scholarly journals Amplifikasi Gen Resistensi Tembaga (Cur) pada Bakteri yang Diisolasi dari Limbah Industri di Surabaya

2020 ◽  
Vol 21 (1) ◽  
pp. 16-22
Author(s):  
Wahyu Irawati
Keyword(s):  
Minimum Inhibitory Concentration ◽  
16S Rdna ◽  
Inhibitory Concentration ◽  
Resistance Mechanism ◽  
Copper Resistance ◽  
Resistant Bacteria ◽  
Universal Primer ◽  
Base Solution ◽  
Lactobacillus Lactis ◽  
Cupriavidus Pauculus

ABSTRACTCopper pollution is one of the serious environmental problems in Indonesia. Higher concentration of copper is toxic so that threat living organism. Bio-remediation using copper resistant bacteria is effective for solving heavy metals pollution because the bacteria adapt easily when applied in environment. Using bacteria containing gene encoded copper resistance could help effort of copper bio-remediation. The purpose of this research is to isolate and characterize copper resistant bacteria from industrial sewage in Surabaya also to amplification of gene encoded resistance to copper (Cur). Bacteria was grown on Salts Base Solution medium with addition of appropriate concentration of copper. Resistance to copper was determined based on Minimum Inhibitory Concentration of CuSO4. Molecular characterization was done based on 16S rDNA gene analysis using universal primer. Amplification of Cur gene was done using specific primer of gene orf3, orf4, orf5 from Lactoccocus lactis. Three highly copper resistant bacteria have been isolated with the MIC of 2.5-7.5 mM CuSO4 encoded IrC1, IrC2, and IrC4. Isolate IrC4 is the highest copper resistant bacteria with the MIC of 7.5 mM. Resistance mechanism may be through accumulation copper inside the cells with the total of 371 mg/g dry weight of cells. The three bacteria have plasmid with the size of 21 kb. Isolate IrC4 have 96.99% similarity with Cupriavidus pauculus. Amplification of copper-resistance (Cur) gene demonstrated that a single band of 0.9 kb was obtained from isolate C4. The finding of indigenous resistant bacteria encoded Cur gene may give better solution for pollution problem in Indonesia.Keywords: bacteria, copper, Isolate IrC4, Lactococcus lactis, resistantABSTRAKPencemaran tembaga di Indonesia merupakan salah satu masalah lingkungan yang serius sehingga perlu diatasi. Tembaga pada konsentrasi yang tinggi bersifat toksik sehingga mengancam kehidupan organisme. Bioremediasi menggunakan bakteri resisten tembaga yang diisolasi dari lingkungan tercemar terbukti efektif mengatasi pencemaran logam berat karena lebih mudah beradaptasi ketika diterapkan di lingkungan. Pemanfaatan bakteri yang mengandung gen penyandi resistensi tembaga dapat menunjang keberhasilan usaha bioremediasi tembaga. Penelitian ini bertujuan untuk mengisolasi dan mengkarakterisasi bakteri resisten tembaga dari limbah industri di Surabaya serta melakukan amplifikasi gen yang menyandi resistensi bakteri tersebut terhadap tembaga (Cur).  Isolasi bakteri dilakukan menggunakan medium Salt Base Solution dengan penambahan CuSO4. Uji resistensi ditentukan berdasarkan nilai Minimum Inhibitory Concentration (MIC) terhadap CuSO4. Karakterisasi molekular dilakukan berdasarkan analisis gen 16S rDNA menggunakan primer universal. Amplifikasi gen Cur dilakukan dengan menggunakan primer yang spesifik terhadap gen orf3, orf4, orf5 dari gen Cur pada Lactobacillus lactis. Hasil penelitian menunjukkan bahwa terdapat tiga isolat bakteri yang paling resisten dengan nilai MIC= 6.5-7.5 mM, yaitu isolat IrC1, IrC2, dan IrC4. isolat IrC4 merupakan isolat bakteri yang paling resisten dengan nilai MIC sebesar 7.5 mM. Mekanisme reistensi isolat IrC4 adalah dengan cara mengakumulasi tembaga di dalam sel selitar 371 mg/g berat kering sel. Ketiga isolat memiliki plasmid berukuran sekitar 21 kb. Analisis gen 16S rDNA menunjukkan bahwa isolat IrC4 memiliki kemiripan 96,99% dengan Cupriavidus pauculus. Gen Cur pada isolat IrC4 sepanjang 0.9 kb berhasil diamplifikasi dengan menggunakan primer spesifik gen orf3 yang menyandikan pengikatan tembaga. Penemuan bakteri indigen indonesia yang menyandikan gen resisten tembaga diharapkan dapat menunjang keberhasilan penanganan masalah pencemaran tembaga di Indonesia.Kata kunci:  bakteri, isolat IrC4, Lactobacillus lactis, resisten, tembaga.

