Single Ribosomal Protein Mutations in Antibiotic-Resistant Bacteria Analyzed by Mass Spectrometry

ABSTRACT Mutations in several ribosomal proteins are known to be related to antibiotic resistance. For several strains of Escherichia coli, the mutated protein is known but the amino acid actually altered has not been documented. Characterization of these determinants for antibiotic resistance in proteins will further the understanding of the precise mechanism of the antibiotic action as well as provide markers for resistance. Mass spectrometry can be used as a valuable tool to rapidly locate and characterize mutant proteins by using a small amount of material. We have used electrospray and matrix-assisted laser desorption ionization–time of flight (MALDI–TOF) mass spectrometry to map out all 56 ribosomal proteins in E. coli based on intact molecular masses. We used this fingerprinting approach to locate variants of ribosomal proteins displaying a change in mass. In particular we have studied proteins responsible for streptomycin, erythromycin, and spectinomycin resistance in three strains of E. coli, and then we characterized each mutation responsible for resistance by analyzing tryptic peptides of these proteins by using MALDI-TOF and nanoelectrospray tandem mass spectrometry. The results provided markers for antibiotic resistance and demonstrated that mass spectrometry can be used to rapidly investigate changes in individual proteins from a complex with picomole amounts of protein.

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A trimethoprim derivative impedes antibiotic resistance evolution

Nature Communications ◽

10.1038/s41467-021-23191-z ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Madhu Sudan Manna ◽

Yusuf Talha Tamer ◽

Ilona Gaszek ◽

Nicole Poulides ◽

Ayesha Ahmed ◽

...

Keyword(s):

Antibiotic Resistance ◽

Resistant Bacteria ◽

Gene Encoding ◽

Antibiotic Resistant ◽

E Coli ◽

Resistance Evolution ◽

Evolutionary Trajectories ◽

Evolution Of Resistance

AbstractThe antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4’-desmethyltrimethoprim (4’-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4’-DTMP than in the presence of TMP. We find that 4’-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.

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Relationship of antibiotic resistance to results of treatment in sepsis patients in Hue Central Hospital 2017 – 2018

Journal of Medicine and Pharmacy ◽

10.34071/jmp.2019.5.7 ◽

2019 ◽

pp. 48-54

Author(s):

Duy Binh Nguyen ◽

Trung Tien Phan ◽

Trong Hanh Hoang ◽

Van Tuan Mai ◽

Xuan Chuong Tran

Keyword(s):

Antibiotic Resistance ◽

Bacterial Infection ◽

Resistant Bacteria ◽

Central Hospital ◽

International Consensus ◽

Antibiotic Resistant ◽

E Coli ◽

Results Of Treatment ◽

Amoxicillin Clavulanic Acid ◽

Relationship Of

Sepsis is a serious bacterial infection. The main treatment is using antibiotics. However, the rate of antibiotic resistance is very high and this resistance is related to the outcome of treatment. Objectives: To evaluate the situation of antibiotic resistance of some isolated bacteria in sepsis patients treated at Hue Central Hospital; to evaluate the relationship of antibiotic resistance to the treatment results in patients with sepsis. Subjects and methods: prospective study of 60 sepsis patients diagnosed according to the criteria of the 3rd International Consensus-Sepsis 3 and its susceptibility patterns from April 2017 to August 2018. Results and Conclusions: The current agents of sepsis are mainly S. suis, Burkhoderiae spp. and E. coli. E. coli is resistant to cephalosporins 3rd, 4th generation and quinolone group is over 75%; resistance to imipenem 11.1%; the ESBL rate is 60%. S. suis resistant to ampicilline 11.1%; no resistance has been recorded to ceftriaxone and vancomycine. Resistance of Burkholderiae spp. to cefepime and amoxicillin/clavulanic acid was 42.9% and 55.6%, resistant to imipenem and meropenem is 20%, resistance to ceftazidime was not recorded. The deaths were mostly dued to E. coli and K. pneumoniae. The mortality for patients infected with antibiotic-resistant bacteria are higher than for sensitive groups. Key words: Sepsis, bacterial infection, antibiotics

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Characterization of ESBL-Producing Enterobacteria from Fruit Bats in an Unprotected Area of Makokou, Gabon

Microorganisms ◽

10.3390/microorganisms8010138 ◽

2020 ◽

Vol 8 (1) ◽

pp. 138 ◽

Cited By ~ 1

Author(s):

Pierre Philippe Mbehang Nguema ◽

Richard Onanga ◽

Guy Roger Ndong Atome ◽

Jean Constant Obague Mbeang ◽

Arsène Mabika Mabika ◽

...

