scholarly journals Three new integration vectors and fluorescent proteins for use in the opportunistic human pathogenStreptococcus pneumoniae

2019 ◽  
Author(s):  
Lance E. Keller ◽  
Anne-Stéphanie Rueff ◽  
Jun Kurushima ◽  
Jan-Willem Veening
Keyword(s):  
Transformation Efficiency ◽  
Fluorescent Proteins ◽  
Inducible Promoter ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Large Set ◽  
Wide Range ◽  
Integration Sites ◽  
Opportunistic Human Pathogen ◽  
Multiple Cloning Sites

AbstractHere we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogenStreptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform, employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate inEscherichia coliand harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously were tested. Two of the three PEP vectors share homology of the integration regions with over half of theS. pneumoniaegenomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

scholarly journals Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 394 ◽  
Author(s):  
Lance E. Keller ◽  
Anne-Stéphanie Rueff ◽  
Jun Kurushima ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transformation Efficiency ◽  
Fluorescent Proteins ◽  
Inducible Promoter ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Large Set ◽  
Human Pathogen ◽  
Wide Range ◽  
Opportunistic Human Pathogen

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

scholarly journals Volatile Oils ofNepeta tenuifolia(Jing Jie) as an Alternative Medicine against Multidrug-Resistant Pathogenic Microbes

2018 ◽  
Vol 2018 ◽  
pp. 1-8 ◽  
Author(s):  
Yi-Hsuan Lee ◽  
Chao-Min Wang ◽  
Po-Yu Liu ◽  
Ching-Chang Cheng ◽  
Zong-Yen Wu ◽  
...  
Keyword(s):  
Essential Oils ◽  
In Vitro Study ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Gram Negative ◽  
E Coli ◽  
Wide Range ◽  
Main Component ◽  
Reference Strains

Essential oils from the dried spikes ofNepeta tenuifolia(Benth) are obtained by steam distillation. Pulegone was identified as the main component in the spikes ofN. tenuifoliathrough analysis, with greater than 85% purity obtained in this study. The essential oils are extremely active against all Gram-positive and some Gram-negative reference bacteria, particularlySalmonella enterica,Citrobacter freundii, andEscherichia coli. The minimum inhibitory concentration was found to be between 0.08 and 0.78% (againstS. enterica), 0.39 and 0.78% (againstC. freundii), and 0.097 and 0.39% (againstE. coli), whereas the minimum bactericidal concentration varied in range from 0.097% to 1.04%. In general, the essential oils show a strong inhibitory action against all tested reference strains and clinical isolates. However, the antibacterial activity of EOs against bothPseudomonas aeruginosareference strains and clinical isolates was relatively lower than other Gram-negative pathogens. The essential oils ofN. tenuifoliaalso displayed bactericidal activities (MBC/MIC < 4) in this study. These findings reflect the bactericidal activity of the essential oils against a wide range of multidrug-resistant clinical pathogens in an in vitro study. In addition, we propose the fragmentation pathways of pulegone and its derivatives by LC-ESI-MS/MS in this study.

scholarly journals Detection of plasmid-mediated AmpC-?-lactamases among clinical isolates:Adiagnostic and therapeutic challenge

Biomedicine ◽  
2021 ◽  
Vol 41 (2) ◽  
pp. 310-314
Author(s):  
Mol P Ronni ◽  
Shanthi Ganesan ◽  
Almarabheh Amer ◽  
Bindayna Khalid M
Keyword(s):  
Clinical Laboratory ◽  
Resistance Mechanism ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Fourth Generation ◽  
Care Hospital ◽  
Cross Sectional ◽  
Chain Reactions ◽  
Genotypic Analysis ◽  
Wide Range