scholarly journals Determination of Drug Efflux Pump Efficiency in Drug-Resistant Bacteria Using MALDI-TOF MS

Antibiotics ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 639 ◽  
Author(s):  
Wen-Jung Lu ◽  
Hsuan-Ju Lin ◽  
Pang-Hung Hsu ◽  
Hong-Ting Victor Lin
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Resistant Bacteria ◽  
Pump Efficiency ◽  
Drug Efflux ◽  
Maldi Tof Ms ◽  
E Coli ◽  
Maldi Tof ◽  
Conventional Methods ◽  
Tof Ms

Multidrug efflux pumps play an essential role in antibiotic resistance. The conventional methods, including minimum inhibitory concentration and fluorescent assays, to monitor transporter efflux activity might have some drawbacks, such as indirect evidence or interference from color molecules. In this study, MALDI-TOF MS use was explored for monitoring drug efflux by a multidrug transporter, and the results were compared for validation with the data from conventional methods. Minimum inhibitory concentration was used first to evaluate the activity of Escherichia coli drug transporter AcrB, and this analysis showed that the E. coli overexpressing AcrB exhibited elevated resistance to various antibiotics and dyes. Fluorescence-based studies indicated that AcrB in E. coli could decrease the accumulation of intracellular dyes and display various efflux rate constants for different dyes, suggesting AcrB’s efflux activity. The MALDI-TOF MS analysis parameters were optimized to maintain a detection accuracy for AcrB’s substrates; furthermore, the MS data showed that E. coli overexpressing AcrB led to increased ions abundancy of various dyes and drugs in the extracellular space at different rates over time, illustrating continuous substrate efflux by AcrB. This study concluded that MALDI-TOF MS is a reliable method that can rapidly determine the drug pump efflux activity for various substrates.

scholarly journals Overview of Quantitative Methodologies to Understand Antimicrobial Resistance via Minimum Inhibitory Concentration

Animals ◽  
2020 ◽  
Vol 10 (8) ◽  
pp. 1405
Author(s):  
Alec Michael ◽  
Todd Kelman ◽  
Maurice Pitesky
Keyword(s):  
Logistic Regression ◽  
Minimum Inhibitory Concentration ◽  
Antimicrobial Resistance ◽  
Model Selection ◽  
Inhibitory Concentration ◽  
Failure Time ◽  
Resistant Bacteria ◽  
Accelerated Failure Time ◽  
Clinical Resistance ◽  
Interval Censored

The development of antimicrobial resistance (AMR) represents a significant threat to humans and food animals. The use of antimicrobials in human and veterinary medicine may select for resistant bacteria, resulting in increased levels of AMR in these populations. As the threat presented by AMR increases, it becomes critically important to find methods for effectively interpreting minimum inhibitory concentration (MIC) tests. Currently, a wide array of techniques for analyzing these data can be found in the literature, but few guidelines for choosing among them exist. Here, we examine several quantitative techniques for analyzing the results of MIC tests and discuss and summarize various ways to model MIC data. The goal of this review is to propose important considerations for appropriate model selection given the purpose and context of the study. Approaches reviewed include mixture models, logistic regression, cumulative logistic regression, and accelerated failure time–frailty models. Important considerations in model selection include the objective of the study (e.g., modeling MIC creep vs. clinical resistance), degree of censoring in the data (e.g., heavily left/right censored vs. primarily interval censored), and consistency of testing parameters (e.g., same range of concentrations tested for a given antibiotic).

