Genomic insights into the diversity, virulence and resistance of Klebsiella pneumoniae extensively drug resistant clinical isolates

Klebsiella pneumoniae has been implicated in wide-ranging nosocomial outbreaks, causing severe infections without effective treatments due to antibiotic resistance. Here, we performed genome sequencing of 70 extensively drug resistant clinical isolates, collected from Brasília’s hospitals (Brazil) between 2010 and 2014. The majority of strains (60 out of 70) belonged to a single clonal complex (CC), CC258, which has become distributed worldwide in the last two decades. Of these CC258 strains, 44 strains were classified as sequence type 11 (ST11) and fell into two distinct clades, but no ST258 strains were found. These 70 strains had a pan-genome size of 10 366 genes, with a core-genome size of ~4476 genes found in 95 % of isolates. Analysis of sequences revealed diverse mechanisms of resistance, including production of multidrug efflux pumps, enzymes with the same target function but with reduced or no affinity to the drug, and proteins that protected the drug target or inactivated the drug. β-Lactamase production provided the most notable mechanism associated with K. pneumoniae . Each strain presented two or three different β-lactamase enzymes, including class A (SHV, CTX-M and KPC), class B and class C AmpC enzymes, although no class D β-lactamase was identified. Strains carrying the NDM enzyme involved three different ST types, suggesting that there was no common genetic origin.

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In vitro synergistic activity of the sulbactam/avibactam combination against extensively drug-resistant Acinetobacter baumannii

Journal of Medical Microbiology ◽

10.1099/jmm.0.001211 ◽

2020 ◽

Vol 69 (7) ◽

pp. 928-931 ◽

Cited By ~ 1

Author(s):

Carlos Hernán Rodriguez ◽

Adriana Brune ◽

Marcela Nastro ◽

Carlos Vay ◽

Angela Famiglietti

Keyword(s):

Acinetobacter Baumannii ◽

Type Species ◽

Synergistic Activity ◽

Drug Resistant ◽

Disc Diffusion ◽

Content Type ◽

Link Type ◽

Extensively Drug Resistant ◽

Synergy Test ◽

Time Kill

Introduction. The therapeutic options to treat Acinetobacter baumannii infections are very limited. Aim. Our aim was to evaluate the activity of sulbactam combined directly with avibactam or the ampicillin-sulbactam/ceftazidime-avibactam combination against extensively drug-resistant A. baumannii isolates. Methodology. Extensively drug-resistant A. baumannii isolates (n=127) collected at several South American hospitals were studied. Synergy with the sulbactam/avibactam combination was assessed in all isolates using the agar dilution method. Avibactam was used at a fixed concentration of 4 mg l−1. A disc diffusion synergy test was also performed. Synergy by a time-kill experiment was performed in a selected isolate. Results. Synergy with sulbactam/avibactam was demonstrated in 124 isolates and it showed MIC values ≤4 mg l−1. This synergy was not detected in the three New Delhi metallo-β-lactamase-harbouring isolates. Similar results were observed with the disc diffusion synergy test of ampicillin-sulbactam/ceftazidime-avibactam. In the time-kill experiments, sulbactam/avibactam showed a rapid synergistic and bactericidal activity in ampicillin-sulbactam-resistant isolates. Conclusions. This study demonstrated that the sulbactam/avibactam combination displayed synergistic activity against A. baumannii isolates. This synergy was observed when both inhibitors were also used as part of the commercially available combinations: ampicillin-sulbactam and ceftazidime-avibactam.

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Complete Genome Sequences of Four Extensively Drug-Resistant Acinetobacter baumannii Isolates from Thailand

Microbiology Resource Announcements ◽

10.1128/mra.00949-20 ◽

2020 ◽

Vol 9 (40) ◽

Author(s):

Peechanika Chopjitt ◽

Thidathip Wongsurawat ◽

Piroon Jenjaroenpun ◽

Parichart Boueroy ◽

Rujirat Hatrongjit ◽

...

