Detection and Assessment of Clarithromycin Inducible Resistant Strains Among Korean
            Mycobacterium abscessus
            Clinical Strains: PCR Methods

Mycobacterium abscessus is a fast growing Mycobacterium species mainly causing skin and respiratory infections in human. M. abscessus is resistant to numerous drugs, which is a major challenge for the treatment. In this study, we have sequenced the genomes of two clinical M. abscessus strains having rough and smooth morphology, using the single molecule real-time and Illumina HiSeq sequencing technology. In addition, we reported the first comparative methylome profiles of a rough and a smooth M. abscessus clinical strains. The number of N4-methylcytosine (4mC) and N6-methyladenine (6mA) modified bases obtained from smooth phenotype were two-fold and 1.6 fold respectively higher than that of rough phenotype. We have also identified 4 distinct novel motifs in two clinical strains and genes encoding antibiotic-modifying/targeting enzymes and genes associated with intracellular survivability having different methylation patterns. To our knowledge, this is the first report about genome-wide methylation profiles of M. abscessus strains and identification of a natural linear plasmid (15 kb) in this critical pathogen harboring methylated bases. The pan-genome analysis of 25 M. abscessus strains including two clinical strains revealed an open pan genome comprises of 7596 gene clusters. Likewise, structural variation analysis revealed that the genome of rough phenotype strain contains more insertions and deletions than the smooth phenotype and that of the reference strain. A total of 391 single nucleotide variations responsible for the non-synonymous mutations were detected in clinical strains compared to the reference genome. The comparative genomic analysis elucidates the genome plasticity in this emerging pathogen. Furthermore, the detection of genome-wide methylation profiles of M. abscessus clinical strains may provide insight into the significant role of DNA methylation in pathogenicity and drug resistance in this opportunistic pathogen.

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Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

Journal of Clinical Microbiology ◽

10.1128/jcm.01144-14 ◽

2014 ◽

Vol 52 (10) ◽

pp. 3573-3582 ◽

Cited By ~ 31

Author(s):

R. M. Davidson ◽

N. A. Hasan ◽

P. R. Reynolds ◽

S. Totten ◽

B. Garcia ◽

...

Keyword(s):

United States ◽

Genome Sequencing ◽

The United States ◽

Mycobacterium Abscessus ◽

Clinical Strains

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A Novel Broad Allele-Specific TaqMan Real-Time PCR Method To Detect Triazole-Resistant Strains of Aspergillus fumigatus, Even with a Very Low Percentage of Triazole-Resistant Cells Mixed with Triazole-Susceptible Cells

Journal of Clinical Microbiology ◽

10.1128/jcm.00604-19 ◽

2019 ◽

Vol 57 (9) ◽

Author(s):

Qian Wang ◽

Dimitrios P. Kontoyiannis ◽

Ruoyu Li ◽

Wei Chen ◽

Dingfang Bu ◽

...

Keyword(s):

Aspergillus Fumigatus ◽

Real Time ◽

Real Time Pcr ◽

High Efficiency ◽

Gene Mutations ◽

Health Concern ◽

Content Type ◽

Clinical Strains ◽

Allele Specific ◽

Resistant Strains

ABSTRACT Invasive aspergillosis caused by triazole-resistant strains of Aspergillus fumigatus is a growing public health concern, as is the occurrence of mixed infections with triazole-resistant and -susceptible A. fumigatus strains. Therefore, it is crucial to develop robust methods to identify triazole-resistant strains of A. fumigatus, even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. In this work, we developed a robust, highly selective, and broad-range allele-specific TaqMan real-time PCR platform consisting of 7 simultaneous assays that detect TR34 (a 34-bp tandem repeat in the promoter region), TR46, G54W (a change of G to W at position 54), G54R, L98H, Y121F, and M220I mutations in the cyp51A gene of A. fumigatus. The method is based on the widely used TaqMan real-time PCR technology and combines allele-specific PCR with a blocking reagent (minor groove binder [MGB] oligonucleotide blocker) to suppress amplification of the wild-type cyp51A alleles. We used this method to detect triazole-resistant clinical strains of A. fumigatus with a variety of cyp51A gene mutations, as well as the triazole-resistant strains in mixtures of triazole-resistant and -susceptible strains of A. fumigatus. The method had high efficiency and sensitivity (300 fg/well, corresponding to about 100 CFU per reaction mixture volume). It could promptly detect triazole resistance in a panel of 30 clinical strains of A. fumigatus within about 6 h. It could also detect cyp51A-associated resistance alleles, even in mixtures containing only 1% triazole-resistant A. fumigatus strains. These results suggest that this method is robustly able to detect cyp51A-associated resistance alleles even in mixtures of triazole-resistant and -susceptible strains of A. fumigatus and that it should have important clinical applications.