Keyword(s):

Resistant Bacteria ◽

Fruit Bats ◽

Antibiotic Resistant ◽

Disk Diffusion Test ◽

Beta Lactamases ◽

E Coli ◽

Terrestrial Mammals ◽

Extended Spectrum Beta Lactamases ◽

First Time

In Gabon, terrestrial mammals of protected areas have been identified as a possible source of antibiotic-resistant bacteria. Some studies on antibiotic resistance in bats have already been carried out. The main goal of our study was to detect extended-spectrum beta-lactamases (ESBLs) that are produced by enterobacteria from bats in the Makokou region in Gabon. Sixty-eight fecal samples were obtained from 68 bats caught in the forests located 1 km from the little town of Makokou. After culture and isolation, 66 Gram-negative bacterial colonies were obtained. The double-disk diffusion test confirmed the presence of ESBLs in six (20.69%) Escherichia coli isolates, four (13.79%) Klebsiella pneumoniae isolates, and one (3.45%) Enterobacter cloacae isolate. The analysis based on the nucleotide sequences of the ESBL resistance genes showed that all cefotaximase-Munichs (CTX-Ms) were CTX-M-15 and that all sulfhydryl variables (SHVs) were SHV-11: 41.67% CTX-M-15-producing E. coli, 16.67% CTX-M-15+SHV-11-producing E. coli, 8.33% CTX-M-15-producing K. pneumoniae, 25% CTX-M-15+SHV-11-producing K. pneumoniae, and 8.33% CTX-M-15-produced E. cloacae. This study shows for the first time the presence of multiresistant ESBL-producing enterobacteria in fruit bats in Makokou.

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Characterization of Antibiotic Resistance E. Coli and Antibiotic Resistance Genes in Aquatic Environment of Taihu Lake, China

Applied Mechanics and Materials ◽

10.4028/www.scientific.net/amm.295-298.630 ◽

2013 ◽

Vol 295-298 ◽

pp. 630-634 ◽

Cited By ~ 1

Author(s):

Ni Ni Han ◽

Song He Zhang ◽

Pei Fang Wang ◽

Chao Wang

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Surface Water ◽

Resistance Genes ◽

Taihu Lake ◽

Antibiotic Resistance Genes ◽

Antibiotic Resistant ◽

E Coli ◽

Realtime Pcr

The aims of this study are to evaluate multiple antibiotic resistant Escherichia coli isolated from surface water and to investigate the presence and distribution antibiotic resistance genes (ARGs) in sediments of Taihu Lake. The results show that the presentence of four ARGs concentrations in the sediments of the lake was in sequence: strB>qnrB>strA>qnrS, as determined by realtime-PCR technique. The southwest and east areas of Taihu Lake were polluted seriously than other areas from all kinds of antibiotics. The screening Escherichia coli had a higher resistance to streptomycin, tetracycline and ampicillin than other four antibiotics, and had a lowest resistance to levofloxacin.

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Antibacterial effects of peptides synthesized based on the sequence of ribosome protein S1

Biomeditsinskaya Khimiya ◽

10.18097/pbmc20216703231 ◽

2021 ◽

Vol 67 (3) ◽

pp. 231-243

Author(s):

S.R. Kurpe ◽

S.Yu. Grishin ◽

A.V. Glyakina ◽

M.V. Slizen ◽

A.V. Panfilov ◽

...

