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The Journal of Antimicrobial... Jan 2018To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae,...
Characterization of the Actinobacillus pleuropneumoniae SXT-related integrative and conjugative element ICEApl2 and analysis of the encoded FloR protein: hydrophobic residues in transmembrane domains contribute dynamically to florfenicol and chloramphenicol efflux.
OBJECTIVES
To characterize ICEApl2, an SXT-related integrative and conjugative element (ICE) found in a clinical isolate of the porcine pathogen Actinobacillus pleuropneumoniae, and analyse the functional nature of the encoded FloR.
METHODS
ICEApl2 was identified in the genome of A. pleuropneumoniae MIDG3553. Functional analysis was done using conjugal transfer experiments. MIDG3553 was tested for susceptibility to the antimicrobials for which resistance genes are present in ICEApl2. Lack of florfenicol/chloramphenicol resistance conferred by the encoded FloR protein was investigated by cloning and site-directed mutagenesis experiments in Escherichia coli.
RESULTS
ICEApl2 is 92660 bp and contains 89 genes. Comparative sequence analysis indicated that ICEApl2 is a member of the SXT/R391 ICE family. Conjugation experiments showed that, although ICEApl2 is capable of excision from the chromosome, it is not self-transmissible. ICEApl2 encodes the antimicrobial resistance genes floR, strAB, sul2 and dfrA1, and MIDG3553 is resistant to streptomycin, sulfisoxazole and trimethoprim, but not florfenicol or chloramphenicol. Cloning and site-directed mutagenesis of the floR gene revealed the importance of the nature of the hydrophobic amino acid residues at positions 160 and 228 in FloR for determining resistance to florfenicol and chloramphenicol.
CONCLUSIONS
Our results indicate that the nature of hydrophobic residues at positions 160 and 228 of FloR contribute dynamically to specific efflux of florfenicol and chloramphenicol, although some differences in resistance levels may depend on the bacterial host species. This is also, to our knowledge, the first description of an SXT/R391 ICE in A. pleuropneumoniae or any member of the Pasteurellaceae.
Topics: Actinobacillus pleuropneumoniae; Animals; Bacterial Proteins; Biological Transport; Chloramphenicol; Conjugation, Genetic; Drug Resistance, Multiple, Bacterial; Hydrophobic and Hydrophilic Interactions; Interspersed Repetitive Sequences; Microbial Sensitivity Tests; Pneumonia; Swine; Swine Diseases; Thiamphenicol
PubMed: 29029160
DOI: 10.1093/jac/dkx342 -
Frontiers in Microbiology 2020Antimicrobial resistance (AMR) is becoming a huge problem in countries all over the world, and new approaches to identifying strains resistant or susceptible to certain...
Antimicrobial resistance (AMR) is becoming a huge problem in countries all over the world, and new approaches to identifying strains resistant or susceptible to certain antibiotics are essential in fighting against antibiotic-resistant pathogens. Genotype-based machine learning methods showed great promise as a diagnostic tool, due to the increasing availability of genomic datasets and AST phenotypes. In this article, Support Vector Machine (SVM) and Set Covering Machine (SCM) models were used to learn and predict the resistance of the five drugs (Tetracycline, Ampicillin, Sulfisoxazole, Trimethoprim, and Enrofloxacin). The SVM model used the number of co-occurring k-mers between the genome of the isolates and the reference genes to learn and predict the phenotypes of the bacteria to a specific antimicrobial, while the SCM model uses a greedy approach to construct conjunction or disjunction of Boolean functions to find the most concise set of k-mers that allows for accurate prediction of the phenotype. Five-fold cross-validation was performed on the training set of the SVM and SCM model to select the best hyperparameter values to avoid model overfitting. The training accuracy (mean cross-validation score) and the testing accuracy of SVM and SCM models of five drugs were above 90% regardless of the resistant mechanism of which were acquired resistant or point mutation in the chromosome. The results of correlation between the phenotype and the model predictions of the five drugs indicated that both SVM and SCM models could significantly classify the resistant isolates from the sensitive isolates of the bacteria ( < 0.01), and would be used as potential tools in antimicrobial resistance surveillance and clinical diagnosis in veterinary medicine.
