Bla Klebsiella Pneumoniae Carbapenemase Research Articles

Genomic epidemiology of carbapenem-resistant Enterobacterales at a New York City hospital over a 10-year period reveals complex plasmid-clone dynamics and evidence for frequent horizontal transfer of bla KPC.

Transmission of carbapenem-resistant Enterobacterales (CRE) in hospitals has been shown to occur through complex, multifarious networks driven by both clonal spread and horizontal transfer mediated by plasmids and other mobile genetic elements. We performed nanopore long-read sequencing on CRE isolates from a large urban hospital system to determine the overall contribution of plasmids to CRE transmission and identify specific plasmids implicated in the spread of bla KPC (the Klebsiella pneumoniae carbapenemase [KPC] gene). Six hundred and five CRE isolates collected between 2009 and 2018 first underwent Illumina sequencing for genome-wide genotyping; 435 bla KPC-positive isolates were then successfully nanopore sequenced to generate hybrid assemblies including circularized bla KPC-harboring plasmids. Phylogenetic analysis and Mash clustering were used to define putative clonal and plasmid transmission clusters, respectively. Overall, CRE isolates belonged to 96 multilocus sequence types (STs) encoding bla KPC on 447 plasmids which formed 54 plasmid clusters. We found evidence for clonal transmission in 66% of CRE isolates, over half of which belonged to four clades comprising K. pneumoniae ST258. Plasmid-mediated acquisition of bla KPC occurred in 23%-27% of isolates. While most plasmid clusters were small, several plasmids were identified in multiple different species and STs, including a highly promiscuous IncN plasmid and an IncF plasmid putatively spreading bla KPC from ST258 to other clones. Overall, this points to both the continued dominance of K. pneumoniae ST258 and the dissemination of bla KPC across clones and species by diverse plasmid backbones. These findings support integrating long-read sequencing into genomic surveillance approaches to detect the hitherto silent spread of carbapenem resistance driven by mobile plasmids.

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.

Carbapenemase investigation with rapid phenotypic test (RESIST-4 O.K.N.V) and comparison with PCR in carbapenem-resistant Enterobacterales strains.

RESİST-4 O.K.N.V. assay is a lateral immunochromatocraphic test for the identification of oxacillinase (OXA)-48-like, Klebsiella pneumoniae carbapenemase (KPC), New Delhi metallo-β-lactamase (NDM), and Verona integron-encoded metallo-β-lactamase (VIM) producing strains. It was aimed to evaluate the performance of the RESIST-4 O.K.N.V. test and to compare it with the reference method polymerase chain reaction (PCR). Also, the objective was to determine the distribution of carbapenemase types of CRE strains isolated in our hospital. Between January 2016-October 2019, 187 strains isolated from clinical samples were included in this study. Bacterial identification was done using MALDI-TOF MS. Antibiotic susceptibility tests were studied with the VITEK-2 automated system. Meropenem minimum inhibitory concentrations (MICs) were determined by the gradient test. All strains were studied with the RESIST-4 O.K.N.V. test and then the strains were selected for the PCR test. bla OXA-48, bla NDM, bla KPC, and bla VIM were investigated with PCR. K. pneumoniae NCTC® 13438 (KWIKSTIKTM, Microbiologics®, USA) was used as the positive control, E. coli ATTC® 25922 TM (Microbiologics®, USA) and three carbapenem-sensitive clinical isolates were also used as the negative control. Meropenem MIC50 and MIC90 values were determined to be >32 mg/L. With PCR bla OXA-48, bla NDM, bla KPC and bla VIM were detected in 79, 63, 20, and 4 strains, respectively. bla OXA-48 and bla NDM were found together in 51 of the isolates. bla OXA-48, bla NDM, bla KPC, and bla VIM were not detected in two strains with carbapenem resistance in susceptibility tests. The sensitivity of the immunochromatographic test was 100% for OXA-48, KPC, and VIM but 84.1% for NDM. Specificity was determined as 100% for OXA-48, NDM, KPC, and VIM. RESIST-4 O.K.N.V. test showed high sensitivity and specificity in detecting OXA-48, KPC, NDM, and VIM type carbapenemases. However, it should be kept in mind that there may be false-negative results related to NDM.