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Type II Topoisomerase Mutations in Ciprofloxacin-Resistant Strains of Pseudomonas aeruginosa

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.43.1.62 ◽

1999 ◽

Vol 43 (1) ◽

pp. 62-66 ◽

Cited By ~ 83

Author(s):

Hyam Mouneimné ◽

Jérome Robert ◽

Vincent Jarlier ◽

Emmanuelle Cambau

Keyword(s):

Pseudomonas Aeruginosa ◽

Novel Mutation ◽

Geometric Mean ◽

Quinolone Resistance ◽

Single Mutation ◽

Type Ii ◽

Wild Type ◽

Clinical Strains ◽

Major Mechanism ◽

Resistant Strains

ABSTRACT We determined the sequences of the quinolone resistance-determining regions of gyrA, gyrB, and parCgenes for 30 clinical strains of Pseudomonas aeruginosaresistant to ciprofloxacin that were previously complemented by wild-type gyrA and gyrB plasmid-borne alleles and studied for their coresistance to imipenem (E. Cambau, E. Perani, C. Dib, C. Petinon, J. Trias, and V. Jarlier, Antimicrob. Agents Chemother. 39:2248–2252, 1995). In the present study, we found mutations in type II topoisomerase genes for all strains. Twenty-eight strains had a missense mutation in gyrA (codon 83 or 87). Ten of them had an additional mutation in parC(codon 80 or 84), including a novel mutation of Ser-80 to Trp, but all were fully complemented by a plasmid-borne wild-type gyrAallele. The remaining two strains harbored the first gyrBmutation described in P. aeruginosa, leading to the substitution of phenylalanine for serine 464. The strains which had two mutations in type II topoisomerase genes (i.e.,gyrA and parC) were significantly more resistant to fluoroquinolones than those with a single mutation ingyrA or gyrB (geometric mean MICs of ciprofloxacin, 39.4 versus 10.9 μg/ml, P < 0.01; geometric mean MICs of sparfloxacin, 64.0 versus 22.6,P < 0.01). No mutant with a parC mutation alone was observed, which favors DNA gyrase being the primary target for fluoroquinolones. These results demonstrate that gyrAmutations are the major mechanism of resistance to fluoroquinolones for clinical strains of P. aeruginosa and that additional mutations in parC lead to a higher level of quinolone resistance.

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Genomic Analysis of Molecular Bacterial Mechanisms of Resistance to Phage Infection

10.21203/rs.3.rs-699717/v1 ◽

2021 ◽

Author(s):

Antón Ambroa ◽

Lucia Blasco ◽

María López ◽

Olga Pacios ◽

Inés Bleriot ◽

...

