MALDI-TOF mass spectrometry and blakpc gene phylogenetic analysis of an outbreak of carbapenem-resistant K. pneumoniae strains.

Carbapenem-resistant Klebsiella pneumoniae isolates are an important cause of nosocomial infections. This study evaluated a rapid cost-saving method based on MALDI-TOF technology, was and compared it with phenotypic, genotypic and epidemiological data for characterization of KPC-Kp strains consecutively isolated during a supposed outbreak. Twenty-five consecutive KPC Klebsiella pneumoniae isolates were identified and clustered by the MALDI Biotyper (Bruker, Daltonics). To display and rank the variance within a data set, principal component analysis (PCA) was performed. ClinProTools models were generated to investigate the highest sum of recognition capability and cross-validation. A Class dendrogram of isolates was constructed using ClinproTool. MLST was performed sequencing gapA, infB, mdh, pgi, rpoB, phoE and tonB genes. blakpc and cps genes were typed. Phylogenetic analysis and genetic distance of the KPC gene were performed using the MEGA6 software. PCA analysis defined two clusters, I and II, which were identified in a dendrogram by both temporal split and different antimicrobial susceptibility profiles. These clusters were composed mostly of strains of the same sequence type (ST512), the most prevalent ST in Italy, and the same cps (type 2). In cluster II, blakpc genotype resulted more variable than in cluster I. Phylogenetic analysis confirmed the genetic diversity in both clusters supported by the epidemiological data. Our study confirms that MALDI-TOF can be a rapid and cost-saving method for epidemiological clustering of KPC K. pneumoniae isolates and its association with blakpc genotyping represents a reliable method to recognize possible clonal strains in nosocomial settings.

CRISPR/Cas13-assisted carbapenem-resistant Klebsiella pneumoniae detection.

BACKGROUND/PURPOSE: Carbapenem-resistant Klebsiella pneumoniae (CRKP) is capable of causing serious community and hospital-acquired infections. However, currently, the identification of CRKP is complex and inefficient. Hence, this study aimed to develop methods for the early and effective identification of CRKP to allow reasonable antimicrobial therapy in a timely manner. METHODS: K. pneumoniae (KP)-, K. pneumoniae carbapenemase (KPC)- and New Delhi metallo-ß-lactamase (NDM)- specific CRISPR RNAs (crRNAs), polymerase chain reaction (PCR) primers and recombinase-aided amplification (RAA) primers were designed and screened in conserved sequence regions. We established fluorescence and lateral flow strip assays based on CRISPR/Cas13a combined with PCR and RAA, respectively, to assist in the detection of CRKP. Sixty-one clinical strains (including 51 CRKP strains and 10 carbapenem-sensitive strains) were collected for clinical validation. RESULTS: Using the PCR-CRISPR assay, the limit of detection (LOD) for KP and the blaKPC and blaNDM genes reached 1 copy/µL with the fluorescence signal readout. Using the RAA-CRISPR assay, the LOD could reach 101 copies/µL with both the fluorescence signal readout and the lateral flow strip readout. Additionally, the positivity rates of CRKP-positive samples detected by the PCR/RAA-CRISPR fluorescence and RAA-CRISPR lateral flow strip methods was 92.16% (47/51). The sensitivity and specificity reached 100% for KP and blaKPC and blaNDM gene detection. For detection in a simulated environmental sample, 1 CFU/cm2 KP could be detected. CONCLUSION: We established PCR/RAA-CRISPR assays for the detection of blaKPC and blaNDM carbapenemase genes, as well as KP, to facilitate the detection of CRKP.

Antimicrobial Resistance Caused by KPC-2 Encoded by Promiscuous Plasmids of the Klebsiella pneumoniae ST307 Strain.

BACKGROUND: A lineage of Klebsiella pneumoniae that produces carbapenemase-2 (KPC-2), sequence type (ST) 307, emerged in 2017. We analyzed the complete sequences of plasmids from KPC-2-producing K. pneumoniae (KPC-Kp) ST307, investigated the antimicrobial resistance conferred by this strain, and confirmed the horizontal interspecies transmission of KPC-carbapenemase-producing Enterobacteriaceae (CPE) characteristics among Enterobacteriaceae. METHODS: We performed antimicrobial susceptibility testing, PCR analysis, multilocus sequence typing, curing tests, and whole-genome sequencing to characterize plasmid-derived KPC-2-producing Enterobacteriaceae clinical isolates. RESULTS: Sequence analysis of KPC-Kp strain ST307 revealed novel plasmid-located virulence factors, including a gene cluster for glycogen synthesis. Three Enterobacteriaceae strains were identified in one patient: K. pneumoniae (CPKp1825), Klebsiella aerogenes (CPEa1826), and Escherichia coli (CPEc1827). The bla KPC-2 gene from K. pneumoniae ST307 was horizontally transmitted between these strains. The plasmids could be transferred through conjugation, because all three strains of bacteria contained the type IV secretion system, pilus genes, and tra genes for conjugal transfer. The bla KPC-2 gene was located on a truncated Tn4401 transposon. Plasmids containing the bla KPC-2 gene could not be artificially removed; thus, the three strains could not be cured. CONCLUSIONS: The ease of horizontal transfer of KPC-Kp ST307 carbapenem resistance has serious public health and epidemiological implications. This study provides a better understanding of the genetic characteristics that can contribute to the growth and spread of KPC-Kp ST307, and their association with antimicrobial resistance genes.

blaKPC gene Detection in Clinical Isolates of Carbapenem Resistant Enterobacteriaceae in a Tertiary Care Hospital.

INTRODUCTION: 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). AIMS AND OBJECTIVES: The present study focused on determining the antibiotic resistance pattern and prevalence of bla KPC gene coding for KPC in carbapenem resistant Enterobacteriaceae. METHODOLOGY: 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.