Keyword(s):

Acinetobacter Baumannii ◽

Complete Genome ◽

Sequence Type ◽

Clinical Isolates ◽

Drug Resistant ◽

Genome Sequences ◽

Content Type ◽

Type Isolate ◽

Resistant Genes ◽

Extensively Drug Resistant

ABSTRACT Here, we report the complete genome sequences of four clinical isolates of extensively drug-resistant Acinetobacter baumannii (XDRAB), isolated in Thailand. These results revealed multiple antimicrobial-resistant genes, each involving two sequence type 16 (ST16) isolates, ST2, and a novel sequence type isolate, ST1479.

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Genomic evolution and local epidemiology of Klebsiella pneumoniae from a major hospital in Beijing, China, over a 15 year period: dissemination of known and novel high-risk clones

Microbial Genomics ◽

10.1099/mgen.0.000520 ◽

2021 ◽

Author(s):

Mattia Palmieri ◽

Kelly L. Wyres ◽

Caroline Mirande ◽

Zhao Qiang ◽

Ye Liyan ◽

...

Keyword(s):

High Risk ◽

Klebsiella Pneumoniae ◽

Type Species ◽

Treatment Options ◽

Multidrug Resistant ◽

Virulence Plasmid ◽

Content Type ◽

Origin And Evolution ◽

Link Type ◽

Beijing China

Klebsiella pneumoniae is a frequent cause of nosocomial and severe community-acquired infections. Multidrug-resistant (MDR) and hypervirulent (hv) strains represent major threats, and tracking their emergence, evolution and the emerging convergence of MDR and hv traits is of major importance. We employed whole-genome sequencing (WGS) to study the evolution and epidemiology of a large longitudinal collection of clinical K. pneumoniae isolates from the H301 hospital in Beijing, China. Overall, the population was highly diverse, although some clones were predominant. Strains belonging to clonal group (CG) 258 were dominant, and represented the majority of carbapenemase-producers. While CG258 strains showed high diversity, one clone, ST11-KL47, represented the majority of isolates, and was highly associated with the KPC-2 carbapenemase and several virulence factors, including a virulence plasmid. The second dominant clone was CG23, which is the major hv clone globally. While it is usually susceptible to multiple antibiotics, we found some isolates harbouring MDR plasmids encoding for ESBLs and carbapenemases. We also reported the local emergence of a recently described high-risk clone, ST383. Conversely to strains belonging to CG258, which are usually associated to KPC-2, ST383 strains seem to readily acquire carbapenemases of different types. Moreover, we found several ST383 strains carrying the hypervirulence plasmid. Overall, we detected about 5 % of simultaneous carriage of AMR genes (ESBLs or carbapenemases) and hypervirulence genes. Tracking the emergence and evolution of such strains, causing severe infections with limited treatment options, is fundamental in order to understand their origin and evolution and to limit their spread. This article contains data hosted by Microreact.

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Emergence of mgrB locus deletion mediating polymyxin resistance in pandemic KPC-producing Klebsiella pneumoniae ST15 lineage

Journal of Medical Microbiology ◽

10.1099/jmm.0.001309 ◽

2021 ◽

Author(s):

Luís Guilherme de Araújo Longo ◽

Herrison Fontana ◽

Viviane Santos de Sousa ◽

Natalia Chilinque Zambão da Silva ◽

Ianick Souto Martins ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Klebsiella Pneumoniae ◽

