Applying Rapid Whole-Genome Sequencing To Predict Phenotypic Antimicrobial Susceptibility Testing Results among Carbapenem-Resistant Klebsiella pneumoniae Clinical Isolates.

Standard antimicrobial susceptibility testing (AST) approaches lead to delays in the selection of optimal antimicrobial therapy. Here, we sought to determine the accuracy of antimicrobial resistance (AMR) determinants identified by Nanopore whole-genome sequencing in predicting AST results. Using a cohort of 40 clinical isolates (21 carbapenemase-producing carbapenem-resistant , 10 non-carbapenemase-producing carbapenem-resistant , and 9 carbapenem-susceptible isolates), three separate sequencing and analysis pipelines were performed, as follows: (i) a real-time Nanopore analysis approach identifying acquired AMR genes, (ii) an assembly-based Nanopore approach identifying acquired AMR genes and chromosomal mutations, and (iii) an approach using short-read correction of Nanopore assemblies. The short-read correction of Nanopore assemblies served as the reference standard to determine the accuracy of Nanopore sequencing results. With the real-time analysis approach, full annotation of acquired AMR genes occurred within 8 h from subcultured isolates. Assemblies sufficient for full resistance gene and single-nucleotide polymorphism annotation were available within 14 h from subcultured isolates. The overall agreement of genotypic results and anticipated AST results for the 40  isolates was 77% (range, 30% to 100%) and 92% (range, 80% to 100%) for the real-time approach and the assembly approach, respectively. Evaluating the patients contributing the 40 isolates, the real-time approach and assembly approach could shorten the median time to effective antibiotic therapy by 20 h and 26 h, respectively, compared to standard AST. Nanopore sequencing offers a rapid approach to both accurately identify resistance mechanisms and to predict AST results for isolates. Bioinformatics improvements enabling real-time alignment, coupled with rapid extraction and library preparation, will further enhance the accuracy and workflow of the Nanopore real-time approach.

Tamma PD ，Fan Y ，Bergman Y ，Pertea G ，Kazmi AQ ，Lewis S ，Carroll KC ，Schatz MC ，Timp W ，Simner PJ ... -  《-》

Effect of Resistance Mechanisms on the Inoculum Effect of Carbapenem in Klebsiella pneumoniae Isolates with Borderline Carbapenem Resistance.

We aimed to examine the effects of resistance mechanisms on several resistance phenotypes among carbapenem-resistant Klebsiella pneumoniae isolates with borderline carbapenem MICs. We compared carbapenemase-negative K. pneumoniae with carbapenemase-producing K. pneumoniae (CPKP) isolates with similar MICs. CPKP isolates exhibited a marked inoculum effect and were more resistant to the bactericidal effect of meropenem. This suggests that MIC measurements alone may not be sufficient in predicting the therapeutic efficacy of carbapenems against CPKP.

Adler A ，Ben-Dalak M ，Chmelnitsky I ，Carmeli Y ... -  《-》

Emergence of Ceftazidime-Avibactam Resistance Due to Plasmid-Borne bla(KPC-3) Mutations during Treatment of Carbapenem-Resistant Klebsiella pneumoniae Infections.

Ceftazidime-avibactam is a novel β-lactam/β-lactamase inhibitor with activity against carbapenem-resistant (CRE) that produce carbapenemase (KPC). We report the first cases of ceftazidime-avibactam resistance to develop during treatment of CRE infections and identify resistance mechanisms. Ceftazidime-avibactam-resistant emerged in three patients after ceftazidime-avibactam treatment for 10 to 19 days. Whole-genome sequencing (WGS) of longitudinal ceftazidime-avibactam-susceptible and -resistant isolates was used to identify potential resistance mechanisms. WGS identified mutations in plasmid-borne , which were not present in baseline isolates. mutations emerged independently in isolates of a novel sequence type 258 sublineage and resulted in variant KPC-3 enzymes. The mutations were validated as resistance determinants by measuring MICs of ceftazidime-avibactam and other agents following targeted gene disruption in , plasmid transfer, and cloning into competent In rank order, the impact of KPC-3 variants on ceftazidime-avibactam MICs was as follows: D179Y/T243M double substitution > D179Y > V240G. Remarkably, mutations reduced meropenem MICs ≥4-fold from baseline, restoring susceptibility in from two patients. Cefepime and ceftriaxone MICs were also reduced ≥4-fold against D179Y/T243M and D179Y variant isolates, but susceptibility was not restored. Reverse transcription-PCR revealed that expression of encoding D179Y/T243M and D179Y variants was diminished compared to expression in baseline isolates. In conclusion, the development of resistance-conferring mutations in within 10 to 19 days of ceftazidime-avibactam exposure is troubling, but clinical impact may be ameliorated if carbapenem susceptibility is restored in certain isolates.

Shields RK ，Chen L ，Cheng S ，Chavda KD ，Press EG ，Snyder A ，Pandey R ，Doi Y ，Kreiswirth BN ，Nguyen MH ，Clancy CJ ... -  《-》

Whole genome sequencing characterization of Slovenian carbapenem-resistant Klebsiella pneumoniae, including OXA-48 and NDM-1 producing outbreak isolates.