Keyword(s):

Ribosomal Proteins ◽

Thermus Thermophilus ◽

Human Fibroblasts ◽

Model Organism ◽

Antimicrobial Effect ◽

Bioinformatic Analysis ◽

Global Scale ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

E Coli

Antibiotic resistance of bacteria is a topical problem on a global scale. Sometimes vigorous human activity leads to an increase in the number of bacteria carrying resistance genes in the environment. Antimicrobial peptides (AMPs) and similar compounds are potential candidates for combating antibiotic-resistant bacteria. Previously, we proposed and successfully tested on Thermus thermophilus a new mechanism of AMP action. This mechanism of directed coaggregation is based on the interaction of a peptide capable of forming fibrils with a target protein. In this work, we discuss the criteria for choosing a target for the targeted action of AMP, describe the features of the “parental” S1 ribosomal proteins T. thermophilus and Escherichia coli and the studied peptides using bioinformatic analysis methods, assess the antimicrobial effect of the synthesized peptides on a model organism of E. coli and cytotoxicity on cells of human fibroblasts. The obtained results will be important for the creation of new AMPs for pathogenic organisms.

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Immobilised Cerium-Doped Zinc Oxide as a Photocatalyst for the Degradation of Antibiotics and the Inactivation of Antibiotic-Resistant Bacteria

Catalysts ◽

10.3390/catal9030222 ◽

2019 ◽

Vol 9 (3) ◽

pp. 222 ◽

Cited By ~ 12

Author(s):

Ian Zammit ◽

Vincenzo Vaiano ◽

Ana Ribeiro ◽

Adrián Silva ◽

Célia Manaia ◽

...

Keyword(s):

Zinc Oxide ◽

Antibiotic Resistance ◽

Sodium Chloride ◽

Reaction Times ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

E Coli ◽

Isotonic Sodium Chloride ◽

Real Wastewater ◽

Autochthonous Bacteria

The threat of antibiotic resistance to the wellbeing of societies is well established. Urban wastewater treatment plants (UWTPs) are recognised sources for antibiotic resistance dissemination in the environment. Herein a novel cerium-doped zinc oxide (Ce-ZnO) photocatalyst is compared to ZnO and the benchmark TiO2-P25 in the immobilised form on a metallic support, to evaluate a photocatalytic process as a possible tertiary treatment in UWTPs. The catalysts were compared for the removal of two antibiotics, trimethoprim (TMP) and sulfamethoxazole (SMX), and for the inactivation of Escherichia coli (E. coli) strain DH5-Alpha in isotonic sodium chloride solution and of autochthonous bacteria in real secondary wastewater. In real wastewater, E. coli and other coliforms were monitored, as well as the respective fractions resistant to ofloxacin and azithromycin. In parallel, Pseudomonas aeruginosa and the respective sub-population resistant to ofloxacin or ciprofloxacin were also monitored. Photocatalysis with both ZnO and Ce-ZnO was faster than using TiO2-P25 at degrading the antibiotics, with Ce-ZnO the fastest against SMX but slower than undoped ZnO in the removal of TMP. Ce-ZnO catalyst reuse in the immobilised form produced somewhat slower kinetics maintained >50% of the initial activity, even after five cycles of use. Approximately 3 log10 inactivation of E. coli in isotonic sodium chloride water was recorded with reproducible results. In the removal of autochthonous bacteria in real wastewater, Ce-ZnO performed better (more than 2 log values higher) than TiO2-P25. In all cases, E. coli and other coliforms, including their resistant subpopulations, were inactivated at a higher rate than P. aeruginosa. With short reaction times no evidence for enrichment of resistance was observed, yet with extended reaction times low levels of bacterial loads were not further inactivated. Overall, Ce-ZnO is an easy and cheap photocatalyst to produce and immobilise and the one that showed higher activity than the industry standard TiO2-P25 against the tested antibiotics and bacteria, including antibiotic-resistant bacteria.

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Investigation of MALDI-TOF Mass Spectrometry of Diverse Synthetic Metalloporphyrins, Phthalocyanines and Multiporphyrin Arrays

Journal of Porphyrins and Phthalocyanines ◽

10.1002/(sici)1099-1409(199904)3:4<283::aid-jpp132>3.0.co;2-f ◽

1999 ◽

Vol 03 (04) ◽

pp. 283-291 ◽

Cited By ~ 94

Author(s):

N. SRINIVASAN ◽

CAROL A. HANEY ◽

JONATHAN S. LINDSEY ◽

WENZHU ZHANG ◽

BRIAN T. CHAIT

Keyword(s):