Type Species ◽

Virulence Genes ◽

Health Concern ◽

Beta Lactamase ◽

Content Type ◽

Link Type ◽

Antimicrobial Resistance Genes ◽

Encoding Gene

Klebsiella pneumoniae causes a diversity of infections in both healthcare and community settings. This pathogen is showing an increased ability to accumulate antimicrobial resistance and virulence genes, making it a public health concern. Here we describe the whole-genome sequence characteristics of an ST15 colistin-resistant K. pneumoniae isolate obtained from a blood culture of a 79-year-old female patient admitted to a university hospital in Brazil. Kp14U04 was resistant to most clinically useful antimicrobial agents, remaining susceptible only to aminoglycosides and fosfomycin. The colistin resistance in this isolate was due to a ~1.3 kb deletion containing four genes, namely mgrB, yebO, yobH and the transcriptional regulator kdgR. The study isolate presented a variety of antimicrobial resistance genes, including the carbapenemase-encoding gene bla KPC-2, the extended-spectrum beta-lactamase (ESBL)-encoding gene bla SHV-28 and the beta-lactamase-encoding gene bla OXA-1. Additionally, Kp14U04 harboured a multiple stress resistance protein, efflux systems and regulators, heavy metal resistance and virulence genes, plasmids, prophage-related sequences and genomic islands. These features revealed the high potential of this isolate to resist antimicrobial therapy, survive in adverse environments, cause infections and overcome host defence mechanisms.

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Emergence of a plasmid-borne tigecycline resistance in Klebsiella pneumoniae in Vietnam

Journal of Medical Microbiology ◽

10.1099/jmm.0.001320 ◽

2021 ◽

Author(s):

Aki Hirabayashi ◽

Van Thi Thu Ha ◽

An Van Nguyen ◽

Son Thai Nguyen ◽

Keigo Shibayama ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Gene Cluster ◽

Type Species ◽

Bacterial Infections ◽

Efflux Pump ◽

Whole Genome Sequence ◽

High Sequence Identity ◽

Pump System ◽

Content Type ◽

Link Type

Tigecycline is a last-resort antimicrobial used to treat multidrug-resistant Gram-negative bacterial infections. One of the common antimicrobial resistance mechanisms is the efflux pump system composed of membrane protein complexes to excrete xenobiotic substrates. Recently, a novel gene cluster, tmexCD1-toprJ1, encoding the resistance–nodulation–cell division (RND) efflux pump was identified on plasmids in Klebsiella pneumoniae isolates in China. TMexCD1-TOprJ1 was found to be capable of excreting multiple antimicrobials, including tigecycline, which contributed to the strain's resistance. In this study, we identified K. pneumoniae isolates harbouring the tmexCD1-toprJ1 genes outside of China for the first time. Two tigecycline-resistant K. pneumoniae isolates belonging to ST273 by multilocus sequence typing were collected from different patients in a medical institution in Hanoi, Vietnam, in 2015. Whole-genome sequence analysis revealed that these isolates harboured a 288.0 kb tmexCD1-toprJ1–carrying plasmid with IncFIB and IncHI1B replicons. The tmexCD1-toprJ1 gene cluster was surrounded by several mobile gene elements, including IS26, and the plasmids had high sequence identity with that of K. pneumoniae isolated in China. Our finding suggests that the horizontal spread of tigecycline resistance mediated by tmexCD1-toprJ1–carrying plasmids has occurred in Vietnam and other countries, and raises concern about the further global dissemination.

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Evaluation of Pyrosequencing for Detecting Extensively Drug-Resistant Mycobacterium tuberculosis among Clinical Isolates from Four High-Burden Countries

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.03614-14 ◽

2014 ◽

Vol 59 (1) ◽

pp. 414-420 ◽

Cited By ~ 20

Author(s):

Kanchan Ajbani ◽

Shou-Yean Grace Lin ◽

Camilla Rodrigues ◽

Duylinh Nguyen ◽

Francine Arroyo ◽

...

