A Recombinase Aided Amplification Assay for Rapid Detection of the Klebsiella pneumoniae Carbapenemase Gene and Its Characteristics in Klebsiella pneumoniae

Klebsiella pneumoniae carbapenemase genes (blaKPC) play an important role in carbapenem-resistant Enterobacteriaceae in China. A rapid detection method for blaKPC genes and investigations into the molecular characteristics of blaKPC positive Klebsiella pneumoniae were necessary. In this study, an easy and rapid recombinase aided amplification assay (RAA) for blaKPC was established. This protocol could be completed at 39°C in 15–20 min. The sensitivity of this assay was determined as 48 copies per reaction, and the specificity was 100%. The blaKPC RAA method could be used for clinical diagnosis and epidemiological investigation. Among 801 fecal samples from inpatients, 34 blaKPC positive isolates were identified from each sample, of which 23 isolates were K. pneumoniae. ST11 with blaKPC-2 was the most prevalent type. All these strains were multidrug resistant and carried various virulence genes. Fecal carriage of blaKPC positive carbapenem-resistant K.pneumoniae poses significant challenges for public health control.

Download Full-text

1189. A Comprehensive Characterization of the Emerging Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates From a Public Hospital in Lima, Peru

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1022 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S359-S360 ◽

Cited By ~ 1

Author(s):

Fiorella Krapp ◽

Catherine Amaro ◽

Karen Ocampo ◽

Lizeth Astocondor ◽

Noemi Hinostroza ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Dna Extraction ◽

Antibiotic Susceptibility ◽

Tertiary Care ◽

Clinical Isolates ◽

Molecular Characteristics ◽

Care Hospital ◽

Carbapenem Resistant ◽

Carbapenemase Gene ◽

String Test

Abstract Background In contrast with other countries in Latin America, Peru had been notoriously spared by the global dissemination of carbapenem-resistant Klebsiella pneumoniae (CR-Kp), until recently. Even though, isolated cases of KPC-producing K. pneumoniae had been reported since 2013, it was not until 2016 that the first outbreak of NDM- producing K. pneumoniae was described in Peru. By 2017, rapid emergence of CR-Kp took place in Hospital Cayetano Heredia (HCH), a tertiary care hospital in Lima. Here, we provide a description of clinical, microbiological and molecular characteristics of CR-Kp isolates recovered at HCH. Methods Retrospective review of all CR-Kp clinical isolates recovered at HCH until December 2017. Antibiotic susceptibility data were obtained during routine care (Vitek or disc diffusion) and was assessed using CLSI breakpoints. DNA extraction was performed by heat shock, and PCR was performed to assess carriage of blaNDM gene. String test was performed to detect hypermucoviscosity. Results The first case of CR-Kp in HCH dated from July 2015. Since then, a total of 69 CR-Kp clinical isolates, from 60 patients have been recovered until December 2017. A significant increase in the number of cases was observed during 2017 (Figure 1). The average age of patients was 55. Urinary, and respiratory sources of infection or colonization were the most common ones (35% and 30%, respectively), followed by blood stream (17%) and intraabdominal (10%) infections. Isolate recovery and DNA extraction was achieved in 40 cases. Of these, 15 (38%) had a positive PCR for blaNDM carbapenemase gene (Figure 2). Antibiotic susceptibility testing revealed that amikacin was the most effective antimicrobial with the rest of antimicrobials having extremely high rates of resistance (Figure 3). String test was positive in two of these isolates, suggesting that hypervirulent CR-KP might be emerging in this region. Conclusion An epidemic of CR-Kp has established in our hospital, representing the first one reported in Peru. The different mechanisms of carbapenem resistance found suggest a polyclonal expansion. Amikacin remains the only active antimicrobial within the routinely tested antibiotics, highlighting the need to add other antimicrobials to the routine panel. Disclosures All authors: No reported disclosures.

