scholarly journals Flow cytometry as a rapid test for detection of tetracycline resistance directly in bacterial cells in Micrococcus luteus

10.5219/1354 ◽  
2020 ◽  
Vol 14 ◽  
pp. 954-959
Author(s):  
Elena Zayko ◽  
Dagmara Bataeva ◽  
Yuliya Yushina ◽  
Anzhelika Makhova ◽  
Mariya Grudistova
Keyword(s):  
Flow Cytometry ◽  
Antibiotic Resistance ◽  
Micrococcus Luteus ◽  
Tetracycline Resistance ◽  
Positive Control ◽  
Living Cells ◽  
Sybr Green ◽  
Live Cells ◽  
Resistant Bacteria ◽  
Speed And Accuracy

Correct effective doses of antibiotics are important in the treatment of infectious diseases. The most frequently used methods for determination of the antibiotic susceptibility of bacterial pathogens are slow. The detection of multidrug-resistant bacteria currently relies on primary isolation followed by phenotypic detection of antibiotic resistance by measuring bacterial growth in the presence of the antibiotic being tested. The basic requirements for methods of detection of resistance to antibiotics include speed and accuracy. We studied the speed and accuracy of flow cytometry for the detection of tetracycline resistance in the Gram-positive bacteria Micrococcus luteus. Detection of cell viability and reliability of antibiotic resistance was carried out on the Guava EasyCyte flow cytometer (Merck Millipore, Germany) with SYBR Green and PI dyes. M. luteus was exposed to tetracycline (at 30, 90, 180 and 270 μg.mL-1) over 24 hours. Concentrations of live and dead cells were measured after 4 and 24 hours of incubation. The results revealed that the use of mixed dyes PI and SYBR Green allowed the division of cells into large subpopulations of live and dead cells and the DNA of destroyed cells. After 4 h exposure to tetracycline 30 μg.mL-1, the subpopulation of live cells decreased by 47% compared to the positive control. Tetracycline at 90 μg.mL-1 decreased the subpopulation of live cells by 59% compared to the positive control. A continued increase in concentration caused a shift in the population and an increase in dead cells, indicating damage to the cells of the microorganism. Incubation of M. luteus with 180 and 270 μg.mL-1 tetracycline decreased the subpopulation of live cells by 82% and 94%, respectively, in comparison with the positive control. After incubation with 30 μg of tetracycline over 24 h the number of living cells decreased by 70% in comparison with the positive control. Tetracycline treatment (90 μg.mL-1 for 24 h) killed 71% of cells. After exposure to 90 μg.mL-1 tetracycline 29% cells were viable. The viability of living cells was confirmed by a microbiological test.

scholarly journals Characterization of antibiotic resistance genes in the species of the rumen microbiota

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Yasmin Neves Vieira Sabino ◽  
Mateus Ferreira Santana ◽  
Linda Boniface Oyama ◽  
Fernanda Godoy Santos ◽  
Ana Júlia Silva Moreira ◽  
...  
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Animal Health ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Ruminal Bacteria ◽  
Genes Encoding

AbstractInfections caused by multidrug resistant bacteria represent a therapeutic challenge both in clinical settings and in livestock production, but the prevalence of antibiotic resistance genes among the species of bacteria that colonize the gastrointestinal tract of ruminants is not well characterized. Here, we investigate the resistome of 435 ruminal microbial genomes in silico and confirm representative phenotypes in vitro. We find a high abundance of genes encoding tetracycline resistance and evidence that the tet(W) gene is under positive selective pressure. Our findings reveal that tet(W) is located in a novel integrative and conjugative element in several ruminal bacterial genomes. Analyses of rumen microbial metatranscriptomes confirm the expression of the most abundant antibiotic resistance genes. Our data provide insight into antibiotic resistange gene profiles of the main species of ruminal bacteria and reveal the potential role of mobile genetic elements in shaping the resistome of the rumen microbiome, with implications for human and animal health.

scholarly journals Changes in Antibiotic-Resistance Genes Induced by the Grazing Effect in Three Cladoceran Species

