan K. Shields ◽  
Cornelius J. Clancy ◽  
Binghua Hao ◽  
Liang Chen ◽  
Ellen G. Press ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Mechanisms Of Resistance ◽  
Content Type ◽  
Klebsiella Pneumoniae Carbapenemase ◽  
Carbapenem Resistant ◽  
Extended Spectrum ◽  
High Level ◽  
Emergence Of Resistance

ABSTRACTAvibactam is a novel β-lactamase inhibitor with affinity forKlebsiella pneumoniaecarbapenemases (KPCs). In combination with ceftazidime, the agent demonstrates activity against KPC-producingK. pneumoniae(KPC-Kp). KPC-Kp strains are genetically diverse and harbor multiple resistance determinants, including defects in outer membrane proteins and extended-spectrum β-lactamases (ESBLs). Mutations in porin geneompK36confer high-level carbapenem resistance to KPC-Kp strains. Whether specific mechanisms of antimicrobial resistance also influence the activity of ceftazidime-avibactam is unknown. We defined the effects of ceftazidime-avibactam against 72 KPC-Kp strains with diverse mechanisms of resistance, including various combinations of KPC subtypes and ESBL andompK36mutations. Ceftazidime MICs ranged from 64 to 4,096 μg/ml and were lowered by a median of 512-fold with the addition of avibactam. All strains exhibited ceftazidime-avibactam MICs at or below the CLSI breakpoint for ceftazidime (≤4 μg/ml; range, 0.25 to 4). However, the MICs were within two 2-fold dilutions of the CLSI breakpoint against 24% of the strains, and those strains would be classified as nonsusceptible to ceftazidime by EUCAST criteria (MIC > 1 μg/ml). Median ceftazidime-avibactam MICs were higher against KPC-3 than KPC-2 variants (P= 0.02). Among KPC-2-Kp strains, the presence of both ESBL and porin mutations was associated with higher drug MICs compared to those seen with either factor alone (P= 0.003 andP= 0.02, respectively). In conclusion, ceftazidime-avibactam displays activity against genetically diverse KPC-Kp strains. Strains with higher-level drug MICs provide a reason for caution. Judicious use of ceftazidime-avibactam alone or in combination with other agents will be important to prevent the emergence of resistance.

scholarly journals Carbapenem-Resistant Strain of Klebsiella oxytoca Harboring Carbapenem-Hydrolyzingβ -Lactamase KPC-2

2003 ◽  
Vol 47 (12) ◽  
pp. 3881-3889 ◽  
Author(s):  
Hesna Yigit ◽  
Anne Marie Queenan ◽  
J. Kamile Rasheed ◽  
James W. Biddle ◽  
Antonio Domenech-Sanchez ◽  
...  
Keyword(s):  
Amino Acid ◽  
Clavulanic Acid ◽  
Outer Membrane Proteins ◽  
Klebsiella Oxytoca ◽  
Carbapenem Resistance ◽  
Kinetic Studies ◽  
Conjugative Plasmid ◽  
Single Amino Acid ◽  
Amino Acid Difference ◽  
Extended Spectrum

ABSTRACT We investigated a Klebsiella oxytoca isolate demonstrating resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The MICs of both imipenem and meropenem were 32μ g/ml. The β-lactamase activity against imipenem and meropenem was inhibited in the presence of clavulanic acid. Isoelectric focusing studies demonstrated five β-lactamases with pIs of 8.2 (SHV-46), 6.7 (KPC-2), 6.5 (unknown), 6.4 (probable OXY-2), and 5.4 (TEM-1). The presence of the bla SHV and bla TEM genes was confirmed by specific PCR assays and DNA sequence analysis. Transformation and conjugation studies with Escherichia coli showed that the β-lactamase with a pI of 6.7, Klebsiella pneumoniae carbapenemase-2 (KPC-2), was encoded on an approximately 70-kb conjugative plasmid that also carried SHV-46, TEM-1, and the β-lactamase with a pI of 6.5. The bla KPC-2 determinant was cloned in E. coli and conferred resistance to imipenem, meropenem, extended-spectrum cephalosporins, and aztreonam. The amino acid sequence of KPC-2 showed a single amino acid difference, S174G, when compared with KPC-1, another carbapenem-hydrolyzing β-lactamase from K. pneumoniae 1534. Hydrolysis studies showed that purified KPC-2 hydrolyzed not only carbapenems but also penicillins, cephalosporins, and aztreonam. KPC-2 had the highest affinity for meropenem. The kinetic studies revealed that KPC-2 was inhibited by clavulanic acid and tazobactam. An examination of the outer membrane proteins of the parent K. oxytoca strain demonstrated that it expressed detectable levels of OmpK36 (the homolog of OmpC) and a higher-molecular-weight OmpK35 (the homolog of OmpF). Thus, carbapenem resistance in K. oxytoca 3127 is due to production of the Bush group 2f, class A, carbapenem-hydrolyzing β-lactamase KPC-2. This β-lactamase is likely located on a transposon that is part of a conjugative plasmid and thus has a very high potential for dissemination.