PubMed: 32117101
DOI: 10.3389/fmicb.2020.00048 -
Pharmacology Research & Perspectives Jun 2022Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias...
Compounds that induce 5-aminolevulinic acid [ALA] synthase-1 and/or cytochromes P-450 may induce acute porphyric attacks in patients with the acute hepatic porphyrias [AHPs]. Currently, there is no simple, robust model used to assess and predict the porphyrogenicity of drugs and chemicals. Our aim was to develop a fluorescence-based in vitro assay for this purpose. We studied four different hepatic cell culture models: HepG2 cells, LMH cells, 3D HepG2 organoids, and 3D organoids of primary liver cells from people without known disease [normal human controls]. We took advantage of the fluorescent properties of protoporphyrin IX [PP], the last intermediate of the heme biosynthesis pathway, performing fluorescence spectrometry to measure the intensity of fluorescence emitted by these cells treated with selected compounds of importance to patients with AHPs. Among the four cell culture models, the LMH cells produced the highest fluorescence readings, suggesting that these cells retain more robust heme biosynthesis enzymes or that the other cell models may have lost their inducibility of ALA synthase-1 [ALAS-1]. Allyl isopropyl acetamide [AIA], a known potent porphyrogen and inducer of ALAS-1, was used as a positive control to help predict porphyrogenicity for tested compounds. Among the tested compounds (acetaminophen, acetylsalicylic acid, β-estradiol, hydroxychloroquine sulfate, alpha-methyldopa, D (-) norgestrel, phenobarbital, phenytoin, sulfamethoxazole, sulfisoxazole, sodium valproate, and valsartan), concentrations greater than 0.314 mM for norgestrel, phenobarbital, phenytoin, and sodium valproate produced fluorescence readings higher than the reading produced by the positive AIA control. Porphyrin accumulation was also measured by HPLC to confirm the validity of the assay. We conclude that LMH cell cultures in multi-well plates are an inexpensive, robust, and simple system to predict the porphyrogenicity of existing or novel compounds that may exacerbate the AHPs.
Topics: Cell Culture Techniques; Heme; Hepatocytes; Humans; Liver; Norgestrel; Phenobarbital; Phenytoin; Porphobilinogen Synthase; Porphyrias, Hepatic; Valproic Acid
PubMed: 35445802
DOI: 10.1002/prp2.951 -
Journal of Dairy Science Apr 2020Klebsiella spp. are important opportunistic pathogens commonly defined as environmental clinical mastitis agents. Despite Klebsiella mastitis being clinically impairing...
Klebsiella spp. are important opportunistic pathogens commonly defined as environmental clinical mastitis agents. Despite Klebsiella mastitis being clinically impairing in cows and costly to the industry, only a few studies describe Klebsiella isolated from mastitis cases. The aim of this work was to characterize species of Klebsiella involved in clinical mastitis cases in Canada. Klebsiella isolated from clinical mastitis cases (n = 53) were identified to the species level using a biochemical test panel and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The rpoB gene sequence was used as the gold standard method and identified Klebsiella pneumoniae (n = 40), Klebsiella oxytoca (n = 9), Raoultella ornithinolytica (n = 2), and Raoultella planticola (n = 2). Raoultella, a genus closely related to Klebsiella, was also accurately identified using mass spectrometry but not via biochemical testing. Using the disc diffusion technique, 31 (58%) isolates were found to be susceptible to all antimicrobials tested (n = 18). The remaining 22 (42%) isolates were resistant to 1 or more of the following antimicrobials: kanamycin (2%), streptomycin (38%), spectinomycin (13%), sulfisoxazole (13%), and tetracycline (19%). The following antimicrobial resistance genes were identified: tetA, tetB, sul1, strA/strB, and aadA. Random amplified polymorphic DNA revealed the majority of our isolates as unrelated and having different patterns, indicating environmental contamination as the primary source of infection. All isolates were shown to be biofilm producers. In conclusion, although antimicrobial resistance was low for both Klebsiella and Raoultella species, genetically related Klebsiella spp. isolates appeared to be more resistant.
