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Martine Boulianne - One of the best experts on this subject based on the ideXlab platform.
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effect of cessation of ceftiofur and substitution with lincomycin Spectinomycin on extended spectrum β lactamase ampc genes and multidrug resistance in escherichia coli from a canadian broiler production pyramid
Applied and Environmental Microbiology, 2019Co-Authors: Luc Verrette, John M Fairbrother, Martine BoulianneAbstract:ABSTRACT Ceftiofur, a third-generation cephalosporin antimicrobial, was used in Canadian hatcheries for many years to prevent early mortality in chicks, leading to a high prevalence of cephalosporin resistance in Escherichia coli in chickens. Preventive use of ceftiofur in hatcheries ceased in 2014. We examined the effect of ceftiofur cessation ( n = 40 flocks with ceftiofur and n = 28 flocks without antimicrobial at hatchery) and its replacement with an antimicrobial combination, lincomycin-Spectinomycin ( n = 32), at the hatchery on the proportion of samples with E. coli positive for extended-spectrum-β-lactamase (ESBL) and AmpC β-lactamase-related genes, and on the multidrug resistance profiles of ESBL/AmpC-positive E. coli in broilers and their associated breeders ( n = 46 samples), at 1 year postcessation. For indicator E. coli from nonenriched media, a significant decrease postcessation in the proportion of samples harboring E. coli isolates positive for bla CMY-2 and/or bla CTX-M was observed. In contrast, following enrichment in medium containing ceftriaxone (1 mg/liter) to facilitate recovery of ESBL/AmpC β-lactamase-producing E. coli colonies, both pre- and postcessation, 99% of the samples harbored E. coli positive for bla CMY-2 or bla CTX-M . Among the 15 tested antimicrobial agents, flocks receiving lincomycin-Spectinomycin after cessation of ceftiofur showed a significantly greater nonsusceptibility to aminoglycosides, folate inhibitors, phenicols, and tetracyclines and a greater proportion of possible extensively drug-resistant E. coli than those receiving ceftiofur or no antimicrobial at hatchery. This study clearly demonstrates an initial decrease in ESBL/AmpC-positive E. coli following the cessation of ceftiofur in the hatchery but an increase in antimicrobial non-β-lactam resistance of ESBL/AmpC-positive E. coli following replacement with lincomycin-Spectinomycin. IMPORTANCE Antimicrobial resistance is a global problem. The antimicrobial ceftiofur has been used worldwide for disease prevention in poultry production, resulting in a greatly increased resistance to this antimicrobial important in poultry and human medicine. Our study examined the impact of ceftiofur cessation and its replacement with the antimicrobial combination lincomycin-Spectinomycin, a common practice in the industry. Our study demonstrated a decrease in ceftiofur resistance after the cessation of ceftiofur use, although the resistance genes remain ubiquitous in all phases of poultry production, showing that poultry remains a reservoir for ceftiofur resistance and requiring continued vigilance. We also observed a decrease in multidrug resistance involving different antimicrobial classes after cessation of ceftiofur but an increase following use of lincomycin-Spectinomycin, indicating that this antimicrobial use should be questioned. Reduced resistance to ceftiofur in poultry may translate to better treatment efficacy, decreased morbidity/mortality, and enhanced food safety for humans.