Mass Spectrometry ◽

Negative Ion ◽

Maldi Tof Ms ◽

Maldi Tof ◽

Flight Mass Spectrometry ◽

Powerful Analytical Tool ◽

Molecular Masses ◽

Dominant Process ◽

Tof Ms

We investigated the utility of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for analyzing porphyrinic compounds using a variety of different synthetic porphyrins, azaporphyrins, phthalocyanines and multiporphyrin arrays. Comparisons of spectra obtained from these analytes deposited either as neat samples or codeposited with neutral or acidic matrices have been made with the goal of identifying conditions that yield minimal demetalation, transmetalation, adduct formation and fragmentation. It was found that the molecular masses of many porphyrins can be successfully measured from neat sample preparations and do not require a matrix to facilitate desorption and ionization, although the measurement of large multiporphyrin arrays was facilitated by the use of matrices. Demetalation of magnesium porphyrins occurred in the presence of acidic matrices, but not with neutral matrices such as 1,4-benzoquinone. Positive ion spectra were obtained for each compound and negative ion spectra were also collected for the azaporphyrins and phthalocyanines. Examination of selected samples (prepared neat, with 1,4-benzoquinone, 2,3,5,6-tetrachloro-1,4-benzoquinone or α-cyano-4-hydroxycinnamic acid) showed that the dominant process of ionization involved oxidation yielding the radical cation M+· rather than the protonated molecule [M+H]+. MALDI-TOF-MS is shown to be a powerful analytical tool for the characterization of diverse synthetic porphyrinic compounds.

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Escherichia coli Antibiotic Resistance Patterns from Co-Grazing and Non-Co-Grazing Livestock and Wildlife Species from Two Farms in the Western Cape, South Africa

Antibiotics ◽

10.3390/antibiotics10060618 ◽

2021 ◽

Vol 10 (6) ◽

pp. 618

Author(s):

Michaela Sannettha van den Honert ◽

Pieter Andries Gouws ◽

Louwrens Christiaan Hoffman

Keyword(s):

Escherichia Coli ◽

South Africa ◽

Antibiotic Resistance ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

E Coli ◽

Wildlife Species ◽

Resistance Patterns ◽

Antibiotic Resistance Patterns ◽

Grazing Livestock

Although limited, studies have found conflicting results on whether co-grazing results in significant antibiotic resistance transfer between species. This type of farming system can act as a vector in the geographical spread of antibiotic-resistant bacteria in the environment. The aim of this study was to determine the antibiotic-resistant patterns between co-grazing and non-co-grazing livestock and wildlife species in South Africa. Escherichia coli was isolated from the faeces of various wildlife and livestock species from two farms in South Africa and was tested for antibiotic resistance using the Kirby–Bauer disk diffusion method against chloramphenicol, nalidixic acid, ampicillin, streptomycin, sulphafurazole, and tetracycline. A selection of some common antibiotic-resistant genes (blaCMY, aadA1, sul1, sul2, tetA, and tetB) were detected using PCR. The E. coli isolates from wildlife and livestock that co-grazed showed no significant differences in antibiotic resistance patterns. However, this was not the case for tetracycline resistance as the livestock isolates were significantly more resistant than the co-grazing wildlife isolates. The E. coli isolates from the non-co-grazing livestock and wildlife had significant differences in their antibiotic susceptibility patterns; the wildlife E. coli isolates were significantly more resistant to sulphafurazole and streptomycin than the livestock isolates, whilst those isolated from the cattle were significantly more resistant to ampicillin than the wildlife and sheep isolates. The results of this study suggest that there could be an exchange of antibiotic-resistant bacteria and genes between livestock and wildlife that co-graze.