Topics: Animals; Anti-Bacterial Agents; Canada; Cattle; Drug Resistance, Bacterial; Female; Klebsiella; Mastitis, Bovine; Microbial Sensitivity Tests
PubMed: 32089315
DOI: 10.3168/jds.2019-17324 -
Veterinaria Italiana Dec 2021Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease...
Salmonellosis is currently the second most common zoonosis in European Union but in the 6-years periods, between 2012 and 2017, there has been a significant decrease trend in the yearly number of infections caused by Salmonella. In Italy, S. Typhimurium and monophasic S. Typhimurium represent the most prevalent serotypes. In this paper, we investigated these two serovars isolated from 2012 to 2017 in Abruzzo and Molise regions. A set of 345 strains isolated from human sporadic cases, surface water, food and animals were collected and analyzed. Monophasic S. Typhimurium strains were found to be resistant to streptomycin, sulfisoxazole, ampicillin, tetracycline and nalidixic acid, while S. Typhimurium isolates showed high levels of resistance to tetracycline, sulfisoxazole and ampicillin. The 5-loci Multilocus Variable-Number Tandem Repeat Analysis (MLVA) identified 88 genotypes (GT), six of which were common for the two serovars. Several MLVA profiles were shared by human and non-human isolates. MLVA had sufficient typing resolution for epidemiological studies of S. Typhimurium but demonstrated poor discriminatory in trace-back study of monophasic S. Typhimurium.
Topics: Ampicillin; Animals; Anti-Bacterial Agents; Italy; Salmonella typhimurium; Sulfisoxazole; Tetracyclines
PubMed: 35593494
DOI: 10.12834/VetIt.2060.12083.1 -
Frontiers in Microbiology 2018This study aimed to investigate the prevalence, serotype distribution, and antibiotic resistance, and to characterize the extended spectrum β-lactamases (ESBLs)...
This study aimed to investigate the prevalence, serotype distribution, and antibiotic resistance, and to characterize the extended spectrum β-lactamases (ESBLs) producing isolates from chicken and pork meats from retail markets in Guangdong province, China. A total of 903 retail meat samples (475 chicken and 428 pork meats) were obtained from six cities (Guangzhou, Shenzhen, Heyuan, Shaoguan, Foshan, and Yunfu) of Guangdong province between May 2016 and April 2017. High levels of contamination were detected in chicken (302/475, 63.6%) and pork (313/428, 73.1%). Thirty-eight serotypes were identified in 615 detected , and the serotypes varied greatly between chicken and pork samples. Agona (55/302, 18.2%), Corvallis (45/302, 14.9%), Kentucky (38/302, 12.6%), Mbandaka (32/302, 10.6%) was the dominant serotypes in chicken samples. However, Typhimurium (78/313, 24.9%), Rissen (67/313, 24.1%), Derby (66/313, 21.1%), and London (48, 15.3%) were the most common in pork samples. High rates of antibiotic resistance were found to sulfisoxazole (468/615, 76.1%), tetracycline (463/615, 75.3%), ampicillin (295/615, 48.0%), and ofloxacin (275/615, 44.7%). Notably, antimicrobial susceptibility tests identified resistance to polymyxin B (12/615, 2.0%) and imipenem (3/615, 0.5%). Multidrug-resistance (MDR) was detected in isolated from chicken (245/302, 81.1%) and pork (229/313, 73.2%). The resistance rate of different serotypes varied widely. Especially, isolates such as Typhimurium, Agona, orvallis and Kentucky exhibited highly resistance to antibiotics. The MDR rate of isolates from chicken was significantly higher than that from pork isolates ( < 0.05). Twenty-one isolates were identified as ESBLs-producing, covering six serotypes and displaying different pulse field gel electrophoresis (PFGE) genotypes. was the dominant ESBLs gene (9/21, 42.9%), followed by (5/21, 23.8%). This study indicated that was widespread in chicken and pork from retail markets in Guangdong province and the isolates showed high multidrug-resistance, especially the known multidrug-resistant serotypes. Therefore, it is important to focus on serotypes and strengthen the long-term monitoring of MDR serotypes in animal-derived foods.