Download Full-text

Prevalence of blaKPC-2, blaKPC-3 and blaKPC-30—Carrying Plasmids in Klebsiella pneumoniae Isolated in a Brazilian Hospital

Pathogens ◽

10.3390/pathogens10030332 ◽

2021 ◽

Vol 10 (3) ◽

pp. 332

Author(s):

Letícia B. Migliorini ◽

Romário O. de Sales ◽

Paula C. M. Koga ◽

Andre M. Doi ◽

Anja Poehlein ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Tertiary Hospital ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

Genomic Context ◽

Multidrug Resistant Bacteria ◽

Klebsiella Pneumoniae Carbapenemase ◽

Carbapenem Resistant ◽

Horizontal Spread ◽

Widespread Dissemination

Klebsiella pneumoniae carbapenemase (KPC) actively hydrolyzes carbapenems, antibiotics often used a last-line treatment for multidrug-resistant bacteria. KPC clinical relevance resides in its widespread dissemination. In this work, we report the genomic context of KPC coding genes blaKPC-2, blaKPC-3 and blaKPC-30 in multidrug-resistant Klebsiellapneumoniae isolates from Brazil. Plasmids harboring blaKPC-3 and blaKPC-30 were identified. Fifteen additional carbapenem-resistant K. pneumoniae isolates were selected from the same tertiary hospital, collected over a period of 8 years. Their genomes were sequenced in order to evaluate the prevalence and dissemination of blaKPC–harboring plasmids. We found that blaKPC genes were mostly carried by one of two isoforms of transposon Tn4401 (Tn4401a or Tn4401b) that were predominantly located on plasmids highly similar to the previously described plasmid pKPC_FCF3SP (IncN). The identified pKPC_FCF3SP-like plasmids carried either blaKPC-2 or blaKPC-30. Two K. pneumoniae isolates harbored pKpQIL-like (IncFII) plasmids, only recently identified in Brazil; one of them harbored blaKPC-3 in a Tn4401a transposon. Underlining the risk of horizontal spread of KPC coding genes, this study reports the prevalence of blaKPC-2 and the recent spread of blaKPC-3, and blaKPC-30, in association with different isoforms of Tn4401, together with high synteny of plasmid backbones among isolates studied here and in comparison with previous reports.

Download Full-text

Genetic determinants of resistance and virulence among carbapenemase-producing Klebsiella pneumoniae from Sri Lanka

10.1101/330951 ◽

2018 ◽

Author(s):

Chendi Zhu ◽

Veranja Liyanapathirana ◽

Carmen Li ◽

V. Pinto ◽

Mamie Hui ◽

...

Keyword(s):

Intensive Care ◽

Klebsiella Pneumoniae ◽

Intensive Care Units ◽

Mobile Element ◽

Multidrug Resistant ◽

Quinolone Resistance ◽

Sri Lankan ◽

Virulence Determinants ◽

Carbapenem Resistant ◽

Carbapenemase Gene

ABSTRACTWhole genome sequencing of carbapenem-resistant Enterobacteriaceae from the intensive care units of a Sri Lankan teaching hospital revealed the presence of carbapenemase gene, blaOXA-181 among isolates of carbapenase-producing Klebsiella pneumoniae belonging to ST437 (2 strains) and ST147 (8 strains) in 2015. blaOXA-181 genes were carried in three variants of ColE-type plasmids. Elevated carbapemen resistance were observed in ompK36 mutant strains. ESBL genes, plasmid–mediated quinolone resistance (PMQR) determinants (qnr, aac(6’)-Ib-cr, oqxAB) and mutations on chromosomal quinolone resistance-determining regions (QRDRs) with substitutions at ser83→I of gyrA and ser80→I of parC were observed. All strains possessed yersiniabactin on the mobile element ICEkp and other virulence determinants. Strict infection control and judicious use of antibiotics are warranted to prevent further spread of multidrug-resistant Klebsiella pneumoniae in the intensive care units.