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effect of ceftiofur cessation and substitution with lincomycin Spectinomycin on extended spectrum beta lactamase ampc genes and multidrug resistance in e coli from a canadian broiler production pyramid
bioRxiv, 2019Co-Authors: Luc Verrette, John M Fairbrother, Martine BoulianneAbstract:Abstract Ceftiofur, a cephalosporin antimicrobial, was used systematically in Canadian hatcheries for many years to prevent early mortality in chicks leading to a high prevalence of cephalosporin resistance in Escherichia coli in chickens. Preventive use of ceftiofur in hatcheries ceased in 2014. We examined the effect of ceftiofur cessation and replacement with lincomycin-Spectinomycin at the hatchery on the proportion of E. coli positive for extended-spectrum β-lactamase (ESBL) and AmpC β-lactamase related genes, and on the multidrug resistance profiles of ESBL/AmpC positive E. coli in broilers and their associated breeders, at one year post-cessation. For indicator E. coli from non-enriched media, a significant decrease post-cessation in the proportion of samples harboring E. coli isolates positive for blaCMY-2 and/or blaCTX-M was observed. In contrast, following enrichment in medium containing ceftriaxone (1mg/L) to facilitate recovery of ESBL/AmpC β-lactamase producing E. coli colonies, both pre- and post-cessation, 99% of the samples harbored E. coli positive for blaCMY-2 or blaCTX-M. Flocks receiving lincomycin-Spectinomycin after cessation of ceftiofur showed a significantly greater non-susceptibility to aminoglycoside, folate inhibitor, phenicol, tetracycline and possible extensively drug resistant E. coli compared to those receiving ceftiofur or no antimicrobial at hatchery. This study clearly demonstrates an initial decrease in ESBL/AmpC positive E. coli following the cessation of ceftiofur in hatchery but an increase in multidrug resistant E. coli following replacement with lincomycin-Spectinomycin. Importance Antimicrobial resistance is a global problem. The antimicrobial ceftiofur has been used worldwide for disease prevention in poultry production resulting in a greatly increased resistance to this antimicrobial important in poultry and human medicine. Our study examines the impact of ceftiofur cessation and its replacement with the antimicrobial combination lincomycin-Spectinomycin, a common practice in the industry. Our study demonstrated a decrease in ceftiofur resistance after the cessation of its use, although the resistance genes remain ubiquitous in all phases of poultry production, showing that poultry remains a reservoir for ceftiofur resistance and requiring continued vigilance. We also observed a decrease in multidrug resistance after cessation of ceftiofur although the contrary finding following use of lincomycin-Spectinomycin indicates that the use of these antimicrobials should be questioned. Reduced resistance to ceftiofur in poultry may translate to better treatment efficacy, decreased morbidity, mortality, duration and cost of hospitalization in humans.
Jeffrey L. Watts - One of the best experts on this subject based on the ideXlab platform.
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in vitro activities of Spectinomycin and comparator agents against pasteurella multocida and mannheimia haemolytica from respiratory tract infections of cattle
Journal of Antimicrobial Chemotherapy, 2004Co-Authors: Stefan Schwarz, Corinna Kehrenberg, Sarah A Salmon, Jeffrey L. WattsAbstract:OBJECTIVES: Prior to the renewal of Spectinomycin licensing for veterinary uses in Germany, 154 Pasteurella multocida and 148 Mannheimia haemolytica strains from respiratory tract infections in cattle were investigated for their MICs of Spectinomycin and other antimicrobial agents. The data obtained should serve as a baseline from which to judge the future development of resistance. Moreover, the in vitro activity of Spectinomycin in comparison with other antimicrobials should be assessed. METHODS: MIC determination for all 302 strains was performed by the broth dilution method and evaluated according to NCCLS standards. MIC(50) and MIC(90) values were calculated. Strains resistant to Spectinomycin were subjected to PCR assays for genes known to mediate Spectinomycin resistance in Gram-negative and Gram-positive bacteria. RESULTS: With the exception of resistance to sulfamethoxazole in P. multocida and M. haemolytica, and resistance to ampicillin in M. haemolytica, an overall low level of resistance was detected. A total of 93.5% of the P. multocida and 98.6% of the M. haemolytica strains were susceptible to Spectinomycin, with MIC(90)s of 32 mg/L. PCR analysis showed that none of the Spectinomycin-resistant strains carried any of the aadA gene subtypes, nor the genes spc or aad(9). CONCLUSIONS: Prior to the renewal of Spectinomycin, only a small number of Spectinomycin-resistant strains was detected among bovine P. multocida and M. haemolytica. The genes responsible for Spectinomycin resistance in these strains seemed to be different from those so far known to occur in other Gram-negative and Gram-positive bacteria.