Keyword(s):

Bacterial Resistance ◽

Phage Therapy ◽

Genomic Analysis ◽

Genomic Islands ◽

Clinical Strain ◽

Phage Resistance ◽

Modification System ◽

Clinical Strains ◽

Restriction Modification System ◽

Resistant Strains

Abstract BackgroundIn order to optimize phage therapy, we need to understand how bacteria evolve against phage attack. One of the main problems of the phage therapy is the appearance of bacterial resistance variants. The use of genomics to track antimicrobial resistance is increasingly developed and used in clinical laboratories. For that reason, it is important to consider, in an emerging future with phage therapy, to detect and avoid phage resistant strains, that can be overcomed by the analysis of metadata provided by WGS. Here, we identified genes associated with phage resistance in 18 Acinetobacter baumannii clinical strain belonging to the ST-2 clonal complex during a decade (Ab2000 vs 2010): 9 from 2000 and 9 from 2010.ResultsThe presence of genes putatively associated to phage resistance were detected. Genes detected were associated with an abortive infection system, restriction-modification system, genes predicted to be associated with defence systems but with unknown function and CRISPR-Cas system. Between 118 and 171 genes were found in the 18 clinical strains. On average, 26% of these genes were detected inside genomic islands (GIs) in the 2000 strains and 32% in 2010 strains. Furthermore, 38 potential CRISPR arrays in 17 of 18 of the strains were found, as well as 705 proteins associated with CRISPR-Cas systems.ConclusionsA moderately higher presence of these genes in the strains of the 2010 in comparison to those of the 2000 were found, especially those related to the R-M system and CRISPR-Cas system. The presence of these genes in GIs in a higher rate in the strains of the 2010 compared to those of the 2000 was also detected. WGS and bioinformatics could be powerful tools to avoid drawbacks when a personalized therapy is applied. In this study, it allows us to take care of the phage resistance in A. baumannii clinical strains to prevent a failure in a possible phage therapy.

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Investigation of the combined use effect of the antiseptic decamethoxin and fluoroquinolones on clinical strains S. aureus

Reports of Vinnytsia National Medical University ◽

10.31393/reports-vnmedical-2020-24(1)-15 ◽

2020 ◽

Vol 24 (1) ◽

pp. 80-83

Author(s):

O.A. Nazarchuk ◽

S.V. Pavliuk ◽

H.H. Nazarchuk ◽

V.M. Mruh ◽

A.O. Dudar ◽

...

Keyword(s):

Antimicrobial Properties ◽

High Sensitivity ◽

Infectious Complications ◽

Chemotherapeutic Agents ◽

Staphylococcal Infection ◽

Dilution Method ◽

Antibiotic Resistant ◽

Clinical Strains ◽

Resistant Strains ◽

Low Sensitivity

Annotation. Postoperative infectious complications in eye microsurgery are most often caused by S. aureus strains, among which resistance to fluoroquinolones, first-line drugs for the prevention of postoperative complications, is common. An alternative to fluoroquinolones is antiseptics. The aim of the study was to investigate the effect of decamethoxin antiseptic on the antimicrobial properties of fluoroquinolones against S. aureus. Studies on the combined effect of the antimicrobial properties of fluoroquinolones and decamethoxin (DCM) were performed on the S. aureus museum strain ATCC 25923, on moderately stable and persistent S. aureus clinical strains (n=42) obtained from patients undergoing eye microsurgical procedures, by serial dilution method. The minimum bacteriostatic concentration (MBsC), the minimum bactericidal concentration (MBcC) of antimicrobials separately in pure form and with the addition of sub-bacteriostatic concentrations (subBsC, 1/4 MBsC) of DCM were determined. Statistical data processing was performed using special and office programs “STATISTICA 6.0”, “Microsoft Excel 2010”. The study found low sensitivity to fluoroquinolones in clinical strains of S. aureus, high sensitivity to DCM (MBsC 0.66±0.1; MBcC 3.19±0.4 μg/ml). The sensitivity of resistant and moderately resistant strains of S. aureus to fluoroquinolones in the presence of subBsC DCM was established: MBsC of ciprofloxacin in the presence of DCM decreased almost 4 times, norfloxacin — 5.5 times, ofloxacin — 6.8 times, of levofloxacin — 6.8 times, moxifloxacin 7.1 times. It was found that MBcC of norfloxacin for clinical resistant S. aureus strains decreased in 5.7 times, ofloxacin — in 9.2 times, levofloxacin — in 6.9 times, ciprofloxacin — in 8.6 times, moxifloxacin — in 7.9 times. The simultaneous use of antiseptic DCM and various fluoroquinolone chemotherapeutic agents provides effective protection against staphylococcal infection, contributes to the fight against antibiotic resistant strains of S. aureus.