The first hospital outbreak of carbapenemase-producing Enterobacteriaceae in Slovenia occurred in 2014-2016. Whole genome sequencing was used to analyse the population of carbapenem-resistant Klebsiella pneumoniae collected in Slovenia in 2014-2017, including OXA-48 and/or NDM-1 producing strains from the outbreak. A total of 32 K. pneumoniae isolates were analysed using short-read sequencing. Multi-locus sequence typing and core genome multi-locus sequence typing were used to infer genetic relatedness. Antimicrobial resistance markers, virulence factors, plasmid content and wzi types were determined. Long-read sequencing was used for six isolates for detailed analysis of plasmids and their possible transmission. Overall, we detected 10 different sequence types (STs), the most common being ST437 (40.6%). Isolates from the initial outbreak belonged to ST437 (12/16) and ST147 (4/16). A second outbreak of four ST15 isolates was discovered. A new ST (ST3390) and two new wzi types (wzi-556, wzi-559) were identified. blaOXA-48 was found in 17 (53.1%) isolates, blaNDM-1 in five (15.6%), and a combination of blaOXA-48/NDM-1 in seven (21.9%) isolates. Identical plasmids carrying blaOXA-48 were found in outbreak isolates sequenced with long-read sequencing technology. Whole genome sequencing of Slovenian carbapenem-resistant K. pneumoniae isolates revealed multiple clusters of STs, two of which were involved in the first hospital outbreak of carbapenem producing K. pneumoniae in Slovenia. Transmission of the plasmid carrying blaOXA-48 between two STs was likely to have occurred. A previously unidentified second outbreak was also discovered, highlighting the importance of whole genome sequencing in detection and/or characterization of hospital outbreaks and surveillance of drug-resistant bacterial clones.

Benulič K ，Pirš M ，Couto N ，Chlebowicz M ，Rossen JWA ，Zorec TM ，Seme K ，Poljak M ，Lejko Zupanc T ，Ružić-Sabljić E ，Cerar T ... -  《PLoS One》

Exploring the resistome, virulome, and mobilome of multidrug-resistant Klebsiella pneumoniae isolates: deciphering the molecular basis of carbapenem resistance.

Klebsiella pneumoniae, a notorious pathogen for causing nosocomial infections has become a major cause of neonatal septicemia, leading to high morbidity and mortality worldwide. This opportunistic bacterium has become highly resistant to antibiotics due to the widespread acquisition of genes encoding a variety of enzymes such as extended-spectrum beta-lactamases (ESBLs) and carbapenemases. We collected Klebsiella pneumoniae isolates from a local tertiary care hospital from February 2019-February 2021. To gain molecular insight into the resistome, virulome, and genetic environment of significant genes of multidrug-resistant K. pneumoniae isolates, we performed the short-read whole-genome sequencing of 10 K. pneumoniae isolates recovered from adult patients, neonates, and hospital tap water samples. The draft genomes of the isolates varied in size, ranging from 5.48 to 5.96 Mbp suggesting the genome plasticity of this pathogen. Various genes conferring resistance to different classes of antibiotics e.g., aminoglycosides, quinolones, sulfonamides, tetracycline, and trimethoprim were identified in all sequenced isolates. The highest resistance was observed towards carbapenems, which has been putatively linked to the presence of both class B and class D carbapenemases, bla and bla, respectively. Moreover, the biocide resistance gene qacEdelta1 was found in 6/10 of the sequenced strains. The sequenced isolates exhibited a broad range of sequence types and capsular types. The significant antibiotic resistance genes (ARGs) were bracketed by a variety of mobile genetic elements (MGEs). Various spontaneous mutations in genes other than the acquired antibiotic-resistance genes were observed, which play an indirect role in making these bugs resistant to antibiotics. Loss or deficiency of outer membrane porins, combined with ESBL production, played a significant role in carbapenem resistance in our sequenced isolates. Phylogenetic analysis revealed that the study isolates exhibited evolutionary relationships with strains from China, India, and the USA suggesting a shared evolutionary history and potential dissemination of similar genes amongst the isolates of different origins. This study provides valuable insight into the presence of multiple mechanisms of carbapenem resistance in K. pneumoniae strains including the acquisition of multiple antibiotic-resistance genes through mobile genetic elements. Identification of rich mobilome yielded insightful information regarding the crucial role of insertion sequences, transposons, and integrons in shaping the genome of bacteria for the transmission of various resistance-associated genes. Multi-drug resistant isolates that had the fewest resistance genes exhibited a significant number of mutations. K. pneumoniae isolate from water source displayed comparable antibiotic resistance determinants to clinical isolates and the highest number of virulence-associated genes suggesting the possible interplay of ARGs amongst bacteria from different sources.

Rahmat Ullah S ，Irum S ，Mahnoor I ，Ismatullah H ，Mumtaz M ，Andleeb S ，Rahman A ，Jamal M ... -  《BMC GENOMICS》