Introduction and Aim: The AmpC enzymes are cephalosporinases that impart resistance to a wide range of ?-lactam, ?-lactam/?-lactamase inhibitor combinations, and monobactams, but are sensitive to fourth generation cephalosporins and carbapenems. Identification techniques for AmpC beta lactamases are not yet adapted for the clinical laboratory, which is likely to underestimate this resistance mechanism. Detection and determination of the magnitude of AmpC is therefore critical for successful treatment and for the prevention and control of these resistant bacteria.The present study was intended to determine the prevalence of plasmid mediated AmpC genotypes among clinical isolates at atertiary care hospital of South India.   Materials and Methods:It was a cross-sectional study. 94 isolates [E. coli (n=31) and K. pneumoniae (n=63)] were recovered between January 2020 and June 2020. Samples underwent an initial cefoxitin screening test and a subsequent genotypic study with multiplex polymerase chain reactions for AmpC subtypes. Antimicrobial susceptibility characteristics of these clinical isolates have also been investigated.   Results:Thirty-seven clinical isolates were cefoxitin-resistant and genotypic analysis showed that 22 cefoxitin-resistant isolates are AmpC positive, respectively. These AmpC producers are multidrug-resistant and Klebsiella pneumoniae is the dominant strain among them. Among them single AmpC production mechanism included blaDHA producers (n=5), blaCIT producers (n=4), blaEBC producers (n=3) and blaFOX producers (n=1) and 9 isolates showed multiple AmpC genes.   Conclusion: AmpC isolates emergence is worrisome and emphasizes the need for further surveillance in this region. It is proposed that hospitals improve the surveillance of AmpC ?-lactamase in clinical isolates and suggest using carbapenems to treat infections caused by AmpC-producing bacteria.

scholarly journals Antimicrobial susceptibility, serotype distribution, virulence profile and molecular typing of piliated clinical isolates of pneumococci from east coast, Peninsular Malaysia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Nurul Diana Dzaraly ◽  
Mohd Nasir Mohd Desa ◽  
AbdulRahman Muthanna ◽  
Siti Norbaya Masri ◽  
Niazlin Mohd Taib ◽  
...  
Keyword(s):  
Phylogenetic Analysis ◽  
Antimicrobial Susceptibility ◽  
East Coast ◽  
Tertiary Hospital ◽  
Multidrug Resistant ◽  
Peninsular Malaysia ◽  
Clinical Isolates ◽  
Serotype Distribution ◽  
Antimicrobial Susceptibility Test ◽  
Conjugate Vaccines

AbstractPilus has been recently associated with pneumococcal pathogenesis in humans. The information regarding piliated isolates in Malaysia is scarce, especially in the less developed states on the east coast of Peninsular Malaysia. Therefore, we studied the characteristics of pneumococci, including the piliated isolates, in relation to antimicrobial susceptibility, serotypes, and genotypes at a major tertiary hospital on the east coast of Peninsular Malaysia. A total of 100 clinical isolates collected between September 2017 and December 2019 were subjected to serotyping, antimicrobial susceptibility test, and detection of pneumococcal virulence and pilus genes. Multilocus sequence typing (MLST) and phylogenetic analysis were performed only for piliated strains. The most frequent serotypes were 14 (17%), 6A/B (16%), 23F (12%), 19A (11%), and 19F (11%). The majority of isolates were resistant to erythromycin (42%), tetracycline (37%), and trimethoprim-sulfamethoxazole (24%). Piliated isolates occurred in a proportion of 19%; 47.3% of them were multidrug-resistant (MDR) and a majority had serotype 19F. This study showed ST236 was the most predominant sequence type (ST) among piliated isolates, which was related to PMEN clone Taiwan19F-14 (CC271). In the phylogenetic analysis, the piliated isolates were grouped into three major clades supported with 100% bootstrap values. Most piliated isolates belonged to internationally disseminated clones of S. pneumoniae, but pneumococcal conjugate vaccines (PCVs) have the potential to control them.

scholarly journals 1280. In-Vitro Activity of Cefiderocol, Imipenem/Relebactam, & Ceftazidime/Avibactam in Ceftolozane/Tazobactam Resistant Strains of Multidrug Resistant Pseudomonas aeruginosa

2020 ◽  
Vol 7 (Supplement_1) ◽  
pp. S655-S655
Author(s):  
Daniel Navas ◽  
Angela Charles ◽  
Amy Carr ◽  
Jose Alexander
Keyword(s):  
Multidrug Resistant ◽  
Resistance Mechanisms ◽  
Vitro Activity ◽  
Clinical Isolates ◽  
Resistance Testing ◽  
Clinical Samples ◽  
In Vitro Activity ◽  
Carbapenemase Gene ◽  
Resistant Strains