2021 ◽  
Vol 9 (9) ◽  
pp. 1959
Author(s):  
Jong-Yun Choi ◽  
Seong-Ki Kim
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Freshwater Ecosystems ◽  
Resistant Bacteria ◽  
Biological Interactions ◽  
Chydorus Sphaericus ◽  
Cladoceran Species ◽  
Grazing Effect

The acquisition of Antibiotic-Resistance Genes (ARGs) by natural bacteria caused by antibiotic abuse is causing serious problems for human and animal welfare. Here, we evaluated the influence of three cladoceran species on Antibiotic-Resistant Bacteria (ARB) and tetracycline-resistance gene (tet(A)) copies, and discussed the effect of these biological interactions on the distribution and diffusion of ARGs in freshwater ecosystems. Bacterial community and tet(A) abundances in water samples collected from wetlands were strongly influenced by cladoceran presence. The presence of Daphnia obtusa dramatically decreased ARB and tet(A) abundance compared to that with other cladoceran species (Chydorus sphaericus and Simocephalus vetulus). Interestingly, we found a high abundance of Flavobacteriales in the microbiomes of cladoceran species. Considering that Flavobacteriales species are potential carriers of the tet(A) gene, their adsorption and assimilation with cladocerans could significantly impact the reduction of tet(A) in water. Field surveys also showed that tet(A) abundance could be low if the dominance of D. obtusa in each wetland was high. This study highlighted the need for ecological interactions and a broad range of niches in the food web when discussing the fate of ARGs in freshwater ecosystems.

Occurrence of antibiotic-resistant bacteria and endotoxin associated with the land application of biosolids

2007 ◽  
Vol 53 (5) ◽  
pp. 616-622 ◽  
Author(s):  
J.P. Brooks ◽  
S.L. Maxwell ◽  
C. Rensing ◽  
C.P. Gerba ◽  
I.L. Pepper
Keyword(s):  
Antibiotic Resistance ◽  
Applied Field ◽  
Tetracycline Resistance ◽  
Land Application ◽  
Resistant Bacteria ◽  
Culturable Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Background Soil

The purpose of this study was to determine the prevalence of antibiotic-resistant bacteria and endotoxin in soil after land application of biosolids. Soil was collected over a 15 month period following land application of biosolids, and antibiotic resistance was ascertained using clinically relevant antibiotic concentrations. Ampicillin, cephalothin, ciprofloxacin, and tetracycline resistance were all monitored separately for any changes throughout the 15 month period. Endotoxin soil concentrations were monitored using commercially available endotoxin analysis reagents. Overall, land application of biosolids did not increase the percentage of antibiotic-resistant culturable bacteria above background soil levels. Likewise, land application of biosolids did not significantly increase the concentration of endotoxin in soil. This study determined and established a baseline understanding of the overall effect that land application of biosolids had on the land-applied field with respect to antibiotic-resistant bacterial and endotoxin soil densities.

scholarly journals Horizontal gene transfer facilitates the spread of extracellular antibiotic resistance genes in soil

2021 ◽  
Author(s):  
Heather A. Kittredge ◽  
Kevin M. Dougherty ◽  
Sarah E. Evans
Keyword(s):  
Antibiotic Resistance ◽  
Gene Transfer ◽  
Horizontal Gene Transfer ◽  
Resistance Genes ◽  
Natural Transformation ◽  
Antibiotic Resistance Genes ◽  
Live Cells ◽  
Resistant Bacteria ◽  
Antibiotic Resistant Bacteria ◽  
Antibiotic Resistant