The type I-E CRISPR-Cas system influences the acquisition of bla KPC-IncF plasmid in Klebsiella pneumonia

ABSTRACT Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) have disseminated worldwide and emerged as major threats to public health. Of epidemiological significance, the international pandemic of KPC-KP is primarily associated with CG258 isolates and bla KPC-IncF plasmids. CRISPR-Cas system is an adaptive immune system that can hinder gene expansion driven by horizontal gene transfer. Because of bla KPC-IncF plasmids are favored by CG258 K. pneumoniae, it was of interest to examine the co-distribution of CRISPR and bla KPC-IncF plasmids in such isolates. We collected 459 clinical K. pneumoniae isolates in China and collected 203 global whole-genome sequences in GenBank to determine the prevalence of CRISPR-Cas systems. We observed that CRISPR-Cas system was significantly scarce in the CG258 lineage and bla KPC-positive isolates. Furthermore, the results of conjugation and plasmid stability assay fully demonstrated the CRIPSR-Cas system in K. pneumoniae could effectively hindered bla KPC-IncF plasmids invasion and existence. Notably, most bla KPC-IncF plasmids were also proved to be good targets of CRISPR owing to carry matched and functional protospacers and PAMs. Overall, our work suggests that type I-E CRISPR-Cas systems could impact the spread of bla KPC in K. pneumoniae populations, and the scarcity of CRISPR-Cas system was one of potential factors leading to the propagation of bla KPC-IncF plasmids in CG258 K. pneumoniae.

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.

Multidrug-resistant Enterobacteriaceae nosocomial uropathogens at Menoufia University Hospitals: phenotypic characterization and detection of resistance genes using real-time PCR

Objectives The aims of this study were as follows: to determine the incidence of multidrug-resistant Enterobacteriaceae isolated from the urine of patients admitted in different departments at Menoufia University Hospitals; to detect the presence of extended spectrum β-lactamases (ESβLs), AmpC β-lactamases, and carbapenemases among the isolated pathogens using phenotypic methods; and to investigate the presence of bla KPC and bla NDM genes using real-time PCR. Background Urinary tract infections are the most common type of nosocomial infections. ESβLs and AmpC β-lactamases are clinically significant because they may confer resistance to multiple classes of antibiotics. Carbapenemases are diverse enzymes that vary in the ability to hydrolyze carbapenems and other β-lactams. Materials and methods This study included 260 urine samples collected from 260 patients. ESβL production was detected on the basis of resistance to third-generation cephalosporins and aztreonam and then confirmed using the combined disk test. AmpC production was detected using the cefoxitin disk test and confirmed using the AmpC disk test. Carbapenemases were detected on the basis of resistance to imipenem, meropenem, and ertapenem, and then confirmed using the modified Hodge test, the phenylboronic acid combined disk test, and the imipenem/EDTA combined disk test. Carbapenemase gene (bla KPC and bla NDM) detection was performed using real-time PCR. Results Carbapenem resistance was 42.5% to ertapenem, 40.8% to meropenem, and 45% to imipenem. Risk factors associated with Hospital aquired infection were hospital stay of 14 days or more, exposure to invasive procedures, and comorbid conditions. ESβL production occurred in 55.8% and AmpC occurred in 30.8% of Enterobacteriaceae spp. Carbapenemase production was detected in 87%, Klebsiella pneumoniae carbapenemase (KPC) was detected in 37%, metallo-β-lactamase was detected in 54%, and bla KPCgene was detected in 24% of imipenem-resistant Enterobacteriaceae spp. However, bla NDM gene was not detected in all tested isolates. Conclusion Surveys of the prevalence, antibacterial susceptibility patterns, and identification of resistance patterns of bacterial isolates are important for determining appropriate empirical therapy for infections in critically ill patients.

32: The Presence of an 11,111 m/z Peak in the Mass Spectrum of Escherichia coli Is Suggestive of Klebsiella pneumoniae Carbapenemase-Mediated Carbapenem Resistance

Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) spectra were examined for any unique spectral features that could expedite the clinical microbiology laboratory’s identification of Klebsiella pneumoniae carbapenemase (KPC)-producing Escherichia coli (KPC-EC). Previously at our institution, the plasmids carrying bla KPC were investigated in 20 KPC-EC isolates, and various plasmids including IncN (4/20), ColE (2/20), and others were …

Detection of bla KPC-2 in Proteus mirabilis in Brazil.

