Evaluating the Genome and Resistome of Extensively Drug-Resistant Klebsiella pneumoniae using Native DNA and RNA Nanopore Sequencing

ABSTRACTKlebsiella pneumoniae frequently harbour multidrug resistance and current methodologies are struggling to rapidly discern feasible antibiotics to treat these infections. While rapid DNA sequencing has been proposed for prediction of resistance profile; the role of rapid RNA sequencing has yet to be fully explored. The MinION sequencer can sequence native DNA and RNA in real-time, providing an opportunity to contrast the utility of DNA and RNA for prediction of drug susceptibility. This study interrogated the genome and transcriptome of four extensively drug-resistant (XDR) K. pneumoniae clinical isolates. The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kbp). DNA sequencing identified most resistance genes (≥70%) within 2 hours of sequencing. Direct RNA sequencing (with a ∼6x slower pore translocation) was able to identify ≥35% of resistance genes, including aminoglycoside, β-lactam, trimethoprim and sulphonamide and also quinolone, rifampicin, fosfomycin and phenicol in some isolates, within 10 hours of sequencing. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to qRT-PCR across 11 resistance genes. Overall, MinION sequencing rapidly detected the XDR K. pneumoniae resistome and direct RNA sequencing revealed differential expression of these genes.

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Evaluating the genome and resistome of extensively drug-resistant Klebsiella pneumoniae using native DNA and RNA Nanopore sequencing

GigaScience ◽

10.1093/gigascience/giaa002 ◽

2020 ◽

Vol 9 (2) ◽

Cited By ~ 2

Author(s):

Miranda E Pitt ◽

Son H Nguyen ◽

Tânia P S Duarte ◽

Haotian Teng ◽

Mark A T Blaskovich ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Real Time ◽

Rna Sequencing ◽

Resistance Genes ◽

Accurate Estimation ◽

Drug Resistant ◽

Expression Levels ◽

Base Calling ◽

Dna And Rna ◽

Extensively Drug Resistant

Abstract Background Klebsiella pneumoniae frequently harbours multidrug resistance, and current diagnostics struggle to rapidly identify appropriate antibiotics to treat these bacterial infections. The MinION device can sequence native DNA and RNA in real time, providing an opportunity to compare the utility of DNA and RNA for prediction of antibiotic susceptibility. However, the effectiveness of bacterial direct RNA sequencing and base-calling has not previously been investigated. This study interrogated the genome and transcriptome of 4 extensively drug-resistant (XDR) K. pneumoniae clinical isolates; however, further antimicrobial susceptibility testing identified 3 isolates as pandrug-resistant (PDR). Results The majority of acquired resistance (≥75%) resided on plasmids including several megaplasmids (≥100 kb). DNA sequencing detected most resistance genes (≥70%) within 2 hours of sequencing. Neural network–based base-calling of direct RNA achieved up to 86% identity rate, although ≤23% of reads could be aligned. Direct RNA sequencing (with ∼6 times slower pore translocation) was able to identify (within 10 hours) ≥35% of resistance genes, including those associated with resistance to aminoglycosides, β-lactams, trimethoprim, and sulphonamide and also quinolones, rifampicin, fosfomycin, and phenicol in some isolates. Direct RNA sequencing also identified the presence of operons containing up to 3 resistance genes. Polymyxin-resistant isolates showed a heightened transcription of phoPQ (≥2-fold) and the pmrHFIJKLM operon (≥8-fold). Expression levels estimated from direct RNA sequencing displayed strong correlation (Pearson: 0.86) compared to quantitative real-time PCR across 11 resistance genes. Conclusion Overall, MinION sequencing rapidly detected the XDR/PDR K. pneumoniae resistome, and direct RNA sequencing provided accurate estimation of expression levels of these genes.

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Sensititre MycoTB Plate Compared to Bactec MGIT 960 for First- and Second-Line Antituberculosis Drug Susceptibility Testing in Tanzania: a Call To Operationalize MICs

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01117-15 ◽

2015 ◽

Vol 59 (11) ◽

pp. 7104-7108 ◽

Cited By ~ 29

Author(s):

Scott K. Heysell ◽

Suporn Pholwat ◽

Stellah G. Mpagama ◽

Saumu J. Pazia ◽

Happy Kumburu ◽

...