Development of ertapenem resistance in a patient with mediastinitis caused by Klebsiella pneumoniae producing an extended-spectrum β-lactamase

2010 ◽  
Vol 59 (1) ◽  
pp. 115-119 ◽  
Author(s):  
David Skurnik ◽  
Sigismond Lasocki ◽  
Sylvie Bremont ◽  
Claudette Muller-Serieys ◽  
Marie Dominique Kitzis ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Resistant Strain ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Carbapenem Resistant ◽  
Extended Spectrum ◽  
Severe Infections ◽  
Resistant Strains ◽  
Selection Of

The aim was to study the clinical and microbiological features associated with a carbapenem-resistant Klebsiella pneumoniae isolate that had been selected in vivo by an ertapenem-containing regimen in a patient with mediastinitis despite high blood and mediastinal levels of ertapenem. Carbapenem resistance was characterized by conjugation, PCR, DNA sequencing and analysis of outer-membrane proteins. The isolates susceptible and resistant to the carbapenems were compared by ribotyping and PFGE. Resistance to all available β-lactams was most probably due to combined production of extended-spectrum β-lactamase (ESBL) CTX-M-15 and loss of OmpK36 porin. The results of ribotyping and PFGE suggest that the carbapenem-resistant strain was a derivative of the original mediastinal isolate rather than a superinfecting isolate. This observation stresses the risk of selection of pan-penem resistant strains of enterobacteria when ertapenem is used for the treatment of severe infections due to ESBL-producing enterobacteria.

scholarly journals OmpK26, a Novel Porin Associated with Carbapenem Resistance in Klebsiella pneumoniae

2011 ◽  
Vol 55 (10) ◽  
pp. 4742-4747 ◽  
Author(s):  
Laura García-Sureda ◽  
Antonio Doménech-Sánchez ◽  
Mariette Barbier ◽  
Carlos Juan ◽  
Joan Gascó ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Outer Membrane Proteins ◽  
Systemic Infection ◽  
Carbapenem Resistance ◽  
Resistant Isolate ◽  
Identical Result ◽  
Sequencing Analysis ◽  
Content Type ◽  
Carbapenem Resistant

ABSTRACTClinical isolates ofKlebsiella pneumoniaeresistant to carbapenems are being isolated with increasing frequency. Loss of the expression of the major nonspecific porins OmpK35/36 is a frequent feature in these isolates. In this study, we looked for porins that could compensate for the loss of the major porins in carbapenem-resistant organisms. Comparison of the outer membrane proteins from twoK. pneumoniaeclinical isogenic isolates that are susceptible (KpCS-1) and resistant (KpCR-1) to carbapenems revealed the absence of OmpK35/36 and the presence of a new 26-kDa protein in the resistant isolate. An identical result was obtained when another pair of isogenic isolates that are homoresistant (Kpn-3) and heteroresistant (Kpn-17) to carbapenems were compared. Mass spectrometry and DNA sequencing analysis demonstrated that this new protein, designated OmpK26, is a small monomeric oligogalacturonate-specific porin that belongs to the KdgM family of porins. Insertion-duplication mutagenesis of the OmpK26 coding gene,yjhA, in the carbapenem-resistant, porin-deficient isolate KpCR-1 caused the expression of OmpK36 and the reversion to the carbapenem-susceptible phenotype, suggesting that OmpK26 is indispensable for KpCR-1 to lose OmpK36 and become resistant to these antibiotics. Moreover, loss of the major porin and expression of OmpK26 reducedin vitrofitness and attenuated virulence in a murine model of acute systemic infection. Altogether, these results indicate that expression of the oligogalacturonate-specific porin OmpK26 compensates for the absence of OmpK35/36 and allows carbapenem resistance inK. pneumoniaebut cannot restore the fitness of the microorganism.