Infections caused by Klebsiella pneumoniae carbapenemase (KPC)-producing isolates pose a major worldwide public health problem today. A carbapenem-resistant Proteus mirabilis clinical isolate was investigated for plasmid profiles and the occurrence of β-lactamase genes. The isolate exhibited resistance to ertapenem and imipenem and was susceptible to meropenem, polymyxin, and tigecycline. Five plasmids were identified in this isolate. DNA sequencing analysis revealed the presence of bla KPC-2 and bla TEM-1 genes. An additional PCR using plasmid DNA confirmed that bla KPC-2 was present in one of these plasmids. We report the detection of bla KPC-2 in P. mirabilis in Brazil for the first time. This finding highlights the continuous transfer of bla KPC between bacterial genera, which presents a serious challenge to the prevention of infection by multidrug-resistant bacteria.

Molecular characterization of carbapenem-resistant Enterobacteriaceae at a tertiary care laboratory in Mumbai.

Carbapenem-hydrolyzing β-lactamases are increasingly reported worldwide, leading to therapeutic failure. In an era where the drug development pipeline is stagnant, it is crucial to preserve current classes of antibiotics to help fight against infection caused by multidrug-resistant organisms (MDROs), by practicing a rational approach for the use of antibiotics. Identifying the mechanisms of resistance gives us much needed insights in this field. A total of 113 consecutive, non-duplicate carbapenem-resistant clinical isolates were collected from July to December 2012. These isolates were subjected to the modified Hodge test (MHT) for phenotypic detection of carbapenemases, an inhibitor-based test employing EDTA for the detection of metallo-β-lactamase (MBL), and phenylboronic acid for the detection of Klebsiella pneumoniae carbapenemase (KPC). A multiplex polymerase chain reaction (PCR) assay that characterized the five most predominant carbapenemases (bla NDM, bla OXA, bla VIM, bla IMP, bla KPC) was designed. The 113 isolates consisted of Klebsiella spp. (46), Enterobacter spp. (32), Escherichia coli (31), Citrobacter spp. (2), Proteus spp. (1), and Morganella spp. (1). bla NDM-1 was the most prevalent carbapenemase and accounted for 75.22% (85/113) of the isolates. This was followed by bla OXA [4.42% (n = 5)]. 18.5% (21/113) of the isolates possessed dual carbapenemase genes. 98.9% concordance was observed between the phenotypic tests and the molecular tests for the detection of MBL. In conclusion, patients infected with resistant bacteria require early appropriate antimicrobial treatment for good clinical outcome. Thus, identifying the resistant mechanisms of suspected pathogens becomes crucial. Also, the high incidence of plasmid-mediated bla NDM-1 calls for the implementation of strict infection control and contact isolation precautions in order to prevent the spread of these organisms.

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.

Bla KPC gene Detection in Clinical Isolates of Carbapenem Resistant Enterobacteriaceae in a Tertiary Care Hospital

Carbapenem resistance among Enterobacteriaceae, especially in Klebsiella pneumoniae and Escherichia coli, is an emerging problem worldwide. A common mechanism of carbapenem resistance is the production of class-A, Klebsiella pneumoniae carbapenemase (KPC). The present study focused on determining the antibiotic resistance pattern and prevalence of bla KPC gene coding for KPC in carbapenem resistant Enterobacteriaceae. Forty six carbapenem resistant isolates belonging to the family Enterobacteriaceae were tested for antibiotic sensitivity pattern. Modified Hodge Test (MHT) and PCR for bla KPC gene detection were performed on these isolates. Of these, 22 were Klebsiella pneumoniae, 21 were Escherichia coli, 2 were Citrobacter species and 1 was Proteus mirabilis Results: Forty three (93.4%) out of the 46 isolates were resistant to Meropenem, 34 (73.9%) were resistant to Imipenem and 30 (65.2%) were resistant to both Imipenem and Meropenem. Modified Hodge Test was positive in 38 (82.6%) out of 46 isolates and blaKPC gene was detected in 31 (67.4%) isolates. bla KPC gene was detected in 28 out of the 38 MHT positive isolates.