PubMed: 30258419
DOI: 10.3389/fmicb.2018.02104 -
Poultry Science Jul 2021The aim of this study was to determine the prevalence, serovar distribution, antimicrobial resistance, and genotypic analyses of the dominating serovars of Salmonella in...
Clonal dissemination of Salmonella enterica serovar albany with concurrent resistance to ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, and nalidixic acid in broiler chicken in Korea.
The aim of this study was to determine the prevalence, serovar distribution, antimicrobial resistance, and genotypic analyses of the dominating serovars of Salmonella in chickens from a national study in Korea. Between 2017 and 2018, a total of 550 chicken samples were collected from the top 12 integrated broiler chicken operations in Korea. Salmonella was isolated from 117 (32.5%) chicken feces and 19 (10.0%) retail chicken meat sources. Ten serovars were identified, and the most common Salmonella serovar was Salmonella ser. Albany (50 isolates, 36.8%), followed by S. Enteritidis (38 isolates, 27.9%), and S. Montevideo (23 isolates, 16.9%) isolated from 6, 10, and 6 operations, respectively. A total of 35 (25.7%) isolates were with the ACSSuTN (ampicillin, chloramphenicol, streptomycin, sulfisoxazole, tetracycline, and nalidixic acid) resistance pattern, with high prevalence of this resistance pattern in S. Albany (29 isolates, 58.0%). A total of 35 PFGE types were identified among Salmonella isolates of the serovars Albany, Enteritidis, Virchow, Montevideo, and Senftenberg, while 11 distinct types of PFGE patterns were found among S. Albany isolates, which showed an overall homology similarity of higher than 85%. Among these 35 PFGE types, 22 PFGE types corresponded to 32 isolates from samples limited to one operation, and the other 13 PFGE types corresponded to 72 isolates from samples widely distributed among different operations. These results highlighted rapid colony dissemination of multidrug-resistant S. Albany in chicken all over Korea after it first appeared in 2016; furthermore, the spread of Salmonella colonies between various integrated operations was common, and several operations played an important role in Salmonella carriage and transmission in Korea.
Topics: Ampicillin; Animals; Anti-Bacterial Agents; Chickens; Chloramphenicol; Drug Resistance, Multiple, Bacterial; Microbial Sensitivity Tests; Nalidixic Acid; Republic of Korea; Salmonella; Salmonella enterica; Serogroup; Streptomycin; Sulfisoxazole; Tetracycline
PubMed: 34089935
DOI: 10.1016/j.psj.2021.101141 -
Drug Metabolism and Disposition: the... Aug 2008We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those...
We have investigated several in silico and in vitro methods to improve our ability to predict potential drug interactions of antibiotics. Our focus was to identify those antibiotics that activate pregnane X receptor (PXR) and induce CYP3A4 in human hepatocytes and intestinal cells. Human PXR activation was screened using reporter assays in HepG2 cells, kinetic measurements of PXR activation were made in DPX-2 cells, and induction of CYP3A4 expression and activity was verified by quantitative polymerase chain reaction, immunoblotting, and testosterone 6beta-hydroxylation in primary human hepatocytes and LS180 cells. We found that in HepG2 cells CYP3A4 transcription was activated strongly (> 10-fold) by rifampin and troleandomycin; moderately (> or = 7-fold) by dicloxacillin, tetracycline, clindamycin, griseofulvin, and (> or = 4-fold) erythromycin; and weakly (> 2.4-fold) by nafcillin, cefaclor, sulfisoxazole, and (> 2-fold) cefadroxil and penicillin V. Similar although not identical results were obtained in DPX-2 cells. CYP3A4 mRNA and protein expression were induced by these antibiotics to differing extents in both liver and intestinal cells. CYP3A4 activity was significantly increased by rifampin (9.7-fold), nafcillin and dicloxacillin (5.9-fold), and weakly induced (2-fold) by tetracycline, sufisoxazole, troleandomycin, and clindamycin. Multiple pharmacophore models and docking indicated a good fit for dicloxacillin and nafcillin in PXR. These results suggest that in vitro and in silico methods can help to prioritize and identify antibiotics that are most likely to reduce exposures of medications (such as oral contraceptive agents) which interact with enzymes and transporters regulated by PXR. In summary, nafcillin, dicloxacillin, cephradine, tetracycline, sulfixoxazole, erythromycin, clindamycin, and griseofulvin exhibit a clear propensity to induce CYP3A4 and warrant further clinical investigation.