scholarly journals Emergence of Serratia marcescens, Klebsiella pneumoniae, and Escherichia coli Isolates Possessing the Plasmid-Mediated Carbapenem-Hydrolyzing β-Lactamase KPC-2 in Intensive Care Units of a Chinese Hospital

2008 ◽  
Vol 52 (6) ◽  
pp. 2014-2018 ◽  
Author(s):  
Jia Chang Cai ◽  
Hong Wei Zhou ◽  
Rong Zhang ◽  
Gong-Xiang Chen
Keyword(s):  
Escherichia Coli ◽  
Intensive Care ◽  
Klebsiella Pneumoniae ◽  
Serratia Marcescens ◽  
Intensive Care Units ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
E Coli ◽  
Insertional Inactivation

ABSTRACT Twenty-one Serratia marcescens, ten Klebsiella pneumoniae, and one Escherichia coli isolate with carbapenem resistance or reduced carbapenem susceptibility were recovered from intensive care units (ICUs) in our hospital. Enterobacterial repetitive intergenic consensus-PCR and pulsed-field gel electrophoresis demonstrated that all the S. marcescens isolates belonged to a clonal strain and the 10 K. pneumoniae isolates were indistinguishable or closely related to each other. The MICs of imipenem, meropenem, and ertapenem for all isolates were 2 to 8 μg/ml, except for K. pneumoniae K10 (MICs of 128, 256, and >256 μg/ml). Isoelectric focusing, PCRs, and DNA sequencing indicated that all S. marcescens isolates produced KPC-2 and a β-lactamase with a pI of 6.5. All K. pneumoniae isolates produced TEM-1, KPC-2, CTX-M-14, and a β-lactamase with a pI of 7.3. The E. coli E1 isolate produced KPC-2, CTX-M-15, and a β-lactamase with a pI of 7.3. Conjugation studies with E. coli (EC600) resulted in the transfer of reduced carbapenem susceptibility compared to that of the original isolates, and only the bla KPC-2 gene was detected in E. coli transconjugants. Plasmid restriction analysis showed identical restriction patterns among all E. coli transconjugants. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and ompK35/36 gene sequence analysis of outer membrane proteins revealed that K. pneumoniae K10 failed to express OmpK36, because of insertional inactivation by an insertion sequence ISEcp1. All these results indicate that KPC-2-producing S. marcescens, K. pneumoniae, and E. coli isolates emerged in ICUs in our hospital. KPC-2 combined with porin deficiency results in high-level carbapenem resistance in K. pneumoniae. The same bla KPC-2-encoding plasmid was spread among the three different genera.

scholarly journals Detection of carbapenem-resistant Escherichia coli and Klebsiella pneumoniae producing NDM-1 in Lebanon

2012 ◽  
Vol 6 (05) ◽  
pp. 457-461 ◽  
Author(s):  
Rima I El-Herte ◽  
George F Araj ◽  
Ghassan M Matar ◽  
Maysa Baroud ◽  
Zeina A Kanafani ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Carbapenem Resistance ◽  
New Delhi ◽  
Beta Lactamase ◽  
The Novel ◽  
First Report ◽  
Carbapenem Resistant ◽  
Lactamase Gene ◽  
Carbapenem Resistant Enterobacteriaceae

Carbapenem resistance has been encountered globally with poor outcome of infected patients. NDM-1 (New Delhi metallo-beta-lactamase) gene containing organisms have emerged and are now spreading in all continents. This is the first report of Iraqi patients referred to Lebanon from whom carbapenem resistant Enterobacteriaceae were recovered. The genes involved in carbapenem resistance were bla-OXA-48   and the novel NDM-1. This report highlights the alarming introduction of such resistance among Enterobacteriaecae to this country.

scholarly journals In Vitro Synergism of Azithromycin Combination with Antibiotics against OXA-48-Producing Klebsiella pneumoniae Clinical Isolates