AbstractAntibiotic resistance genes (ARGs) are ubiquitous in the environment and pose a serious risk to human and veterinary health. While many studies focus on the spread of live antibiotic resistant bacteria throughout the environment, it is unclear whether extracellular ARGs from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we inoculate antibiotic-free soil with extracellular ARGs (eARGs) from dead Pseudeononas stutzeri cells and track the evolution of antibiotic resistance via natural transformation – a mechanism of horizontal gene transfer involving the genomic integration of eARGs. We find that transformation facilitates the rapid evolution of antibiotic resistance even when eARGs occur at low concentrations (0.25 μg g-1 soil). However, when eARGs are abundant, transformation increases substantially. The evolution of antibiotic resistance was high under soil moistures typical in terrestrial systems (5%-30% gravimetric water content) and was only inhibited at very high soil moistures (>30%). While eARGs transformed into live cells at a low frequency, exposure to a low dose of antibiotic allowed a small number of transformants to reach high abundances in laboratory populations, suggesting even rare transformation events pose a risk to human health. Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance, and that disinfection alone is insufficient to stop the spread of antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish at low frequencies in the absence of antibiotic selection.ImportanceOver the last decade, antibiotics in the environment have gained increasing attention because they can select for drug-resistant phenotypes that would have otherwise gone extinct. To counter this effect, bacterial populations exposed to antibiotics often undergo disinfection. However, the release of extracellular antibiotic resistance genes (eARGs) into the environment following disinfection can promote the transfer of eARGs through natural transformation. This phenomenon is well-documented in wastewater and drinking water, but yet to be investigated in soil. Our results directly demonstrate that eARGs from dead bacteria are an important, but often overlooked source of antibiotic resistance in soil. We conclude that disinfection alone is insufficient to prevent the spread of ARGs. Special caution should be taken in releasing antibiotics into the environment, even if there are no live antibiotic resistant bacteria in the community, as transformation allows DNA to maintain its biological activity past microbial death.

Retail Ready-to-Eat Food as a Potential Vehicle for Staphylococcus spp. Harboring Antibiotic Resistance Genes

2014 ◽  
Vol 77 (6) ◽  
pp. 993-998 ◽  
Author(s):  
WIOLETA CHAJĘCKA-WIERZCHOWSKA ◽  
ANNA ZADERNOWSKA ◽  
BEATA NALEPA ◽  
MAGDA SIERPI´NSKA ◽  
ŁUCJA ŁANIEWSKA-TROKENHEIM
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Meca Gene ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Multidrug Resistant ◽  
Resistant Bacteria ◽  
Antibiotic Resistant ◽  
Cured Meats ◽  
Staphylococcus Spp

Ready-to-eat (RTE) food, which does not need thermal processing before consumption, could be a vehicle for the spread of antibiotic-resistant microorganisms. As part of general microbiological safety checks, staphylococci are routinely enumerated in these kinds of foods. However, the presence of antibiotic-resistant staphylococci in RTE food is not routinely investigated, and data are only available from a small number of studies. The present study evaluated the pheno- and genotypical antimicrobial resistance profile of Staphylococcus spp. isolated from 858 RTE foods (cheeses, cured meats, sausages, smoked fishes, salads). Of 113 strains isolated, S. aureus was the most prevalent species, followed by S. xylosus, S. saprophyticus, and S. epidermidis. More than half (54.9%) of the isolates were resistant to at least one class of tested antibiotic; of these, 35.4% of the strains were classified as multidrug resistant. Most of the isolates were resistant to cefoxitin (49.6%), followed by clindamycin (39.3%), tigecycline (27.4%), quinupristin-dalfopristin (22.2%), rifampin (20.5%), tetracycline (17.9%), and erythromycin (8.5%). All methicillin-resistant staphylococci harbored the mecA gene. Among the isolates resistant to at least one antibiotic, 38 harbored tetracycline resistance determinant tet(M), 24 harbored tet(L), and 9 harbored tet(K). Of the isolates positive for tet(M) genes, 34.2% were positive for the Tn916-Tn1545–like integrase family gene. Our results indicated that retail RTE food could be considered an important route for the transmission of antibiotic-resistant bacteria harboring multiple antibiotic resistance genes.

scholarly journals Tetracycline-resistant bacteria and ribosomal protection protein genes in soils from selected agricultural fields and livestock farms

2021 ◽  
Vol 64 (1) ◽  
Author(s):  
Kathyleen Nogrado ◽  
Tatsuya Unno ◽  
Hor-Gil Hur ◽  
Ji-Hoon Lee
Keyword(s):  
Antibiotic Resistance ◽  
Copy Number ◽  
Agricultural Soils ◽  
Gene Copy Number ◽  
Tetracycline Resistance ◽  
Gene Copy ◽  
Resistant Bacteria ◽  
Bacterial Isolates ◽  
Animal House ◽  
Ribosomal Protection Protein