Download Full-text

Molecular Analysis of Polymyxin Resistance among Carbapenemase-Producing Klebsiella pneumoniae in Colombia

Antibiotics ◽

10.3390/antibiotics10030284 ◽

2021 ◽

Vol 10 (3) ◽

pp. 284

Author(s):

Elsa De La De La Cadena ◽

María Fernanda Mojica ◽

Juan Carlos García-Betancur ◽

Tobías Manuel Appel ◽

Jessica Porras ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Sequence Type ◽

Molecular Characteristics ◽

Amino Acid Substitutions ◽

Resistant Clone ◽

Broth Microdilution ◽

Carbapenem Resistant ◽

High Level ◽

Level Resistance

Polymyxin resistance in Klebsiella pneumoniae has been attributed to mutations in mgrB, phoPQ, pmrAB, and crrAB and to the presence of mcr plasmid-mediated genes. Herein, we describe the molecular characteristics of 24 polymyxin- and carbapenem-resistant K. pneumoniae isolates recovered from six Colombian cities between 2009 and 2019. Minimum inhibitory concentrations (MICs) to polymyxin were confirmed by broth microdilution, and whole-genome sequencing was performed to determine sequence type, resistome, and mutations in the genes related to polymyxin resistance, as well the presence of mcr. The results showed high-level resistance to polymyxin (MICs ≥ 4 μg/mL). blaKPC-3 was present in the majority of isolates (17/24; 71%), followed by blaKPC-2 (6/24; 25%) and blaNDM-1 (1/24; 4%). Most isolates belonged to the CG258 (17/24; 71%) and presented amino acid substitutions in PmrB (22/24; 92%) and CrrB (15/24; 63%); mutations in mgrB occurred in only five isolates (21%). Additional mutations in pmrA, crrA, and phoPQ nor any of the mcr resistance genes were identified. In conclusion, we found clonal dissemination of polymyxin and carbapenem-resistant K. pneumoniae isolates in Colombia, mainly associated with CG258 and blaKPC-3. Surveillance of this multidrug-resistant clone is warranted due to the limited therapeutic options for the treatment of carbapenem-resistant K. pneumoniae infections.

Download Full-text

Genotypes and prevalence of carbapenemase-producing Enterobacteriaceae and Pseudomonas aeruginosa in a hospital in Saudi Arabia

Transactions of the Royal Society of Tropical Medicine and Hygiene ◽

10.1093/trstmh/trab055 ◽

2021 ◽

Author(s):

Jaffar A Al-Tawfiq ◽

Ali A Rabaan ◽

Justin V Saunar ◽

Ali M Bazzi

Keyword(s):

Pseudomonas Aeruginosa ◽

Saudi Arabia ◽

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Resistant Bacteria ◽

New Delhi ◽

Beta Lactam ◽

Klebsiella Pneumoniae Carbapenemase ◽

Carbapenem Resistant ◽

Carbapenem Resistant Enterobacteriaceae

Abstract Background The molecular epidemiology of resistance of carbapenem-resistant Enterobacteriaceae (CRE) and Pseudomonas aeruginosa are important in the study of multidrug-resistant bacteria. We evaluate the prevalence of the different mechanisms of CRE in a hospital in Saudi Arabia. Methods Carbapenem non-susceptible isolates of Enterobacteriaceae and Pseudomonas aeruginosa were tested by real-time PCR for the detection of genes responsible for beta-lactam resistance. Results There were a total of 200 isolates with carbapenem non-susceptibility and these were Klebsiella pneumoniae (n=96, 48%), Escherichia coli (n=51, 25.5%) and Pseudomonas aeruginosa (n=45, 22.5%). The detected carbapenemases were oxacillinase-48 (OXA-48) (n=83, 41.5%), New Delhi metallo-β-lactamase (NDM) (n=19, 2.5%) and both NDM and OXA-48 (n=5, 2.5%). The other carbapenemases were imipenemase (n=1, 0.5%), Verona integrin encoded metallo-β-lactamase (n=6, 3%) and Klebsiella pneumoniae carbapenemase (n=1, 0.5%), but none were detected in 86 isolates (43%). Conclusion The most common carbapenemases were OXA-48 and a significant percentage had no detectable genes. These data will help in the selection of new antimicrobial therapies.