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Antibiotic resistance in Acinetobacter baumannii as agent of surgery infection and approaches to its overcoming by means of deca-methoxinum antiseptic

Perioperaciina Medicina ◽

10.31636/prmd.v1i2.2 ◽

2019 ◽

Vol 1 (2) ◽

pp. 18-22

Author(s):

O A Nazarchuk ◽

V I Nahaichuk

Keyword(s):

Antibiotic Resistance ◽

Antibiotic Sensitivity ◽

Antibiotic Resistant ◽

Clinical Strains ◽

Minimal Inhibitory Concentrations ◽

Hospital Acquired Infections ◽

Causative Agents ◽

Resistant Strains ◽

Burn Disease ◽

Hospital Acquired

Introduction. Non-fermenting Gram-negative bacilli are known as one of the most frequent causative agents of hospital-acquired infections. Acinetobacter baumannii, as causative agent of infection complications of different localization, has obtained recently high resistance to anti-biotics and has belonged to ESKAPE group of pathogens. Antimicrobials, recommended for the prophylaxis and therapy of hospital-acquired infections, have been failing in their effectiveness and lead to selection of antibiotic resistant strains of A. baumannii. The aim of this research was to substantiate the way of overcoming of resistance in clinical strains of A. baumannii, by means of synergic antimicrobial activity of antibiotics and antiseptic decamethoxinum®. Material and methods. The research was carried out on 190 clinical strains of A. baumannii, isolated from patients with burn disease during the period 2011–2015. The sensitivity of clinical strains of A. baumannii was determined to such antibiotics as ampicillin/sulbactam, cefoperazone, cefoperazone/sulbactam, meropenem, imipenem, amikacin, ciprofloxacin, gatifloxacin and antiseptic decamethoxinum® (DCM; Registration certificate No UA/14444/01/01 since 24.06.2015. Order of the Ministry of Health of Ukraine No 373). The sensitivity of A. baumannii to antibiotics and DCM was determined by means of disk diffusion test and serial dilution (Order of the Ministry of Health of Ukraine No167 since 05.04.2007; EUCAST expert rules).The study of the influence of antiseptic DCM on the sensitivity of acinetobacteria to antibiotics was studied on 35 clinical strains of A. baumannii, drafted from the general number of isolates enrolled in the research. For this, the sensitivity of A. baumannii to antibiotics in the presence of sub-minimal inhibitory concentrations (subMIC) of DCM was identified. The received experimental data were analyzed by “Statistica 6.0”. Results and discussion. The changes of antibiotic sensitivity profile of A. baumannii for five years were shown. It was found that the sensitivity of A. baumannii to majority of antibiotics, selected for study, decreased significantly. But the only ampicillin/sulbactam was found to have vice versa tendency. We found the rising quantity of antibiotic resistant strains of A. baumannii. At the same time, high resistance of acinetobacteria to fluoroquinolones (ciprofloxacin– 96,1%; gatifloxacin– 95,8%) was found in 2015. The in vitro research of combined activity of DCM antiseptic remedy and early mentioned antibiotics against clinical strains of A. baumannii demonstrated the reveal antibiotic effectiveness. As follows, minimal inhibitory concentrations of antibiotics decreased in 1.5–4 times in the mediums which contained subMIC of DCM. Especially this tendency was found in resistant clinical strains. Conclusion. Under selective influence of antibiotics protected by β-lactamase inhibitors, carbapenems, fluoroquinolones aminoglycosides increase the antibiotic resistance in A. baumannii, causative agents of infectious complications in patients with burn disease. The antiseptic remedy decamethoxinum® helps to improve antibiotic sensitivity in resistant A. baumannii.