scholarly journals Evaluation of the Sensitivity of Staphylococcus aurous Isolated from Nasal Swabs to Natural Honey

2020 ◽  
pp. 56-64
Author(s):  
A Molanaei ◽  
SA Seyedoshohadaei ◽  
S Hasani ◽  
P Sharifi ◽  
M Rashidian ◽  
...  
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Antibacterial Agents ◽  
Bacterial Resistance ◽  
Inhibitory Concentration ◽  
Antibacterial Properties ◽  
Resistant Bacteria ◽  
Nasal Swabs ◽  
Vancomycin Resistant ◽  
Dilution Tube ◽  
Natural Honey

Introduction: Bacterial resistance to antibacterial agents is a very serious threat to public health. Where some antibacterial agents prove ineffective, the antibacterial properties of honey have been shown to be highly efficacious against several human bacterial pathogens. The purpose of this study is to investigate the sensitivity of Staphylococcus aureus isolated from the nursing staff of a hospital to natural honey. Methods: In this study, 35 strains of methicillin-resistant S. aurous samples were selected from hospital staff's nasal swabs. Two strains were vancomycin-resistant. The serial dilution tube test methodwas used to determine minimum inhibitory concentration (MIC) .The susceptibility of each strain of staph bacteria to natural honey without wax was determined and compared with that of a glucose solution with the same density. Results: In all strains, except for the two strains resistant to vancomycin, MIC level was < 8.3% (v/v). The MIC of glucose as dense as honey was four times higher. The two vancomycin-resistant strains were completely resistant to natural honey. Conclusions: This study has therefore demonstrated that inhibiting bacterial growth is not merely done by purely natural honey not because of osmolality, but vancomycin-resistant bacteria are not sensitive to natural honey. Keywords: sensitivity, Staphylococcus aurous, natural honey, minimum inhibitory concentration

scholarly journals Upregulation of gluconeogenesis leads to increased tolerance to antibiotics targeting the MreB elongosome in E. coli

2020 ◽  
Author(s):  
Brody Barton ◽  
Addison Grinnell ◽  
Randy M. Morgenstein
Keyword(s):  
Antibiotic Resistance ◽  
Minimal Inhibitory Concentration ◽  
Bacterial Infections ◽  
Inhibitory Concentration ◽  
Resistance Mechanism ◽  
Tca Cycle ◽  
Resistant Bacteria ◽  
Antibiotic Development ◽  
New Antibiotics ◽  
Global Threat

AbstractAntibiotic resistant bacteria are a global threat to human health. One way to combat the rise of antibiotic resistance is to make new antibiotics that target previously ignored proteins. The bacterial actin homolog, MreB, is highly conserved among rod-shaped bacteria and essential for growth, making MreB a good focus for antibiotic targeting. Therefore, it is imperative to understand mechanisms that can give rise to resistance to MreB targeting drugs. Using the MreB targeting drug, A22, we show that changes to central metabolism through deletion of TCA cycle genes, leads to the upregulation of gluconeogenesis resulting in cells with an increased minimal inhibitory concentration to A22. This phenotype can be recapitulated through the addition of glucose to the media. Finally, we show that this increase in minimal inhibitory concentration is not specific to A22 but can be seen in other cell wall targeting antibiotics, such as mecillinam.ImportanceThe spread of antibiotic resistance has made bacterial infections harder to treat. Finding new targets for antibiotic development is critical to overcoming the variety of resistance mechanism that are already crippling our ability to treat infections with current antibiotics. The bacterial actin homolog MreB is a good target for new antibiotic development because it is essential for growth and highly conserved among rod-shaped pathogens. The significance of this research is in understanding the mechanisms cells can develop toward the inhibition of MreB to better understand how to make MreB targeting antibiotics in the future.

scholarly journals Colistin-resistant Escherichia coli clinical isolate harbouring the mcr-1 gene in Ecuador

2016 ◽  
Vol 144 (14) ◽  
pp. 2967-2970 ◽  
Author(s):  
D. ORTEGA-PAREDES ◽  
P. BARBA ◽  
J. ZURITA
Keyword(s):  
Escherichia Coli ◽  
Minimum Inhibitory Concentration ◽  
Clinical Isolate ◽  
Inhibitory Concentration ◽  
Resistance Mechanism ◽  
Sequence Type ◽  
Colistin Resistance ◽  
Gram Negative ◽  
E Coli ◽  
Commensal Strain