Keyword(s):

Drug Resistance ◽

Mycobacterium Tuberculosis ◽

The Philippines ◽

Drug Susceptibility Testing ◽

Clinical Isolates ◽

Drug Resistant ◽

Second Line ◽

Additional Advantage ◽

Content Type ◽

Extensively Drug Resistant

ABSTRACTReliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance inMycobacterium tuberculosisisolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identifyM. tuberculosis(via the IS6110marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets includedkatG, theinhApromoter and theahpC-oxyRintergenic region for isoniazid (INH) resistance; therpoBcore region for rifampin (RIF) resistance;gyrAfor fluoroquinolone (FQ) resistance; andrrsfor amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

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Antimicrobial Activities of Aztreonam-Avibactam and Comparator Agents against Contemporary (2016) Clinical Enterobacteriaceae Isolates

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01856-17 ◽

2017 ◽

Vol 62 (1) ◽

Cited By ~ 28

Author(s):

Helio S. Sader ◽

Rodrigo E. Mendes ◽

Michael A. Pfaller ◽

Dee Shortridge ◽

Robert K. Flamm ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Multidrug Resistant ◽

Antimicrobial Activities ◽

Drug Resistant ◽

Content Type ◽

Medical Centers ◽

Extensively Drug Resistant ◽

Mic Value

ABSTRACT A total of 10,451 contemporary (2016) Enterobacteriaceae isolates from 84 U.S. medical centers and 116 metallo-β-lactamase- and/or OXA-48-like-producing Enterobacteriaceae isolates from other countries were tested against aztreonam-avibactam and comparators. All U.S. isolates were inhibited at aztreonam-avibactam MICs of ≤8 μg/ml (MIC50, ≤0.03 μg/ml; MIC90, 0.12 μg/ml), including Klebsiella pneumoniae carbapenemase-producing isolates (n = 102; MIC50, 0.25 μg/ml; MIC90, 0.5 μg/ml), multidrug-resistant isolates (n = 876; MIC50, 0.06 μg/ml; MIC90, 0.25 μg/ml), and extensively drug-resistant isolates (n = 111; MIC50, 0.12 μg/ml; MIC90, 0.5 μg/ml). The highest aztreonam-avibactam MIC value among ex-U.S. isolates was 4 μg/ml.

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Genomic investigation of a suspected Klebsiella pneumoniae outbreak in a neonatal care unit in sub-Saharan Africa

Microbial Genomics ◽

10.1099/mgen.0.000703 ◽

2021 ◽

Vol 7 (11) ◽

Author(s):

Jennifer Cornick ◽

Patrick Musicha ◽

Chikondi Peno ◽

Ezgi Seager ◽

Pui-Ying Iroh Tam ◽

...

Keyword(s):

Klebsiella Pneumoniae ◽

Resistance Genes ◽

Type Species ◽

Bloodstream Infections ◽

Sub Saharan Africa ◽

Content Type ◽

Link Type ◽

Sub Saharan ◽

Neonatal Care Unit ◽

Suspected Outbreak

A special-care neonatal unit from a large public hospital in Malawi was noted as having more frequent, difficult-to-treat infections, and a suspected outbreak of multi-drug-resistant Klebsiella pneumoniae was investigated using genomic characterisation. All K. pneumoniae bloodstream infections (BSIs) from patients in the neonatal ward (n=62), and a subset of K. pneumoniae BSI isolates (n=38) from other paediatric wards in the hospital, collected over a 4 year period were studied. After whole genome sequencing, the strain sequence types (STs), plasmid types, virulence and resistance genes were identified. One ST340 clone, part of clonal complex 258 (CC258) and an ST that drives hospital outbreaks worldwide, harbouring numerous resistance genes and plasmids, was implicated as the likely cause of the outbreak. This study contributes molecular information necessary for tracking and characterizing this important hospital pathogen in sub-Saharan Africa.

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Accuracy of an amplicon-sequencing nanopore approach to identify variants in tuberculosis drug-resistance-associated genes

Microbial Genomics ◽

10.1099/mgen.0.000740 ◽

2021 ◽

Vol 7 (12) ◽

Author(s):

Carla Mariner-Llicer ◽

Galo A. Goig ◽

Laura Zaragoza-Infante ◽

Manuela Torres-Puente ◽

Luis Villamayor ◽

...