Keyword(s):

Susceptibility Testing ◽

Acquired Resistance ◽

Drug Susceptibility ◽

Drug Susceptibility Testing ◽

Drug Resistant ◽

Second Line ◽

Drug Resistant Tuberculosis ◽

Content Type ◽

Resistant Tuberculosis ◽

Extensively Drug Resistant

ABSTRACTMIC testing forMycobacterium tuberculosisis now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first- and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.

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Multidrug-Resistant Klebsiella Pneumoniae: A Retrospective Study in Manaus, Brazil

10.21203/rs.3.rs-1028514/v1 ◽

2021 ◽

Author(s):

Rafael Nakamura-Silva ◽

Louise Cerdeira ◽

Mariana Oliveira-Silva ◽

Karen Regina Carim da Costa ◽

Elder Sano ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Opportunistic Pathogen ◽

Multidrug Resistant ◽

Drug Resistant ◽

Extensively Drug Resistant ◽

Antimicrobial Resistance Genes ◽

Northern Brazil ◽

The City

Abstract Klebsiella pneumoniae is an opportunistic pathogen that can cause several infections, mainly in hospitalised or immunocompromised individuals. The spread of K. pneumoniae emerging virulent and multidrug-resistant clones is a worldwide concern and its identification is crucial to control these strains especially in hospitals. This article reports data related to multi-resistant K. pneumoniae strains, isolated from inpatients in the city of Manaus, Brazil, harbouring virulence and antimicrobial resistance genes, including high-risk international clones belonging to clonal group (CG) 258. Twenty-one strains isolated from different patients admitted to four hospitals in the city of Manaus, located in the state of Amazonas, Northern Brazil (Amazon Rainforest region) were evaluated. The majority of strains (61.9 % n = 13) were classified as multidrug-resistant (MDR), and five strains (23.8 %) as extensively drug-resistant (XDR). Several virulence and antimicrobial resistance genes were found among the strains and eight strains (38.1 %) presented the hypermucoviscous phenotype. MLST analysis demonstrated a great diversity of STs among the strains, totaling 12 different STs (ST11, ST23, ST198, ST277, ST307, ST340, ST378, ST462, ST502, ST3991, ST3993 and ST5209). Four of these (ST11, ST23, ST307 and ST340) belong to CG258.

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Genomic insights into multi-drug and extensively drug resistant Klebsiella pneumoniae from India

International Journal of Infectious Diseases ◽

10.1016/j.ijid.2020.09.069 ◽

2020 ◽

Vol 101 ◽

pp. 12-13

Author(s):

C. Shankar ◽

P. Mathur ◽

J.J. Jacob ◽

C. Rodrigues ◽

K. Walia ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Drug Resistant ◽

Extensively Drug Resistant

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Application of "Precision Medicine" Through the Molecular Characterization of Extensively Drug Resistant (XDR) Klebsiella pneumoniae in a Multivisceral Transplant Candidate

Open Forum Infectious Diseases ◽

10.1093/ofid/ofw172.1580 ◽

2016 ◽

Vol 3 (suppl_1) ◽

Cited By ~ 1

Author(s):

Rossana Rosa ◽

Susan D. Rudin ◽

Laura J. Rojas ◽

Armando Perez-Cardona ◽

Laura Aragon ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Precision Medicine ◽

Molecular Characterization ◽

Drug Resistant ◽

Extensively Drug Resistant ◽

Transplant Candidate

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The Biology and Role of Interleukin-32 in Tuberculosis

Journal of Immunology Research ◽

10.1155/2018/1535194 ◽

2018 ◽

Vol 2018 ◽

pp. 1-9 ◽

Cited By ~ 1

Author(s):

Wu Li ◽

Wanyan Deng ◽

Jianping Xie

Keyword(s):