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Antimicrobial susceptibility of Staphylococcus hyicus isolated from exudative epidermitis in pigs.
Journal of clinical microbiology, 1994Co-Authors: Henrik Caspar Wegener, Jeffrey L. Watts, S. A. Salmon, R.j. YanceyAbstract:Exudative epidermitis or greasy pig syndrome is caused by the coagulase-variable staphylococcal species Staphylococcus hyicus. Treatment of this disease is problematic because of the limited number of antimicrobial agents available for this purpose. Thirteen antimicrobial agents were evaluated for their activities against 100 S. hyicus strains isolated from pigs with exudative epidermitis. Novobiocin was the most active compound tested, with an MIC for 90% of the strains tested (MIC90) of 32.0 micrograms/ml. Initial testing with sulfadiazine-trimethoprim yielded an MIC90 of > 64.0 micrograms/ml, but subsequent testing with thymidine phosphorylase-supplemented medium yielded an MIC90 of 0.06 microgram/ml. Both lincomycin and Spectinomycin were relatively inactive against the S. hyicus strains tested, with MIC90s of > 64.0 and > 128.0 micrograms/ml, respectively. However, the combination of the two compounds at ratios of 1:2 (lincomycin to Spectinomycin) and 1:8 were more active, with MIC90s of 16.0 and 4.0 micrograms/ml, respectively. These results indicate that novobiocin and sulfadiazine-trimethoprim were the most active compounds tested against the S. hyicus strains isolated from pigs with exudative epidermitis. Furthermore, the combination of lincomycin and Spectinomycin was more active than the individual compounds against the strains tested.
Niko S Radulovic - One of the best experts on this subject based on the ideXlab platform.
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development and validation of liquid chromatography tandem mass spectrometry methods for the determination of gentamicin lincomycin and Spectinomycin in the presence of their impurities in pharmaceutical formulations
Journal of Pharmaceutical and Biomedical Analysis, 2011Co-Authors: K Vucicevicprcetic, R Cservenak, Niko S RadulovicAbstract:Liquid chromatography with tandem mass spectrometry (LC/MS/MS) methods for the determination of gentamicin, lincomycin and Spectinomycin in the presence of their impurities were developed and tested. Chromatographic separations were achieved using gradient elution on a C18 column. All components were ionized by positive-ion electrospray and detected by multi reaction monitoring (MRM) with an LC-tandem mass spectrometer. Calibration curves were linear with correlation coefficients better than 0.99. The developed method for the determination of gentamicin provides complete base line separation of components C1, C1a, C2, C2a and C2b mentioned in the European and British Pharmacopoeias. The second developed method makes possible a simultaneous analysis of the active compounds of both lincomycin and Spectinomycin. Additionally, all impurities defined in the pharmacopoeias for all three active components were determined and their identities confirmed. The methods were tested in routine quality control analysis.
Stefan Schwarz - One of the best experts on this subject based on the ideXlab platform.