Abstract Background The activity of imipenem/relebactam (I/R), ceftazidime/avibactam (CZA) and cefiderocol (FDC) were evaluated against clinical isolates of multidrug resistant (MDR) strains of P. aeruginosa which was resistant to ceftolozane/tazobactam (C/T). The recent increase of MDR P. aeruginosa strains isolated from clinical samples has prompted research and development of new antimicrobials that can withstand its multiple resistance mechanisms. C/T is an effective option for treatment of MDR P. aeruginosa in our facility with only 10% of resistance in MDR strains, but the emergence of resistance may occur due to the presence of a carbapenemase gene or an ampC mutation. Methods Antimicrobial susceptibility testing for C/T Etest® (bioMérieux, Inc.) were performed on all MDR strains initially screened by the VITEK2® (bioMérieux, Inc.). 10% (n=20) of all MDR isolates were resistant to C/T by the CLSI 2019 breakpoints. These resistant isolates were tested for presence of a carbapenemase gene using the GeneXpert CARBA-R (Cepheid®) PCR and against CZA Etest® (bioMérieux, Inc.) I/R gradient strips (Liofilchem®) and FDC broth microdilution (Thermo Scientific™ Sensititre™). Results A total of 20 clinical isolates of MDR P. aeruginosa resistant to C/T were tested following standardized CLSI protocols and techniques. All 20 isolates were screened for the presence of a carbapenemase gene (blaVIM, blaNDM, blaKPC, blaOXA-48, blaIMP). A blaVIM gene was detected in 6 (30%) out of 20 isolates. FDC demonstrated the greatest activity with 85% (n=17) of susceptible isolates (CLSI MIC &lt;4µg/dL). CZA (CLSI MIC &lt;8µg/dL) and I/R (FDA MIC &lt;2µg/dL) showed 15% (n=3) and 10% (n=2) of susceptible isolates respectively. FDC was active against all 6 blaVIM isolates, where all 6 strains were resistant to CZA and I/R as expected. 3 isolates tested non-susceptible against FDC; additional characterization was not performed at this time. Conclusion Based on these results, FDC demonstrated the greatest in-vitro activity against C/T resistant strains of MDR P. aeruginosa. FDC also demonstrated activity against all 6 MDR P. aeruginosa carrying blaVIM gene. FDC is a strong option to consider on MDR P. aeruginosa strains based on a resistance testing algorithm and a cost/effective protocol. Disclosures All Authors: No reported disclosures

Prevalence of Enterobacteriaceae spp. and its multidrug-resistant rates in clinical isolates: A two-center cross-sectional study

2021 ◽  
Author(s):  
Bahman Mirzaei ◽  
Ryhane Babaei ◽  
Zahra Norouzi Bazgir ◽  
Hamid Reza Goli ◽  
Shima Keshavarzi ◽  
...  
Keyword(s):  
Multidrug Resistant ◽  
Cross Sectional Study ◽  
Clinical Isolates ◽  
Sectional Study ◽  
Cross Sectional

scholarly journals Detection of mutations associated with isoniazid resistance in multidrug-resistant Mycobacterium tuberculosis clinical isolates

2014 ◽  
Vol 69 (9) ◽  
pp. 2369-2375 ◽  
Author(s):  
Tomasz Jagielski ◽  
Zofia Bakuła ◽  
Katarzyna Roeske ◽  
Michał Kamiński ◽  
Agnieszka Napiórkowska ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Isoniazid Resistance ◽  
Detection Of Mutations

scholarly journals Molecular characterization of multidrug resistant Klebsiella pneumoniae clinical isolates recovered from King Abdulaziz Specialist Hospital at Taif City, Saudi Arabia

2021 ◽  
Vol 14 (1) ◽  
pp. 143-151
Author(s):  
Rihab Lagha ◽  
Fethi Ben Abdallah ◽  
Asmaa A.H. ALKhammash ◽  
Nabil Amor ◽  
Mohamed M. Hassan ◽  
...  
Keyword(s):  
Saudi Arabia ◽  
Klebsiella Pneumoniae ◽  
Molecular Characterization ◽  
Multidrug Resistant ◽  
Clinical Isolates
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