SUMMARYColistin resistance mediated by the mcr-1 gene has been reported worldwide, but to date not from the Andean region, South America. We report the first clinical isolate of Escherichia coli harbouring the mcr-1 gene in Ecuador. The strain was isolated from peritoneal fluid from a 14-year-old male with acute appendicitis, and subjected to molecular analysis. The minimum inhibitory concentration of colistin for the strain was 8 mg/ml and it was susceptible to carbapenems but resistant to tigecycline. The strain harboured mcr-1 and blaCTX-M-55 genes and was of sequence type 609. The recognition of an apparently commensal strain of E. coli harbouring mcr-1 serves as an alert to the presence in the region of this recently described resistance mechanism to one of the last line of drugs available for the treatment of multi-resistant Gram-negative infections.

scholarly journals Isolation and Identification of Multi-drug Resistant Bacteria from various sources and Antimicrobial Activity of Terminalia chebula Retz against isolated Multi-drug Resistant Bacteria

2021 ◽  
Vol 11 (4) ◽  
pp. 7338-7344
Author(s):  
Tamalika Chakraborty ◽  
Kanchan Chettri ◽  
Sumana Chatterjee ◽  
Lopamudra Datta ◽  
Abhijit Sengupta
Keyword(s):  
Drug Resistance ◽  
Minimum Inhibitory Concentration ◽  
Inhibitory Concentration ◽  
Minimum Bactericidal Concentration ◽  
Bacterial Activity ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Terminalia Chebula ◽  
Isolation And Identification ◽  
Drug Resistant Bacteria

Drug resistance is a threat to civilization, which results from over-prescription and irrational use of antibiotics. This has led to an increased demand for novel leads of herbal origin to overcome drug resistance. The present work involves the screening of various antibiotics against isolated Staphylococcus sp. from Hospital Effluent and the Minimum Inhibitory concentration for antibiotics namely Vancomycin, Erythromycin and Oxacillin were found to be 7.33+0.6 µg/ml 25.33+0.6 µg/ml and 27.33+0.6 µg/ml respectively whereas Minimum bactericidal concentration of Vancomycin, Erythromycin and oxacillin was found to be 180µg/ml; 146.67 + 0.3 µg/ml and 96.66 + 0.6 µg/ml respectively. Thus, the isolated bacteria were proved to be Multi-Drug Resistant. Haritaki (Terminalia chebula Retz) is given potential importance in Ayurveda for its properties to cure and prevent diseases. Terminalia chebula Retz is often known as “King of Medicines” and enlisted in Ayurveda for its extraordinary therapeutic contribution. The proved Multi-Drug Resistant bacteria was further subjected to a crude extract of Haritaki. Minimum Inhibitory Concentration for Terminalia chebula was found to be 1.33 +0.3 mg/ml and thus proved to be exhibiting potential anti-bacterial activity against isolated Multi-Drug Resistant Staphylococcus sp.

scholarly journals Effective inhibition of Clostridioides difficile by the novel peptide CM-A

PLoS ONE ◽  
2021 ◽  
Vol 16 (9) ◽  
pp. e0257431
Author(s):  
Sirirak Arthithanyaroj ◽  
Surang Chankhamhaengdecha ◽  
Urai Chaisri ◽  
Ratchaneewan Aunpad ◽  
Amornrat Aroonnual
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Cell Membrane ◽  
Inhibitory Concentration ◽  
Cell Permeability ◽  
Resistant Bacteria ◽  
Microscopy Imaging ◽  
Antibiotic Resistant ◽  
Clostridioides Difficile ◽  
Novel Approach ◽  
Cell Depolarization