Download Full-text

Klebsiella pneumoniae Carbapenemase (KPC)–Producing K. pneumoniae Contamination of an In-Room Sink in a New Bed Tower

Antimicrobial Stewardship & Healthcare Epidemiology ◽

10.1017/ash.2021.143 ◽

2021 ◽

Vol 1 (S1) ◽

pp. s73-s73

Author(s):

Bobby Warren ◽

Becky Smith ◽

Sarah Lewis ◽

Deverick Anderson ◽

Bechtler Addison

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Klebsiella Pneumoniae Carbapenemase ◽

Carbapenem Resistant ◽

Routine Surveillance ◽

Colony Forming Units ◽

Time Of Admission ◽

Uv C ◽

New Strategies ◽

Carbapenem Resistant Enterobacteriaceae

Group Name: Duke Center for Antimicrobial Stewardship and Infection PreventionBackground: Wastewater drains in hospital patient rooms have been identified as environmental reservoirs for multidrug-resistant organisms, and they have been linked to outbreaks of carbapenem-resistant Enterobacteriaceae (CRE). We studied the colonization of wastewater drains in a new hospital bed tower. Methods: A patient care unit in a new bed tower opened on July 18, 2020. In-room sinks were located in each hospital room opposite the patient head wall. Patients admitted to this unit underwent weekly rectal cultures to survey for carbapenemase-producing CRE. Additionally, infection preventionists performed routine surveillance of all clinical cultures for CRE. Cultures were performed from all patient room sinks in this unit monthly beginning September 14, 2020. Samples were obtained from the drain cover, handles, and top of bowl using sponges soaked in neutralizing buffer and processed using the stomacher technique. The tail-pipe was sampled using a flocked mini-tip swab soaked in neutralizing buffer; the P-trap water was sampled with sterile tubing attached to a 50-mL syringe. All samples were plated on HARDYCHROM-ESBL and KPC Colorex media and were incubated at 37°C for 24 hours. Results: The first identified CRE-positive patient was admitted to the new unit on December 4, 2020; urine culture obtained at the time of admission grew KPC–producing Klebsiella pneumoniae (KPC-KP). The sink in this patient’s room had been sampled 3 prior times (most recently on November 9, 2020) and was negative for CRE. On December 7, 2020, KPC-KP was found on the drain cover (6,750 colony-forming units, CFU) and in the sink’s P-trap (1,840 CFU) of the index patient’s room during routine sink surveillance. Additional samples from other room surfaces were taken on December 9, 2020, and KPC-KP was recovered from the computer keyboard (452 CFU) and patient bedrails (880 CFU). The patient was discharged from this room December 13, 2020, and the room underwent enhanced terminal room cleaning including UV-C light. On the next routine sink sampling on January 4, 2021, KPC-KP was recovered again in the index room sink P-trap (9,800 CFU) but at no additional sites. MLST was performed, and all isolates were ST-258. Conclusions: In a new bed tower with no prior evidence of CRE-positive patients, the first identified case of a CRE (KPC-KP) in a patient resulted in widespread environmental contamination of the room after only 3 days of hospitalization and contamination of the in-room sink drain that persisted after 1 month. Given the ease with which CRE colonizes wastewater drains, new strategies are needed to mitigate drain colonization and to prevent CRE transmission to subsequent patients.Funding: NoDisclosures: None

Download Full-text

A multi-institutional outbreak of New Delhi metallo-β-lactamase–producing Escherichia coli with subsequent acquisition of the Klebsiella pneumoniae carbapenemase gene

Infection Control and Hospital Epidemiology ◽

10.1017/ice.2020.1361 ◽

2020 ◽

pp. 1-4

Author(s):

Ester Solter ◽

Jason C. Kwong ◽

Aaron Walton ◽

Norelle Sherry ◽

Benjamin P. Howden ◽

...

Keyword(s):

Escherichia Coli ◽

Klebsiella Pneumoniae ◽

Common Ancestor ◽

Sequence Type ◽

Second Phase ◽

New Delhi ◽

Genetic Sequencing ◽

Klebsiella Pneumoniae Carbapenemase ◽

Carbapenemase Gene

Abstract We characterized 57 isolates from a 2-phase clonal outbreak of New Delhi metallo-β-lactamase–producing Eschericha coli, involving 9 Israeli hospitals; all but 1 isolate belonged to sequence-type (ST) 410. Most isolates in the second phase harbored blaKPC-2 in addition to blaNDM-5. Genetic sequencing revealed most dual-carbapenemase–producing isolates to be monophyletically derived from a common ancestor.