AbstractAntibiotic resistance in soil environment has eminently been compared and studied between agricultural and pristine soils, and the role of concentrated animal feeding operations has markedly been recognized as one of the major sources of antibiotic resistance. This study described the tetracycline resistance in small-scale farms in pursuit of presenting its possible role and contribution to the persistence of antibiotic resistance in the environment. Results of the study would render additional information on the occurrence of the ribosomal protection protein (RPP) tet genes among the isolated bacteria from the selected agricultural soils. Four tetracycline resistance and RPP genes were determined in two different agricultural soil settings. Both the culture and molecular method were used to determine and measure tetracycline resistance in soils from arable land and animal house. Results revealed a significantly higher number of culturable antibiotic-resistant bacteria in animal houses than arable lands which was suggestive of higher antibiotic resistance in areas where there was direct administration of the antibiotics. However, quantification of the gene copy numbers in the agricultural soils indicated a different result. Higher gene copy number of tetO was determined in one animal house (IAH-3), while the two other tet genes tetQ and tetW were found to be higher in arable lands. Of the total 110 bacterial isolates, tetW gene was frequently detected, while tetO gene was absent in any of the culturable bacterial isolates. Principal component analysis of occurrence and gene copy number of RPP tet genes tetO, tetQ, and tetW also revealed highest abundance of RPP tet genes in the manure and arable soils. Another important highlight of this study was the similarity of the RPP tet genes detected in the isolated bacteria from the agricultural soils to the identified RPP tet genes among pathogenic bacteria. Some of the tetracycline-resistant bacterial isolates were also multidrug resistant as it displayed resistance to tetracycline, erythromycin, and streptomycin using disk diffusion testing.

scholarly journals Drug resistance and plasmid profile of bacterial isolates from automated teller machine keypads in Benin city

2019 ◽  
Vol 54 (3) ◽  
pp. 231-240
Author(s):  
FI Akinnibosun ◽  
JA Adetitun
Keyword(s):  
Escherichia Coli ◽  
Drug Resistance ◽  
Antibiotic Resistance ◽  
Micrococcus Luteus ◽  
Plasmid Profile ◽  
Resistant Bacteria ◽  
Bacterial Isolates ◽  
Benin City ◽  
Automated Teller Machine ◽  
Mar Index

The purpose of this study was to investigate the drug resistance and plasmid profile of bacterial isolates from automated teller machine (ATM) keypads in Benin City, Nigeria. Samples were collected from the keypads of ATMs situated in banking premises and open access areas using stratified random sampling method at 5 different locations. Isolated bacteria were characterized by extensive phenotypic and biochemical identification procedures. Antibiotic susceptibility pattern and plasmid profile of the isolates were carried out using standard methods. The bacteria isolated were: Klebsiella sp., Pseudomonas aeruginosa, Proteus vulgaris, Escherichia coli, Micrococcus luteus, Bacillus subtilis, Staphylococcus aureus, Corynebacteria sp., Staphylococcus epidermidis and Streptococcus pyogenes. Each isolate exhibited marked resistance to the antibiotics, with multiple antibiotic resistance (MAR) index > 0.2. Escherichia coli showed the highest resistance (MAR index, 1) while Micrococcus luteus exhibited the least resistance (MAR index, 0.5) to the antibiotics used. Plasmid profile of the isolates revealed the presence of plasmids in the isolates, with a reduction in antibiotic resistance after plasmid curing in some isolates. This indicated plasmid-mediated resistance among majority of the isolates. From the results obtained, it is pertinent therefore, to adopt proper measures aimed at preventing outbreak of diseases associated with these multi-drug resistant bacteria isolated from ATM keypads. Bangladesh J. Sci. Ind. Res.54(3), 231-240, 2019

Transport of antibiotic-resistant bacteria and resistance-carrying plasmids through porous media

2006 ◽  
Vol 54 (11-12) ◽  
pp. 363-370 ◽  
Author(s):  
M. Rysz ◽  
P.J.J. Alvarez
Keyword(s):  
Porous Media ◽  
Antibiotic Resistance ◽  
Environmental Pollutants ◽  
Tetracycline Resistance ◽  
Surface Characteristics ◽  
Medical Problem ◽  
Size Exclusion ◽  
Resistant Bacteria ◽  
Electrostatic Repulsion ◽  
Silica Bead