Multidrug Resistant ◽

Drug Resistant ◽

Host Molecule ◽

Tuberculosis Control ◽

Promising Alternative ◽

Extensively Drug Resistant ◽

Important Host ◽

Resistant Strains ◽

Drug Resistant Strains

Tuberculosis, caused by Mycobacterium tuberculosis, remains a leading cause of morbidity and mortality globally, with nearly 10.4 million new cases of incidence and over 1.7 million deaths annually. Drug-resistant M. tuberculosis strains, especially multidrug-resistant or extensively drug-resistant strains, have further intensified the problem associated with tuberculosis control. Host-directed therapy is a promising alternative for tuberculosis control. IL-32 is increasingly recognized as an important host molecule against tuberculosis. In this review, we highlight the proinflammatory properties of IL-32 and the mode of action of IL-32 in mycobacterial infections to inspire the development of novel immunity-based countermeasures and host-directed therapies against tuberculosis.

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Role of embB in natural and acquired resistance to ethambutol in mycobacteria.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.41.10.2270 ◽

1997 ◽

Vol 41 (10) ◽

pp. 2270-2273 ◽

Cited By ~ 80

Author(s):

F Alcaide ◽

G E Pfyffer ◽

A Telenti

Keyword(s):

Nontuberculous Mycobacteria ◽

Practical Implication ◽

Acquired Resistance ◽

Fold Increase ◽

Drug Susceptibility ◽

Natural Resistance ◽

Intrinsic Resistance ◽

Resistant Strains ◽

High Level

The mycobacterial embCAB operon encodes arabinosyl transferases, putative targets of the antimycobacterial agent ethambutol (EMB). Mutations in embB lead to resistance to EMB in Mycobacterium tuberculosis. The basis for natural, intrinsic resistance to EMB in nontuberculous mycobacteria (NTM) is not known; neither is the practical implication of resistance to EMB in the absence of embB mutations in M. tuberculosis well understood. The conserved embB resistance-determining region (ERDR) of a collection of 13 strains of NTM and 12 EMB-resistant strains of M. tuberculosis was investigated. Genotypes were correlated with drug susceptibility phenotypes. High-level natural resistance to EMB (MIC, . or =64 microg/ml) was associated with a variant amino acid motif in the ERDR of M. abscessus, M. chelonae, and M. leprae. Transfer of the M. abscessus emb allele to M. smegmatis resulted in a 500-fold increase in the MICs. In M. tuberculosis, embB mutations were associated with MICs of > or =20 microg/ml while resistance not associated with an ERDR mutation generally resulted in MICs of < or =10 microg/ml. These data further support the notion that the emb region determines intrinsic and acquired resistance to EMB and might help in the reassessment of the current recommendations for the screening and treatment of infections with EMB-resistant M. tuberculosis and NTM.

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Extensively Drug-Resistant Hypervirulent Klebsiella pneumoniae From a Series of Neonatal Sepsis in a Tertiary Care Hospital, India

Frontiers in Medicine ◽

10.3389/fmed.2021.645955 ◽

2021 ◽

Vol 8 ◽

Author(s):

Tuhina Banerjee ◽

Jayalaxmi Wangkheimayum ◽

Swati Sharma ◽

Ashok Kumar ◽

Amitabha Bhattacharjee

Keyword(s):