Download Full-text

First description of NDM-1-, KPC-2-, VIM-2- and IMP-4-producing Klebsiella pneumoniae strains in a single Chinese teaching hospital

Epidemiology and Infection ◽

10.1017/s0950268814000995 ◽

2014 ◽

Vol 143 (2) ◽

pp. 376-384 ◽

Cited By ~ 27

Author(s):

Y. LIU ◽

L.-G. WAN ◽

Q. DENG ◽

X.-W. CAO ◽

Y. YU ◽

...

Keyword(s):

16S Rrna ◽

Klebsiella Pneumoniae ◽

Teaching Hospital ◽

Multidrug Resistant ◽

The Other ◽

Quinolone Resistance ◽

Resistance Determinants ◽

Carbapenem Resistant ◽

Extended Spectrum ◽

Chinese Teaching

SUMMARYA total of 180 non-duplicate carbapenem-resistant Klebsiella pneumoniae isolates were recovered from patients hospitalized between December 2010 and January 2012 at a Chinese hospital. Eight KPC-2, four NDM-1, one VIM-2, and five KPC-2 plus IMP-4 producers were identified and all were multidrug resistant due to the presence of other resistance determinants, including extended-spectrum β-lactamases (CTX-M-15, SHV-12), 16S rRNA methylases (armA, rmtB) and plasmid-mediated quinolone-resistance determinants (qnrA, B, S, aac(6′)-Ib-cr). Nine K. pneumoniae clones (Kpn-A1/ST395, Kpn-A3/ST11, Kpn-A2/ST134, Kpn-B/ST263, Kpn-C/ST37, Kpn-D/ST39, Kpn-E/ST1151, Kpn-F/ST890, Kpn-G/ST1153) were identified. blaKPC-2 was located on transferable ~65 kb IncL/M (ST395, ST11, ST134, ST39) and ~100 kb IncA/C (ST37, ST1153, ST890) plasmids, respectively. On the other hand, blaNDM-1 was associated with a ~70 kb IncA/C plasmid (ST263). However, non-typable plasmids of ~40 kb containing blaVIM-2 were detected in the ST1151 clone. This work reports the first co-occurrence of four diverse types of carbapenemase of K. pneumoniae clones from a single hospital in China. IncA/C, IncL/M, and other successful plasmids may be important for the dissemination of carbapenemases, producing a complex epidemiological picture.

Download Full-text

1348. In vitro Activity of Plazomicin, a Next-Generation Aminoglycoside, Against Carbapenemase-Producing Klebsiella pneumoniae

Open Forum Infectious Diseases ◽

10.1093/ofid/ofy210.1179 ◽

2018 ◽

Vol 5 (suppl_1) ◽

pp. S412-S413

Author(s):

Michael R Jacobs ◽

Caryn E Good ◽

Ayman M Abdelhamed ◽

Daniel D Rhoads ◽

Kristine M Hujer ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Carbapenem Resistance ◽