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Inhibition of MreB and ftsZ proteins to minimize E. coli biofilms formation

10.1101/523167 ◽

2019 ◽

Author(s):

Emile Charles

Keyword(s):

United States ◽

Antibiotic Resistance ◽

Cell Division ◽

Mortality Rate ◽

Cell Shape ◽

The United States ◽

Resistant Bacteria ◽

Antibiotic Resistant ◽

E Coli ◽

Biofilm Development

AbstractIn the United States, more than two million individuals become infected by antibiotic-resistant bacteria, resulting in over 23,000 deaths annually. Bacterial biofilms, one of the major causes of this resistance, form a complex extracellular matrix that physically block antibiotic treatment. Within planktonic bacteria, two proteins, MreB and ftsZ, play a key role in bacterial cell growth and development. MreB regulates this development through maintaining the rod-like shape of gram-negative bacteria, while ftsZ regulates the timing and location of cell division. The present study compared the effects of two protein-inhibitors on biofilm formation of E. coli; the inhibitors, A22 Hydrochloride and PC190723, inhibit MreB (cell shape) and ftsZ (cell division), respectively. Efficacy was measured with a crystal violet staining assay. Four experiments were designed testing 1) the minimum inhibitory concentration of the inhibitors, 2) the synergistic effect of the inhibitors, 3) the microscopic effects of the inhibitors, and 4) the effect of the inhibitors on antibiotic susceptibility. A mid-level dosage of A22 significantly decreased biofilm density while there was no response to PC190732. The effect of A22 was verified microscopically, observing the change from bacilli cells to coccoid ones via the inhibition of MreB. In the second experiment, with conjunct inhibition, no interaction was found. Lastly, A22 was as effective as Amoxicillin in disrupting biofilms. The inhibition of MreB was found to have a key role in biofilm development. A model is proposed for biofilm density based on cell shape as affected by MreB.ImportanceEach year, more than 2 million Americans acquire antibiotic-resistant infection and 23,000 of them die (CDC, 2013). In a study done by Barsoumian et. al (2015), there was a 16% mortality rate pertaining to biofilm-related infections while non-biofilm infection caused a 5% mortality rate. These casualties aren’t limited to the United States. Abroad, antibiotic resistance is a huge issue: 25,000 deaths estimated in the EU; 38,000 deaths in Thailand; and 58,000 deaths in India, among infants alone (CDC, 2012). It is these statistics that inform us that antibiotic resistance must be addressed.

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Plasmidome AMR screening (PAMRS) workflow: a rapid screening workflow for phenotypic characterization of antibiotic resistance in plasmidomes

AAS Open Research ◽

10.12688/aasopenres.13111.1 ◽

2021 ◽

Vol 4 ◽

pp. 18

Author(s):

Kwabena Obeng Duedu ◽

Joana Qwansima Mends ◽

Reuben Ayivor-Djanie ◽

Priscilla Efua Essandoh ◽

Emmanuel Mawuli Nattah ◽

...

Keyword(s):

Antibiotic Resistance ◽

Virulence Genes ◽

Rapid Screening ◽

Phenotypic Characterization ◽

Antibiotic Resistant ◽

E Coli ◽

Genomic Tools ◽

Plasmid Extraction ◽

Bottle Neck

Background: Phenotypic characterization of antimicrobial resistance (AMR) in bacteria has remained the gold standard for investigation and monitoring of what resistance is present in an organism. However, the process is laborious and not attractive for screening multiple plasmids from a microbial community (plasmidomes). Instead, genomic tools are used, but a major bottle neck that presence of genes does not always translate into phenotypes. Methods: We designed the plasmidome AMR screening (PAMRS) workflow to investigate the presence of antibiotic resistant phenotypes in a plasmidome using Escherichia coli as a host organism. Plasmidomes were extracted from the faecal matter of chicken, cattle and humans using commercial plasmid extraction kits. Competent E. coli cells were transformed and evaluated using disk diffusion. Thirteen antibiotic resistant phenotypes were screened. Results: Here, we show that multiple antibiotic resistant phenotypes encoded by plasmids can be rapidly screened simultaneously using the PAMRS workflow. E. coli was able to pick up to 7, 5 or 8 resistant phenotypes from a single plasmidome from chicken, cattle or humans, respectively. Resistance to ceftazidime was the most frequently picked up phenotype in humans (52.6%) and cattle (90.5%), whereas in chickens, the most picked up resistant phenotype was resistance to co-trimoxazole, ceftriaxone and ampicillin (18.4% each). Conclusions: This workflow is a novel tool that could facilitate studies to evaluate the occurrence and expression of plasmid-encoded antibiotic resistance in microbial communities and their associated plasmid-host ranges. It could find application in the screening of plasmid-encoded virulence genes.

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