Topics: Anti-Bacterial Agents; Base Sequence; Cell Line; Cytochrome P-450 CYP3A; DNA Primers; Enzyme Induction; Genes, Reporter; Humans; In Vitro Techniques; Intestines; Liver; Pregnane X Receptor; RNA, Messenger; Receptors, Steroid
PubMed: 18505790
DOI: 10.1124/dmd.108.020701 -
Biology Jul 2021Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment...
Clinical management of cancer-associated cachexia, a multi-organ wasting syndrome, has been challenging without effective treatment strategies. An effective treatment that directly targets cancer-induced wasting is desperately needed to improve the quality of life and the survival of cancer patients. Recently, an antibiotic SFX was shown to have anti-tumour and anti-metastatic effects in mouse models of breast cancer. Hence, in this study, we examined the efficacy of SFX in the treatment of cancer-induced cachexia. C26 cachexic mice models were administered with SFX, and the tumour volume and body weight were regularly measured. Blood glucose, skeletal muscles, and adipose tissue were examined at the endpoint. Contrary to a previous study, SFX did not reduce the tumour volume in mice bearing C26 cells. Administration of SFX neither revealed any survival benefit nor rescued C26 cachectic mice from muscle wasting. Interestingly, SFX administration partially rescued (~10%) tumour-induced weight loss by preserving both the subcutaneous and intestinal fat mass. Together, these results suggest that the administration of SFX could partially rescue cancer-induced weight loss by inhibiting lipolysis. As anti-cachexia therapies are scarce, the results could facilitate the design of combinatorial therapies involving SFX, standard-of-care chemotherapeutics, and drugs that inhibit muscle atrophy for the treatment of cancer cachexia.
PubMed: 34439933
DOI: 10.3390/biology10080700 -
Nature Communications Mar 2019Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer...
Inhibitors of the secretion of cancer exosomes, which promote cancer progression and metastasis, may not only accelerate exosome biology research but also offer therapeutic benefits for cancer patients. Here we identify sulfisoxazole (SFX) as an inhibitor of small extracellular vesicles (sEV) secretion from breast cancer cells through interference with endothelin receptor A (ETA). SFX, an FDA-approved oral antibiotic, showed significant anti-tumor and anti-metastatic effects in mouse models of breast cancer xenografts, the reduced expression of proteins involved in biogenesis and secretion of sEV, and triggered co-localization of multivesicular endosomes with lysosomes for degradation. We demonstrate the important role of ETA, as target of SFX, by gain- and loss-of-function studies of the ETA protein, through a direct binding assay, and pharmacological and genetic approaches. These findings may provide a foundation for sEV-targeted cancer therapies and the mechanistic studies on sEV biology.
Topics: Animals; Anti-Infective Agents; Breast Neoplasms; Cell Line, Tumor; Endosomes; Extracellular Vesicles; Female; Humans; Lysosomes; MCF-7 Cells; Male; Mice; Mice, Nude; Neoplasm Metastasis; Organelle Biogenesis; Receptor, Endothelin A; Sulfisoxazole; Xenograft Model Antitumor Assays
PubMed: 30918259
DOI: 10.1038/s41467-019-09387-4