Rapid and accurate detection of aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases by targeted LC-MS/MS

New and rapid diagnostic methods are needed for the detection of antimicrobial resistance to aid in the curbing of drug-resistant infections. Targeted liquid chromatography-tandem mass spectrometry (LC-MS/MS) is a method that could serve this purpose, as it can detect specific peptides of antimicrobial resistance mechanisms with high accuracy. In the current study, we developed an accurate and rapid targeted LC-MS/MS assay based on parallel reaction monitoring for detection of the most prevalent aminoglycoside modifying enzymes and 16S ribosomal RNA methyltransferases in E. coli and K. pneumoniae that confer resistance to aminoglycosides. Specific tryptic peptides needed for detection were selected and validated for AAC(3)-Ia, AAC(3)-II, AAC(3)-IV, AAC(3)-VI, AAC(6’)-Ib, AAC(6’)-Ib-cr, ANT(2”)-I, APH(3’)-VI, ArmA, RmtB, RmtC and RmtF. In total, 205 isolates containing different aminoglycoside resistance mechanisms that consisted mostly of E. coli and K. pneumoniae were selected for assay development and evaluation. Mass spectrometry results were automatically analyzed and were compared to whole genome sequencing results. Of the 2460 isolate and resistance mechanism combinations tested, 2416 combinations matched. Discrepancies were further analyzed by repeating LC-MS/MS analysis and performing additional PCRs. Mass spectrometry results were also used to predict resistance and susceptibility to gentamicin, tobramycin and amikacin in only the E. coli and K. pneumoniae isolates (n=191). The category interpretations were correctly predicted for gentamicin in 97.4% of the isolates, for tobramycin in 97.4% of the isolates, and for amikacin in 82.7% of the isolates. Targeted LC-MS/MS can be applied for accurate and rapid detection of aminoglycoside resistance mechanisms.

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Development and Persistence of Antimicrobial Resistance in Pseudomonas aeruginosa: a Longitudinal Observation in Mechanically Ventilated Patients

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01278-06 ◽

2007 ◽

Vol 51 (4) ◽

pp. 1341-1350 ◽

Cited By ~ 47

Author(s):

Anita Reinhardt ◽

Thilo Köhler ◽

Paul Wood ◽

Peter Rohner ◽

Jean-Luc Dumas ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Antimicrobial Resistance ◽

Selective Pressure ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Antimicrobial Treatment ◽

Aminoglycoside Resistance ◽

Prolonged Intubation ◽

Treatment Regimens ◽

Underlying Mechanisms

ABSTRACT Intubated patients frequently become colonized by Pseudomonas aeruginosa, which is subsequently responsible for ventilator-associated pneumonia. This pathogen readily acquires resistance against available antimicrobials. Depending on the resistance mechanism selected for, resistance might either be lost or persist after removal of the selective pressure. We investigated the rapidity of selection, as well as the persistence, of antimicrobial resistance and determined the underlying mechanisms. We selected 109 prospectively collected P. aeruginosa tracheal isolates from two patients based on their prolonged intubation and colonization periods, during which they had received carbapenem, fluoroquinolone (FQ), or combined β-lactam-aminoglycoside therapies. We determined antimicrobial resistance phenotypes by susceptibility testing and used quantitative real-time PCR to measure the expression of resistance determinants. Within 10 days after the initiation of therapy, all treatment regimens selected resistant isolates. Resistance to β-lactam and FQ was correlated with ampC and mexC gene expression levels, respectively, whereas imipenem resistance was attributable to decreased oprD expression. Combined β-lactam-aminoglycoside resistance was associated with the appearance of small-colony variants. Imipenem and FQ resistance persisted for prolonged times once the selecting antimicrobial treatment had been discontinued. In contrast, resistance to β-lactams disappeared rapidly after removal of the selective pressure, to reappear promptly upon renewed exposure. Our results suggest that resistant P. aeruginosa is selected in less than 10 days independently of the antimicrobial class. Different resistance mechanisms lead to the loss or persistence of resistance after the removal of the selecting agent. Even if resistant isolates are not evident upon culture, they may persist in the lung and can be rapidly reselected.

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A Resistance Mechanism in Non- mcr Colistin-Resistant Escherichia coli in Taiwan: R81H Substitution in PmrA Is an Independent Factor Contributing to Colistin Resistance

Microbiology Spectrum ◽

10.1128/spectrum.00022-21 ◽

2021 ◽

Author(s):

Ching-Hsun Wang ◽

L. Kristopher Siu ◽

Feng-Yee Chang ◽

Sheng-Kang Chiu ◽

Jung-Chung Lin

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Molecular Epidemiology ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Surveillance Program ◽

Independent Factor ◽

Colistin Resistance ◽

Content Type ◽

E Coli

The molecular epidemiology and resistance mechanisms of mcr -negative colistin-resistant E. coli are not well described. In this study, a total of 11 mcr -negative colistin-resistant E. coli isolates were selected from a nationwide antimicrobial resistance surveillance program in Taiwan for further investigation.