Keyword(s):

Drug Resistance ◽

Type Species ◽

Variant Calling ◽

Targeted Sequencing ◽

Clinical Samples ◽

Drug Resistant ◽

Content Type ◽

Link Type ◽

Resistant Strains ◽

Drug Resistant Strains

A rapid and accurate diagnostic assay represents an important means to detect Mycobacterium tuberculosis , identify drug-resistant strains and ensure treatment success. Currently employed techniques to diagnose drug-resistant tuberculosis include slow phenotypic tests or more rapid molecular assays that evaluate a limited range of drugs. Whole-genome-sequencing-based approaches can detect known drug-resistance-conferring mutations and novel variations; however, the dependence on growing samples in culture, and the associated delays in achieving results, represents a significant limitation. As an alternative, targeted sequencing strategies can be directly performed on clinical samples at high throughput. This study proposes a targeted sequencing assay to rapidly detect drug-resistant strains of M. tuberculosis using the Nanopore MinION sequencing platform. We designed a single-tube assay that targets nine genes associated with drug resistance to seven drugs and two phylogenetic-determining regions to determine strain lineage and tested it in nine clinical isolates and six sputa. The study’s main aim is to calibrate MinNION variant calling to detect drug-resistance-associated mutations with different frequencies to match the accuracy of Illumina (the current gold-standard sequencing technology) from both culture and sputum samples. After calibrating Nanopore MinION variant calling, we demonstrated 100% agreement between Illumina WGS and our MinION set up to detect known drug resistance and phylogenetic variants in our dataset. Importantly, other variants in the amplicons are also detected, decreasing the recall. We identify minority variants and insertions/deletions as crucial bioinformatics challenges to fully reproduce Illumina WGS results.

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Antibacterial activity of high-dose nitric oxide against pulmonary Mycobacterium abscessus disease

Access Microbiology ◽

10.1099/acmi.0.000154 ◽

2020 ◽

Vol 2 (9) ◽

Cited By ~ 1

Author(s):

Kristijan Bogdanovski ◽

Trisha Chau ◽

Chevalia J. Robinson ◽

Sandra D. MacDonald ◽

Ann M. Peterson ◽

...

Keyword(s):

Nitric Oxide ◽

Antibacterial Activity ◽

Type Species ◽

Mycobacterium Abscessus ◽

Clinical Isolates ◽

Content Type ◽

Link Type ◽

Susceptibility Tests ◽

Inhaled No

Introduction. Mycobacterium abscessus is an emerging pulmonary pathogen with limited treatment options. Nitric oxide (NO) demonstrates antibacterial activity against various bacterial species, including mycobacteria. In this study, we evaluated the effect of adjunctive inhaled NO therapy, using a novel NO generator, in a CF patient with pulmonary M. abscessus disease, and examined heterogeneity of response to NO in vitro. Methods. In the compassionate-use treatment, a 24-year-old CF patient with pulmonary M. abscessus was treated with two courses of adjunctive intermittent NO, first at 160 p.p.m. for 21 days and subsequently by escalating the dose up to 240 p.p.m. for 8 days. Methemoglobin, pulmonary function, 6 min walk distance (6MWD), qualify of life and sputum microbiology were assessed. In vitro susceptibility tests were performed against patient’s isolate and comparison clinical isolates and quantified by Hill’s slopes calculated from time–kill curves. Results. M. abscessus lung infection eradication was not achieved, but improvements in selected qualify of life domains, lung function and 6MWD were observed during the study. Inhaled NO was well tolerated at 160 p.p.m. Dosing at 240 p.p.m. was stopped due to adverse symptoms, although methemoglobin levels remained within safety thresholds. In vitro susceptibility tests showed a dose-dependent NO effect on M. abscessus susceptibility and significant heterogeneity in response between M. abscessus clinical isolates. The patient’s isolate was found to be the least susceptible strain in vitro. Conclusion. These results demonstrate heterogeneity in M. abscessus susceptibility to NO and suggest that longer treatment regimens could be required to see the reduction or eradication of more resistant pulmonary strains.

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