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Clomiphene Citrate Shows Effective and Sustained Antimicrobial Activity against Mycobacterium abscessus

International Journal of Molecular Sciences ◽

10.3390/ijms222011029 ◽

2021 ◽

Vol 22 (20) ◽

pp. 11029

Author(s):

Da-Gyum Lee ◽

Yoo-Hyun Hwang ◽

Eun-Jin Park ◽

Jung-Hyun Kim ◽

Sung-Weon Ryoo

Keyword(s):

Clomiphene Citrate ◽

Treatment Success ◽

In Vitro Study ◽

Drug Efficacy ◽

Mycobacterium Abscessus ◽

Pulmonary Infections ◽

Wild Type ◽

Antimicrobial Drugs ◽

Resistant Strains

Mycobacterium abscessus (M. abscessus) causes chronic pulmonary infections and is the most difficult non-tuberculous mycobacteria (NTM) to treat due to its resistance to current antimicrobial drugs, with a treatment success rate of 45.6%. Thus, novel treatment drugs are needed, of which we identified the drug clomiphene citrate (CC), known to treat infertility in women, to exhibit inhibitory activity against M. abscessus. To assess the potential of CC as a treatment for M. abscessus pulmonary diseases, we measured its efficacy in vitro and established the intracellular activity of CC against M. abscessus in human macrophages. CC significantly inhibited the growth of not only wild-type M. abscessus strains but also clinical isolate strains and clarithromycin (CLR)-resistant strains of M. abscessus. CC’s drug efficacy did not have cytotoxicity in the infected macrophages. Furthermore, CC worked in anaerobic non-replicating conditions as well as in the presence of biofilm. The results of this in vitro study on M. abscessus activity suggest the possibility of using CC to develop new drug hypotheses for the treatment of M. abscessus infections.

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Detection of Colistin Resistance in Pseudomonas aeruginosa Using the MALDIxin Test on the Routine MALDI Biotyper Sirius Mass Spectrometer

Frontiers in Microbiology ◽

10.3389/fmicb.2021.725383 ◽

2021 ◽

Vol 12 ◽

Author(s):

Katy Jeannot ◽

Katheryn Hagart ◽

Laurent Dortet ◽

Markus Kostrzewa ◽

Alain Filloux ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Lipid A ◽

Response Regulator ◽

Multidrug Resistant ◽

Positive Control ◽

Colistin Resistance ◽

Clinical Strains ◽

Efficient Detection ◽

Maldi Biotyper ◽

Resistant Strains

Colistin is frequently a last resort treatment for Pseudomonas aeruginosa infections caused by multidrug-resistant (MDR) and extensively drug resistant (XDR) strains, and detection of colistin resistance is essential for the management of infected patients. Therefore, we evaluated the recently developed MALDIxin test for the detection of colistin resistance in P. aeruginosa clinical strains using the routine matrix-assisted laser desorption ionization (MALDI) Biotyper Sirius system. The test is based on the detection by mass spectrometry of modified lipid A by the addition of 4-amino-l-arabinose (l-ara4N) molecules on one or two phosphate groups, in strains resistant to colistin. Overproduction of l-Ara4N molecules is mainly due to the constitutive activation of the histidine kinase (PmrB) or the response regulator (PmrA) following an amino-acid substitution in clinical strains. The performance of the test was determined on a panel of 14 colistin-susceptible and 14 colistin-resistant P. aeruginosa clinical strains, the reference strain PAO1 and positive control mutants PmrB (V28G), PmrB (D172), PhoQ (D240–247), and ParR (M59I). In comparison with the broth microdilution (BMD) method, all the susceptible strains (n=14) and 8/14 colistin-resistant strains were detected in less than 1h, directly on whole bacteria. The remaining resistant strains (n=6) were all detected after a short pre-exposure (4h) to colistin before sample preparation. Validation of the method on a larger panel of strains will be the next step before its use in diagnostics laboratories. Our data showed that the MALDIxin test offers rapid and efficient detection of colistin resistant P. aeruginosa and is thus a valuable diagnostics tool to control the spread of these emerging resistant strains.

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