Microbial antibiotic resistance has emerged as not only a major medical problem but also as an environmental engineering challenge, with antibiotic resistance genetic elements as environmental pollutants. The transport characteristics of a tetracycline-resistant bacterium (B. cepacia) and plasmids carrying tetracycline-resistance genes were investigated using flow-through columns packed with porous media. Higher influent cell concentrations (1.1×108 CFU mL−1) resulted in higher breakthrough (C/C0=0.596±0.055) than a solution with lower cell concentration (2.0×106 CFU mL−1, C/C0=0.461±0.037). This decreased extent of filtration suggests fast initial cell deposition and strong subsequent blocking of binding sites, resulting in less-hindered microbial transport through the sandy medium. The addition of a bromide tracer (NaBr) prior to the plasmid solution resulted in DNA retardation and increased filtration in a zirconia-silica bead matrix. Apparently, Na+ binding to the beads decreased electrostatic repulsion between the negatively charged DNA and zirconia-silica surface. In contrast, plasmid breakthrough preceded that of the tracer when the plasmids were added first, possibly due to size exclusion chromatography coupled with stronger electrostatic repulsion. This implies that efforts to characterize the dynamics of resistance vector propagation in aquifers should consider the effect of groundwater chemistry and the surface characteristics of the porous media on vector transport.

scholarly journals Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry

1999 ◽  
Vol 65 (1) ◽  
pp. 45-52 ◽  
Author(s):  
Dominique Marie ◽  
Corina P. D. Brussaard ◽  
Runar Thyrhaug ◽  
Gunnar Bratbak ◽  
Daniel Vaulot
Keyword(s):  
Flow Cytometry ◽  
Natural Waters ◽  
Sybr Green ◽  
Sybr Green I ◽  
Epifluorescence Microscopy ◽  
Transmission Electron ◽  
Marine Viruses ◽  
Highly Correlated ◽  
Duplicate Sample ◽  
Speed And Accuracy

ABSTRACT Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using thePhaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80°C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, andr = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.

scholarly journals Honeybees and Tetracycline Resistance

mBio ◽  
2013 ◽  
Vol 4 (1) ◽  
Author(s):  
Stuart B. Levy ◽  
Bonnie M. Marshall
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Multiple Drug Resistance ◽  
Single Agent ◽  
Antibiotic Resistance Genes ◽  
Tetracycline Resistance ◽  
Resistant Bacteria ◽  
Multiple Drug ◽  
Selective Pressures ◽  
Multiple Drugs

ABSTRACT Like animals and people, insects can serve as both collectors and disseminators of antibiotic resistance genes, as exquisitely demonstrated by a recent study (B. Tian, N. H. Fadhil, J. E. Powell, W. K. Kwong, and N. A. Moran, mBio 3[6]:e00377-12, doi:10.1128/mBio.00377-12, 2012). Notably, the relatively confined ecosystem of the honeybee gut demonstrates a large propensity for harboring a diverse set of tetracycline resistance genes that reveal the environmental burden resulting from the long-time selective pressures of tetracycline use in the honeybee industry. As in humans and animals, these genes have become established in the native, nonpathogenic flora of the insect gut, adding credence to the concept that commensal floras provide large reservoirs of resistance genes that can readily move into pathogenic species. The homology of these tetracycline resistance determinants with those found in tetracycline-resistant bacteria associated with animals and humans strongly suggests a dissemination of similar or identical genes through shared ecosystems. The emergence of linked coresistances (ampicillin and tetracycline) following single-antibiotic therapy mirrors reports from other studies, namely, that long-term, single-agent therapy will result in resistance to multiple drugs. These results contrast with the marked absence of diverse, single- and multiple-drug resistance genes in wild and domestic bees that are not subjected to such selective pressures. Prospective studies that simultaneously track both resistance genes and antibiotic residues will go far in resolving some of the nagging questions that cloud our understanding of antibiotic resistance dissemination.

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