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1551
Author(s):  
Uthaibhorn Singkham-in ◽  
Netchanok Muhummudaree ◽  
Tanittha Chatsuwan
Keyword(s):  
Escherichia Coli ◽  
Klebsiella Pneumoniae ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Resistance Mechanisms ◽  
In Vitro Activity ◽  
Bacterial Regrowth ◽  
Antibiotic Development ◽  
Carbapenem Resistant

Carbapenem-resistant Klebsiella pneumoniae has globally emerged as an urgent threat leading to the limitation for treatment. K. pneumoniae carrying blaOXA-48, which plays a broad magnitude of carbapenem susceptibility, is widely concerned. This study aimed to characterize related carbapenem resistance mechanisms and forage for new antibiotic combinations to combat blaOXA-48-carrying K. pneumoniae. Among nine isolates, there were two major clones and a singleton identified by ERIC-PCR. Most isolates were resistant to ertapenem (MIC range: 2–>256 mg/L), but two isolates were susceptible to imipenem and meropenem (MIC range: 0.5–1 mg/L). All blaOXA-48-carrying plasmids conferred carbapenem resistance in Escherichia coli transformants. Two ertapenem-susceptible isolates carried both outer membrane proteins (OMPs), OmpK35 and OmpK36. Lack of at least an OMP was present in imipenem-resistant isolates. We evaluated the in vitro activity of an overlooked antibiotic, azithromycin, in combination with other antibiotics. Remarkably, azithromycin exhibited synergism with colistin and fosfomycin by 88.89% and 77.78%, respectively. Bacterial regrowth occurred after exposure to colistin or azithromycin alone. Interestingly, most isolates were killed, reaching synergism by this combination. In conclusion, the combination of azithromycin and colistin may be an alternative strategy in dealing with blaOXA-48-carrying K. pneumoniae infection during a recent shortage of newly effective antibiotic development.

High prevalence of metallo-β-lactamase among carbapenem-resistant Klebsiella pneumoniae in a teaching hospital in China

2014 ◽  
Vol 60 (10) ◽  
pp. 691-695 ◽  
Author(s):  
Bin Li ◽  
Xiao-hong Xu ◽  
Zhi-chang Zhao ◽  
Mei-hua Wang ◽  
Ying-ping Cao
Keyword(s):  
Klebsiella Pneumoniae ◽  
Teaching Hospital ◽  
Carbapenem Resistance ◽  
Broth Culture ◽  
Consensus Sequences ◽  
Carbapenem Resistant ◽  
Extended Spectrum ◽  
Screening And Surveillance ◽  
High Prevalence

The aim of this study was to characterize the carbapenemases in carbapenem-resistant Klebsiella pneumoniae (CR-KP) from a Chinese teaching hospital. A total of 40 CR-KPs were screened for the presence of carbapenemases. Minimum inhibitory concentrations were determined by agar dilution. The modified Hodge test was used for the detection of carbapenemase production. Carbapenemase, extended-spectrum β-lactamase, and AmpC genes were detected using polymerase chain reaction (PCR) and sequencing. A conjugation test was performed using a broth culture mating method, transferred plasmids were typed by PCR-based replicon typing, and clonal relatedness was investigated by enterobacterial repetitive intergenic consensus sequences PCR (ERIC–PCR) and multilocus sequence typing (MLST). The results revealed that modified Hodge test was positive for 28 CR-KPs, and CR-KPs exhibited high resistance rates against various antibiotics, except colistin (5.0%) and tigecycline (22.5%). ERIC and MLST profiles showed no clonal outbreak. PCR demonstrated a high prevalence rate (55.0%, 22/40) of metallo-β-lactamases (MBLs) in CR-KPs. IMP-4, IMP-8, NDM-1, and KPC-2 were identified in 14 (35.0%), 7 (17.5%), 2 (5.0%), and 7 (17.5%) isolates, respectively. Notably, 2 CR-KPs coproduced 2 carbapenemases simultaneously (IMP-8/NDM-1 and IMP-4/KPC-2). In vitro transfer of carbapenem resistance was successful for 11 MBL-producing CR-KPs. The extended spectrum β-lactamase genes were detected in 30 (75.0%) of these CR-KPs. To the best of our knowledge, this is the first report focusing on carbapenem resistance in K. pneumoniae due to metalloenzymes in China. Screening and surveillance of MBLs in Enterobacteriaceae is urgently needed in this region to control and prevent the spread of these resistance determinants.