Klebsiella Pneumoniae ◽

Neonatal Sepsis ◽

Tertiary Care Hospital ◽

Tertiary Care ◽

Plasmid Profile ◽

Care Hospital ◽

Drug Resistant ◽

Maternal Complications ◽

Extensively Drug Resistant ◽

Recent Emergence

The recent emergence of multidrug-resistant (MDR) Klebsiella pneumoniae with hypervirulent traits causing severe infections and considerable mortality is a global cause for concern. The challenges posed by these hypermucoviscous strains of K. pneumoniae with regard to their optimal treatment, management, and control policies are yet to be answered. We studied a series of extensively drug-resistant (XDR) and hypervirulent K. pneumoniae ST5235 isolates with resistance to carbapenems and polymyxins causing neonatal sepsis in a tertiary care hospital in India. A total of 9 K. pneumoniae isolates from 9 cases of neonatal sepsis were studied with respect to their clinical relevance, antimicrobial susceptibility profile, presence of extended spectrum β lactamase (ESBL) production, and responsible genes, carbapenemases (classes A, B, and D), and aminoglycoside-resistant genes. Hypervirulence genes encoding hypermucoid nature, iron uptake, and siderophores were detected by multiplex PCR. The plasmid profile was studied by replicon typing. Isolates were typed by multilocus sequence typing (MLST) and enterobacterial repetitive intergenic consensus (ERIC) PCR to study the sequence types (STs) and clonal relation, respectively. The neonates in the studied cases had history of pre-maturity or low birth weight with maternal complications. All the cases were empirically treated with piperacillin–tazobactam and amikacin followed by imipenem/meropenem and vancomycin and polymyxin B as a last resort. However, all the neonates finally succumbed to the condition (100%). The studied isolates were XDR including resistance to polymyxins harboring multiple ESBL genes and carbapenemase genes (blaNDM and blaOXA−48). Hypervirulence genes were present in various combinations with rmpA/A2 genes present in all the isolates. IncFI plasmids were detected in these isolates. All belonged to ST5235. In ERIC PCR, 6 different clusters were seen. The study highlighted the emergence and burden of XDR hypervirulent isolates of K. pneumoniae causing neonatal sepsis in a tertiary care hospital.

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High Frequency of Multidrug-resistant (Mdr) Klebsiella Pneumoniae Harboring Several β-lactamase and Integron Genes Collected From Several Hospitals in the North of Iran

10.21203/rs.3.rs-260577/v1 ◽

2021 ◽

Author(s):

Mojgan Farhadi ◽

Mohammad Ahanjan ◽

Hamid Reza Goli ◽

Mohammad Reza Haghshenas ◽

Mehrdad Gholami

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Antimicrobial Susceptibility ◽

Hospitalized Patients ◽

Clinical Samples ◽

High Morbidity ◽

Drug Resistant ◽

The North ◽

Agar Diffusion Test ◽

North Of Iran

Abstract Background: Klebsiella pneumoniae is one of the leading causes of hospital outbreaks worldwide. Also, antibiotic-resistant K. pneumoniae is progressively being involved in invasive infections with high morbidity and mortality. The aim of the current study was to determine antimicrobial susceptibility patterns and the incidences of resistance genes (integron types and β-lactamase-encoded genes) among clinical isolates of K. pneumoniae. Methods: In this cross-sectional study, a total of 100 clinical samples were obtained from hospitalized patients in three teaching hospitals in the north of Iran, from November 2018 and October 2019. Antimicrobial susceptibility testing was performed using disk agar diffusion test in line with CLSI recommendation. For colistin, minimum inhibitory concentration (MIC) was determined using broth microdilution. Based on antibiogram, multi-drug resistant (MDR) and extensive-drug resistant (XDR) strains were detected. Finally, integron types and β-lactamase resistance genes were identified using polymerase chain reaction technique.Results. The most and least clinical samples were related to the urine and bronchoalveolar lavage, respectively. Based on the antibiogram results, amikacin and gentamicin exhibited good activity against K. pneumoniae strains in vitro. High resistance rate (93%) to ampicillin/sulbactam also predict the limited efficacy of this antibiotic. Among all the 100 isolates, the frequency of MDR and XDR strains were 58% and 13%, respectively, while no pan-drug resistant (PDR) isolates were found. The prevalence of blaSHV, blaTEM, blaCTX-M-15, blaKPC, blaOXA-48, blaNDM β-lactamase genes were 91.4%, 82.7%, 79.3%, 29.3%, 36.2% and 6.9%, respectively, however 58% of the isolates were carrying intI gene. Class II and III integrons were not detected in any isolates. Conclusion: The MDR K. pneumoniae is becoming a serious problem in hospitals, with many strains developing resistance to most available antimicrobials. Our results indicate co-presence of a series of β-lactamase and integron types on the MDR strains recovered from hospitalized patients. The increasing rate of these isolates emphasizes the importance of choosing an appropriate antimicrobial regimen based on antibiotic susceptibility pattern.

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