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Structural Differences between the 16S Ribosomal RNA of E. coli and its Precursor

Nature New Biology ◽

10.1038/newbio232052a0 ◽

1971 ◽

Vol 232 (28) ◽

pp. 52-54 ◽

Cited By ~ 29

Author(s):

C. V. LOWRY ◽

J. E. DAHLBERG

Keyword(s):

Ribosomal Rna ◽

16S Ribosomal Rna ◽

E Coli ◽

Structural Differences

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Antimicrobial Resistance and Association with Class 1 Integrons in Escherichia coli Isolated from Turkey Meat Products

Journal of Food Protection ◽

10.4315/0362-028x-71.8.1679 ◽

2008 ◽

Vol 71 (8) ◽

pp. 1679-1684 ◽

Cited By ~ 5

Author(s):

M. L. KHAITSA ◽

J. OLOYA ◽

D. DOETKOTT ◽

R. KEGODE

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Antimicrobial Agents ◽

Population Attributable Fraction ◽

Meat Products ◽

Resistance Mechanisms ◽

E Coli ◽

Class 1 Integrons ◽

Turkey Meat ◽

Class 1

The objective of this study was to quantify the role of class 1 integrons in antimicrobial resistance in Escherichia coli isolated from turkey meat products purchased from retail outlets in the Midwestern United States. Of 242 E. coli isolates, 41.3% (102 of 242) tested positive for class 1 integrons. A significant association was shown between presence of class 1 integrons in E. coli isolates and the resistance to tetracycline, ampicillin, streptomycin, gentamicin, sulfisoxazole, and trimethoprim-sulfamethoxazole. Attributable risk analysis revealed that for every 100 E. coli isolates carrying class 1 integrons, resistance was demonstrated for ampicillin (22%), gentamycin (48%), streptomycin (29%), sulfisoxazole (40%), trimethoprimsulfamethoxazole (7%), and tetracycline (26%). Non–integron-related antimicrobial resistance was demonstrated for ampicillin (65%), gentamycin (16.9%), streptomycin (42.1%), sulfisoxazole (35.8%), and tetracycline (49.7%). Population-attributable fraction analysis showed that class 1 integrons accounted for the following resistances: gentamycin, 71% (50 of 71), amoxicillin–clavulanic acid, 19.6% (6 of 33), nalidixic acid, 34% (7 of 21), streptomycin, 28% (30 of 107), sulfisoxazole, 38% (40 of 106), and tetracycline, 14%, (26 of 185). In conclusion, although class 1 integrons have been implicated in resistance to antimicrobial agents, other non–integron resistance mechanisms seem to play an important part.

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Struggle To Survive: the Choir of Target Alteration, Hydrolyzing Enzyme, and Plasmid Expression as a Novel Aztreonam-Avibactam Resistance Mechanism

mSystems ◽

10.1128/msystems.00821-20 ◽

2020 ◽

Vol 5 (6) ◽

Author(s):

Ke Ma ◽

Yu Feng ◽

Alan McNally ◽

Zhiyong Zong

Keyword(s):