Vitro Activity ◽

In Vitro Activity ◽

Next Generation ◽

Aminoglycoside Resistance ◽

Carbapenem Resistant ◽

Carbapenemase Gene ◽

Research Grant

Abstract Background Plazomicin is a next-generation aminoglycoside with in vitro activity against multidrug-resistant Gram-negative species, including carbapenem-resistant isolates. The Consortium on Resistance Against Carbapenems in Klebsiella and other Enterobacteriaceae (CRACKLE) is a federally funded, prospective multicenter consortium of 20 hospitals from nine US healthcare systems to track carbapenem-resistant Enterobacteriaceae. Methods Minimum inhibitory concentrations (MICs) of plazomicin were determined by broth microdilution according to current CLSI guidelines against a collection of 697 carbapenem-resistant Klebsiella pneumoniae with defined carbapenem resistance mechanisms, including KPC and OXA carbapenemases. Isolates were submitted by participating CRACKLE centers. Results Carbapenemases present in study isolates included KPC-2 (n = 323), KPC-3 (n = 364), KPC-4 (n = 2), OXA-48 like (n = 7), and NDM (n = 1). Plazomicin MICs ranged from ≤0.12 to >32 mg/L, with MIC50 and MIC90 values of 0.25 and 1 mg/L, respectively (figure). MICs of 689 (98.8%) isolates were ≤4 mg/L, while MICs of the remaining eight isolates were >32 mg/L. Plazomicin MICs were related to specific carbapenemases present in isolates: of eight isolates with MICs >32 mg/L, seven contained OXA-48 like and one contained KPC-3, suggesting that these isolates possess an aminoglycoside-resistance mechanism on the same plasmid as their carbapenemase gene, such as a 16S ribosomal RNA methyltransferase, against which plazomicin is not active. Conclusion Plazomicin has good in vitro potency against a collection of carbapenemase-producing K. pneumoniae, with MIC90 value of 1 mg/L and MICs of ≤4 mg/L for 98.9% of isolates. Disclosures M. R. Jacobs, Achaogen: Investigator, Research grant. Shionogi: Investigator, Research grant. L. Connolly, Achaogen, Inc.: Consultant, Consulting fee. K. M. Krause, Achaogen: Employee, Salary. S. S. Richter, bioMerieux: Grant Investigator, Research grant. BD Diagnostics: Grant Investigator, Research grant. Roche: Grant Investigator, Research grant. Hologic: Grant Investigator, Research grant. Diasorin: Grant Investigator, Research grant. Accelerate: Grant Investigator, Research grant. Biofire: Grant Investigator, Research grant. D. Van Duin, achaogen: Scientific Advisor, Consulting fee. shionogi: Scientific Advisor, Consulting fee. Allergan: Scientific Advisor, Consulting fee. Astellas: Scientific Advisor, Consulting fee. Neumedicine: Scientific Advisor, Consulting fee. Roche: Scientific Advisor, Consulting fee. T2 Biosystems: Scientific Advisor, Consulting fee.

Download Full-text

Carbapenemase Production and Epidemiological Characteristics of Carbapenem-Resistant Klebsiella pneumoniae in Western Chongqing, China

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.775740 ◽

2022 ◽

Vol 11 ◽

Author(s):

Wan Huang ◽

Jisheng Zhang ◽

Lingyi Zeng ◽

Chengru Yang ◽

Lining Yin ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Klebsiella Pneumoniae ◽

Virulence Genes ◽

Resistance Rate ◽

Molecular Characteristics ◽

Chain Reaction ◽

Detection Rates ◽

Carbapenem Resistant ◽

Polymerase Chain ◽

Epidemiological Characteristics

BackgroundThis study aimed to determine the molecular characteristics of carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates in a hospital in western Chongqing, southwestern China.MethodsA total of 127 unique CRKP isolates were collected from the Yongchuan Hospital of Chongqing Medical University, identified using a VITEK-2 compact system, and subjected to microbroth dilution to determine the minimal inhibitory concentration. Enterobacteriaceae intergenic repeat consensus polymerase chain reaction and multilocus sequence typing were used to analyze the homology among the isolates. Genetic information, including resistance and virulence genes, was assessed using polymerase chain reaction. The genomic features of the CRKP carrying gene blaKPC-2 were detected using whole-genome sequencing.ResultsST11 was the dominant sequence type in the homology comparison. The resistance rate to ceftazidime-avibactam in children was much higher than that in adults as was the detection rate of the resistance gene blaNDM (p < 0.0001). Virulence genes such as mrkD (97.6%), uge (96.9%), kpn (96.9%), and fim-H (84.3%) had high detection rates. IncF (57.5%) was the major replicon plasmid detected, and sequencing showed that the CRKP063 genome contained two plasmids. The plasmid carrying blaKPC-2, which mediates carbapenem resistance, was located on the 359,625 base pair plasmid IncFII, together with virulence factors, plasmid replication protein (rep B), stabilizing protein (par A), and type IV secretion system (T4SS) proteins that mediate plasmid conjugation transfer.ConclusionOur study aids in understanding the prevalence of CRKP in this hospital and the significant differences between children and adults, thus providing new ideas for clinical empirical use of antibiotics.

Download Full-text