scholarly journals Molecular Characterization of Klebsiella pneumoniae Clinical Isolates with Elevated Resistance to Carbapenems

2017 ◽  
Vol 11 (1) ◽  
pp. 152-159 ◽  
Author(s):  
Rasha Barwa ◽  
Mona Shaaban
Keyword(s):  
Random Amplified Polymorphic Dna ◽  
Outer Membrane Proteins ◽  
Carbapenem Resistance ◽  
Microdilution Method ◽  
Carbapenem Resistant ◽  
Copy Numbers ◽  
Broth Microdilution Method ◽  
High Level ◽  
Urine Specimens

Background: Emergence of carbapenems-resistant K. pneumoniae represents a serious challenge for antimicrobial therapy. Objective: The aim of this research is to determine different mechanisms mediating the emergence of K. pneumoniae isolates with high-level carbapenem resistance. Method: A total of 80 K. pneumoniae isolates were purified from sputum and urine specimens. The minimum inhibitory concentrations (MICs) of imipenem and meropenem were determined by broth microdilution method. Carbapenemases were detected by Modified Hodge test and PCR. Additionally, the copy numbers of the identified genes (blaVIM-1, blaNDM-1 and blaOXA-48) were quantified by RT-PCR. The outer membrane proteins OmpK35 and OmpK36 of the resistant isolates were analyzed. Results: Eight isolates were resistant to carbapenems; six of these isolates possessed elevated MICs to imipenem and meropenem (≥16 µg/ml). Carbapenem resistant isolates harbored blaNDM-1 (n=5), blaVIM-1 (n=4) and blaOXA-48 (n=1) with some isolates had multiple carbapenemases genes. Six isolates with high MICs to imipenem contained multi-copies of the carbapenemases genes along with the lack of OmpK35. Isolates with intermediate resistance to carbapenems (MIC; 4-8 µg/ml) did not exhibit multiple carbapenemases but lacked the OmpK35. Random amplified polymorphic DNA exhibited three different patterns and indicated that five isolates encoded the same pattern P1. Conclusion: This study elucidated that multiple carbapenemases genes, high copy number of carbapenemases and loss of the porin OmpK35 could collectively contribute to the emergence of K. pneumoniae isolates with high resistance to carbapenems. Hence, more restrictions should be applied on the use of carbapenems to reduce the emergence of the resistant clones.

scholarly journals Acquisition of the Conjugative Virulence Plasmid From a CG23 Hypervirulent Klebsiella pneumoniae Strain Enhances Bacterial Virulence

2021 ◽  
Vol 11 ◽  
Author(s):  
Dongxing Tian ◽  
Weiwen Wang ◽  
Meng Li ◽  
Wenjie Chen ◽  
Ying Zhou ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Biofilm Formation ◽  
Evidence Base ◽  
Serum Resistance ◽  
Virulence Plasmid ◽  
E Coli ◽  
Virulence Plasmids ◽  
Carbapenem Resistant ◽  
High Level

The emergence of hypervirulent and carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) has become a hot topic and confounding problem for clinicians and researchers alike. Conjugative virulence plasmids have the potential to cause more threatening dissemination of hv-CRKP strains. We previously identified K2606, a CG23 clinical hypervirulent strain of Klebsiella pneumoniae harboring a conjugative virulence plasmid designated pK2606. In this study we examined hypervirulence levels using assays of biofilm formation, serum resistance, and wax larvae and mouse in vivo infection models. Moreover, to define the transfer ability of pK2606 and whether this confers hypervirulence to other strains we performed plasmid transconjugation experiments between K2606 and the ST11 CRKP strain HS11286 along with E. coli J53. We found that although biofilm formation and serum resistance were not significantly increased, the transconjugants acquired the ability of produce high level of siderophores and also caused high mortality of wax larvae and mice. Furthermore, we identified pK2606-like conjugative virulence plasmids in GenBank, providing evidence that such plasmids may have begun to spread throughout China. These findings provide an evidence base for the possible mechanisms of the emergence of hv-CRKP strains and highlight the potential of pK2606-like conjugative virulence plasmids to spread worldwide.

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