Clostridioides difficile infection is the most common cause of nosocomial and antibiotic-associated diarrhea. C. difficile treatment is increasingly likely to fail, and the recurrence rate is high. Antimicrobial peptides are considered an alternative treatment for many infectious diseases, including those caused by antibiotic resistant bacteria. In the present study, we identified a CM peptide, a hybrid of cecropin A and melittin, and its derivative which possesses potent antimicrobial activity against C. difficile strain 630. CM peptide exhibited antibacterial activity with minimum inhibitory concentration of 3.906 μg/ml (2.21 μM). A modified derivative of CM, CM-A, exhibited even greater activity with a minimum inhibitory concentration of 1.953 μg/ml (1.06 μM) and a minimum bactericidal concentration of 7.8125 μg/ml (4.24 μM), which indicates that CM-A peptide is more efficient than its parent peptide. A fluorescence-activated cell sorter analysis revealed that the membrane of C. difficile 630 could be an important target for CM-A. This peptide induced high levels of cell depolarization and cell permeability on C. difficile cell membrane. Moreover, electron microscopy imaging showed that CM-A interferes with the C. difficile cell membrane. Hence, the antimicrobial peptide CM-A may represent a promising novel approach for the treatment of C. difficile infections.

Conjugation of Silver Nanoparticles with De Novo Engineered Cationic Antimicrobial Peptides: Proposal for Study (Preprint)

2021 ◽  
Author(s):  
Alvin Hu
Keyword(s):  
Antimicrobial Activity ◽  
Silver Nanoparticles ◽  
Minimum Inhibitory Concentration ◽  
Antimicrobial Peptides ◽  
Inhibitory Concentration ◽  
De Novo ◽  
Resistant Bacteria ◽  
Drug Resistant ◽  
Cationic Antimicrobial Peptides ◽  
Drug Resistant Bacteria

BACKGROUND Cationic antimicrobial peptides have broad antimicrobial activity and provide a novel way of targeting multi drug resistant bacteria in an era of increasing antimicrobial resistance. Current developments show positive prospects for both antimicrobial peptides and silver nanoparticles individually. OBJECTIVE The primary objective is to propose another method of enhancing antimicrobial activity by conjugating silver nanoparticles with cationic antimicrobial peptides for a subsequent preliminary assessment on studying the minimum inhibitory concentration of multi drug resistant bacteria. The secondary objective would be to evaluate the safety of the conjugated compound to assess viability for in vivo use. METHODS The proposition is planned for approximately 3 overarching stages. Firstly, I propose synthesis of wlbu2c, a modified version of antimicrobial peptide wlbu2 with an added cysteine group, using standard Fmoc procedure. This will subsequently be attempted to stably conjugate with silver nanoparticles ideally through photochemical means. Secondly, the conjugate wlbu2c-AgNP will be tested for antimicrobial activity following Clinical & Laboratory Standards Institute Manual on standard minimum inhibitory concentration testing. If all of the above is completed the experiment can progress to the assessment of cytotoxicity using cell lysis assays. RESULTS I-TASSER simulation revealed that our modified peptide wlbu2c has similar secondary structure to original wlbu2 peptide. No other results have been obtained at this time other than aforementioned theoretical propositions. CONCLUSIONS The addition of silver nanoparticles to already developing de novo engineered antimicrobial peptides provide a second degree of freedom toward the development of potent antimicrobials. Future prospects include emergency last line therapy, treatment for current difficult to eradicate bacterial colonization such as in cystic fibrosis, implantable medical devices, cancer and immunotherapy. This proposal is intended to be provided to the public as I do not anticipate funding at this time.

scholarly journals Re-Potentiation of β-Lactam Antibiotic by Synergistic Combination with Biogenic Copper Oxide Nanocubes against Biofilm Forming Multidrug-Resistant Bacteria

Molecules ◽  
2019 ◽  
Vol 24 (17) ◽  
pp. 3055 ◽  
Author(s):  
Ruby Celsia Arul Selvaraj ◽  
Mala Rajendran ◽  
Hari Prasath Nagaiah
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Copper Oxide ◽  
Inhibitory Concentration ◽  
Copper Oxide Nanoparticles ◽  
Dermal Fibroblasts ◽  
Resistant Bacteria ◽  
Oxide Nanoparticles ◽  
Device Infection ◽  
Synergistic Combination ◽  
Time Kill