Antimicrobial Resistance ◽

Antisense Rna ◽

Antimicrobial Agents ◽

Binding Protein ◽

Resistance Mechanism ◽

Resistance Mechanisms ◽

Penicillin Binding Protein ◽

Plasmid Copy ◽

Copy Numbers

ABSTRACT Aztreonam-avibactam is a promising antimicrobial combination against multidrug-resistant organisms, such as carbapenemase-producing Enterobacterales. Resistance to aztreonam-avibactam has been found, but the resistance mechanism remains poorly studied. We recovered three Escherichia coli isolates of an almost identical genome but exhibiting varied aztreonam-avibactam resistance. The isolates carried a cephalosporinase gene, blaCMY-42, on IncIγ plasmids with a single-nucleotide variation in an antisense RNA-encoding gene, inc, of the replicon. The isolates also had four extra amino acids (YRIK) in penicillin-binding protein 3 (PBP3) due to a duplication of a 12-nucleotide (TATCGAATTAAC) stretch in pbp3. By cloning and plasmid-curing experiments, we found that elevated CMY-42 cephalosporinase production or amino acid insertions in PBP3 alone mediated slightly reduced susceptibility to aztreonam-avibactam, but their combination conferred aztreonam-avibactam resistance. We show that the elevated CMY-42 production results from increased plasmid copy numbers due to mutations in inc. We also verified the findings using in vitro mutation assays, in which aztreonam-avibactam-resistant mutants also had mutations in inc and elevated CMY-42 production compared with the parental strain. This choir of target modification, hydrolyzing enzyme, and plasmid expression represents a novel, coordinated, complex antimicrobial resistance mechanism and also reflects the struggle of bacteria to survive under selection pressure imposed by antimicrobial agents. IMPORTANCE Carbapenemase-producing Enterobacterales (CPE) is a serious global challenge with limited therapeutic options. Aztreonam-avibactam is a promising antimicrobial combination with activity against CPE producing serine-based carbapenemases and metallo-β-lactamases and has the potential to be a major option for combatting CPE. Aztreonam-avibactam resistance has been found, but resistance mechanisms remain largely unknown. Understanding resistance mechanisms is essential for optimizing treatment and developing alternative therapies. Here, we found that either penicillin-binding protein 3 modification or the elevated expression of cephalosporinase CMY-42 due to increased plasmid copy numbers does not confer resistance to aztreonam-avibactam, but their combination does. We demonstrate that increased plasmid copy numbers result from mutations in antisense RNA-encoding inc of the IncIγ replicon. The findings reveal that antimicrobial resistance may be due to concerted combinatorial effects of target alteration, hydrolyzing enzyme, and plasmid expression and also highlight that resistance to any antimicrobial combination will inevitably emerge.

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Structural organization of the 16S ribosomal RNA from E. coli. Topography and secondary structure

Nucleic Acids Research ◽

10.1093/nar/9.9.2153 ◽

1981 ◽

Vol 9 (9) ◽

pp. 2153-2172 ◽

Cited By ~ 57

Author(s):

Patrick Stiegler ◽

Philippe Carbon ◽

Michael Zuker ◽

Jean-Pierre Ebel ◽

Chantal Ehresmann

Keyword(s):

Secondary Structure ◽

Ribosomal Rna ◽

Structural Organization ◽

16S Ribosomal Rna ◽

E Coli

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Antimicrobial resistance and metallo-beta-lactamase producing among commensal Escherichia coli isolates from healthy children of Khuzestan and Fars provinces; Iran

10.21203/rs.3.rs-45361/v3 ◽

2020 ◽

Author(s):

Fahimeh Mahmoodi ◽

Seyedeh Elham Rezatofighi ◽

Mohammad Reza Akhoond

Keyword(s):

Escherichia Coli ◽

Antimicrobial Resistance ◽

Pathogenic Bacteria ◽

Gastrointestinal Diseases ◽

Resistance Mechanisms ◽

Multi Drug Resistance ◽

Healthy Children ◽

Great Ability ◽

E Coli ◽

Plasmid Profiling

Abstract Background: The emergence of metallo-β-lactamase (MBL)-producing isolates is alarming since they carry mobile genetic elements with great ability to spread; therefore, early detection of these isolates, particularly their reservoir, is crucial to prevent their inter- and intra-care setting dissemination and establish suitable antimicrobial therapies. The current study was designed to evaluate the frequency of antimicrobial resistance (AMR), MBL producers and identification of MBL resistance genes in Escherichia coli strains isolated from fecal samples of the healthy children under three years old. A total of 412 fecal E. coli isolates were collected from October 2017 to December 2018. The study population included healthy infants and children aged <3 years who did not exhibit symptoms of any diseases, especially gastrointestinal diseases. E. coli isolates were assessed to determine the pattern of AMR. E. coli isolates were assessed to determine the pattern of AMR, the production of extended spectrum β-lactamase (ESBL) and MBL by phenotypic methods. Carbapenem-resistant isolates were investigated for the presence of MBL and carbapenemase genes, plasmid profiling, and the ability of conjugation.Results: In sum, AMR, multi-drug resistance (MDR) and ESBL production were observed in more than 54.9%, 36.2% and 11.7% of commensal E. coli isolates, respectively. Out of six isolates resistant to imipenem and meropenem, four isolates were phenotypically detected as MBL producers. Two and one E. coli strains carried the blaNDM-1 and blaVIM-2 genes, respectively and were able to transmit imipenem resistance through conjugation. Conclusion: Our findings showed that children not exposed to antibiotics can be colonized by E. coli isolates resistant to the commonly used antimicrobial compounds and can be a good indicator for the occurrence and prevalence of AMR in the community. These bacteria can act as a potential reservoir of AMR genes including MBL genes of pathogenic bacteria and lead to the dissemination of resistance mechanisms to other bacteria.

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