Biofilm-associated tissue and device infection is a major threat to therapy. The present work aims to potentiate β-lactam antibiotics with biologically synthesized copper oxide nanoparticles. The synergistic combination of amoxyclav with copper oxide nanoparticles was investigated by checkerboard assay and time-kill assay against bacteria isolated from a burn wound and a urinary catheter. The control of biofilm formation and extracellular polymeric substance production by the synergistic combination was quantified in well plate assay. The effect of copper oxide nanoparticles on the viability of human dermal fibroblasts was evaluated. The minimum inhibitory concentration and minimum bactericidal concentration of amoxyclav were 70 μg/mL and 140 μg/mL, respectively, against Proteus mirabilis and 50 μg/mL and 100 μg/mL, respectively, against Staphylococcus aureus. The synergistic combination of amoxyclav with copper oxide nanoparticles reduced the minimum inhibitory concentration of amoxyclav by 16-fold against P. mirabilis and 32-fold against S. aureus. Above 17.5 μg/mL, amoxyclav exhibited additive activity with copper oxide nanoparticles against P. mirabilis. The time-kill assay showed the efficacy of the synergistic combination on the complete inhibition of P. mirabilis and S. aureus within 20 h and 24 h, respectively, whereas amoxyclav and copper oxide nanoparticles did not inhibit P. mirabilis and S. aureus until 48 h. The synergistic combination of amoxyclav with copper oxide nanoparticles significantly reduced the biofilm formed by P. mirabilis and S. aureus by 85% and 93%, respectively. The concentration of proteins, carbohydrates, and DNA in extracellular polymeric substances of the biofilm was significantly reduced by the synergistic combination of amoxyclav and copper oxide nanoparticles. The fibroblast cells cultured in the presence of copper oxide nanoparticles showed normal morphology with 99.47% viability. No cytopathic effect was observed. Thus, the study demonstrated the re-potentiation of amoxyclav by copper oxide nanoparticles.

scholarly journals Characterization and partial purification of an antibacterial agent from halophilic actinomycetes Kocuria sp. strain rsk4

Bioimpacts ◽  
2018 ◽  
Vol 8 (4) ◽  
pp. 253-261 ◽  
Author(s):  
Ravi Ranjan Kumar ◽  
Vasantba J Jadeja
Keyword(s):  
Minimum Inhibitory Concentration ◽  
Broad Spectrum ◽  
Antibacterial Agent ◽  
Pathogenic Bacteria ◽  
Inhibitory Concentration ◽  
Flash Chromatography ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Antibacterial Compound ◽  
Halophilic Actinomycetes

Introduction: The inevitable rise of antibiotic-resistant bacteria is a global health problem. These pathogens erode the utility of available antibiotics. Staphylococcus aureus is one of the major causes of community-acquired infections. The aim of work was to evaluate the marine actinomycetes for production of the antibacterial agent against pathogens. Methods: Halophilic actinomycetes were isolated, characterized and screened for production of antibacterial agent against pathogenic bacteria. The antibacterial compounds were extracted by solvent extraction and separated by TLC based bioautography. Antibacterial compound was further purified by flash chromatography followed by high-performance liquid chromatography (HPLC) techniques. The active fraction was characterized by spectroscopy techniques. The minimum inhibitory concentration of antibiotic was determined against pathogens. Results: A new halophilic actinomycetes strain rsk4 was isolated from marine water. It was designated as Kocuria sp. based on the physiological, biochemical and 16S rDNA sequence-based characters. It was able to produce broad-spectrum antibacterial compound and exhibited significant inhibitory activities against antibiotic-resistant S. aureus. The antibacterial compound was secreted optimally at 5% NaCl and neutral pH in the starch casein medium during stationary phase. The crude ethyl acetate extract was separated by chloroform-methanol, 24:1, v/v having Rf value 0.45. Bioassay of HPLC fractions confirms the presence of antibiotics picks at retention time: 3.24 minutes. The UV-Visible and mass spectra of the compound revealed that the active compound was different from other known antibiotics. The lowest minimum inhibitory concentration was recorded against S. aureus (30 µg/mL). Conclusion: The result suggests that a broad-spectrum antibacterial compound obtained from halophilic actinomycetes is effective against pathogenic bacteria. This compound may be a good alternative treatment against antibiotic-resistant pathogen S. aureus.

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