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identification of the novel Spectinomycin resistance gene spw in methicillin resistant and methicillin susceptible staphylococcus aureus of human and animal origin
Journal of Antimicrobial Chemotherapy, 2013Co-Authors: Sarah Wendlandt, Carmen Lozano, Carmen Torres, Stefan SchwarzAbstract:Sir, Spectinomycin resistance in staphylococci is mostly mediated by Spectinomycin 9-O-adenyltransferase encoded by the spc gene. This gene is located together with the macrolide–lincosamide– streptogramin B resistance gene erm(A) on transposon Tn554. The analysis of methicillin-resistant Staphylococcus aureus (MRSA) CC398 from cases of bovine mastitis showed that all Spectinomycin-resistant isolates carried the gene spc along with erm(A), whereas MRSA CC398 isolates from turkey meat and turkey meat products occasionally exhibited Spectinomycin resistance, but lacked the spc gene. This observation pointed towards the existence of other as yet unknown Spectinomycin resistance genes in staphylococci. Most recently, the analysis of MRSA ST398 and methicillin-susceptible S. aureus (MSSA) ST9 isolates of human origin from Spain, but also MRSA ST9 isolates from swine in China, revealed the presence of multiresistance gene clusters that were most likely of enterococcal origin. – 7 So far, two different types of these multiresistance gene clusters have been identified, both of which carry the resistance genes aadE (streptomycin resistance), lnu(B) (lincosamide resistance) and lsa(E) (pleuromutilin– lincosamide–streptogramin A resistance). A closer look at these clusters revealed the presence of a reading frame for a putative Spectinomycin resistance gene between aadE and lsa(E). This reading frame is 810 bp in size, has an unusual start codon (TTG) and codes for an adenyltransferase protein of 269 amino acids. Database searches identified identical or closely related proteins in Enterococcus faecium (e.g. GenBank accession number EFF20873), Enterococcus faecalis (e.g. GenBank accession number EEU82264) and Lactobacillus johnsonii (e.g. GenBank accession number EGP12870) (Figure 1). The corresponding proteins were referred to as streptomycin 3′′-adenylyltransferase, Spectinomycin 9-O-adenylyltransferase, putative toxin-antitoxin system or a hypothetical protein in the respective database entries. Moreover, three database entries (GenBank accession numbers AAL05551, AFM38045 and AFU35063) reported a protein that was 27 amino acids shorter. This was due to the annotation of a wrong start codon (ATG) upstream of which no ribosome binding site was found. Analysis of the corresponding nucleotide sequences (GenBank accession numbers AF408195, JQ861959 and JX560992, respectively) confirmed the presence of the larger correct reading frame of 810 bp. To determine whether or not this protein confers resistance to streptomycin or Spectinomycin, we decided to clone the gene and express it in an S. aureus host. For this, the recently developed PCR5 assay (forward: 5′-TTGGATTGCAGCATTATTGG-3′, reverse: 5′-ATTTGGTCGAAGCCTT GTTG-3′; annealing temperature 568C) was used to generate an amplicon of 1985 bp from the cloned 17.5 kb segment of plasmid pV7037 (GenBank accession number JX560992) from the porcine MRSA ST9 isolate SA7037. This amplicon included the entire reading frame for the putative resistance gene and 239 bp in the upstream and 936 bp in the downstream parts. The amplicon was initially cloned into pCR-Blunt II-TOPO (Life Technologies GmbH, Darmstadt, Germany), and the recombinant vector was transformed into Escherichia coli recipient strain TOP10. The amplicon was then cut off from this vector by EcoRI digestion and inserted into the single EcoRI site of the E. coli–S. aureus shuttle vector pLI50. This recombinant shuttle vector was first introduced by electrotransformation into E. coli TOP10 and subsequently into S. aureus RN4220. Antimicrobial susceptibility testing was performed by broth macrodilution for Spectinomycin and streptomycin according to CLSI document M31-A3 with S. aureus ATCC 29213 as the quality control strain. The MICs of streptomycin were 4 mg/L for S. aureus RN4220, S. aureus RN4220 carrying pLI50 and S. aureus RN4220 carrying the recombinant pLI50, suggesting that the cloned gene does not confer streptomycin resistance. In contrast, the MICs of Spectinomycin were 32 mg/L for S. aureus RN4220 and S. aureus RN4220 carrying pLI50, but were distinctly higher at 4096 mg/L for S. aureus RN4220 carrying the recombinant pLI50 with the cloned gene. This observation confirmed that the gene in question was a Spectinomycin resistance gene, which was then designated spw. Besides the occurrence of spw in E. faecium, E. faecalis and L. johnsonii, a gene was detected in Streptococcus suis that encoded a protein of 262 amino acids (GenBank accession number AER19630) that showed 93.9% amino acid identity to Spw. In contrast, the Spw protein exhibited only 64.7% identity to the Tn554-associated Spc protein (Figure 1). To see whether spw was present in other staphylococci, a PCR assay (forward: 5′-CGGCAGTAATGGGTGGTTTA-3′, reverse: 5′-CAGCCACC TCAGATTCCATT-3′; annealing temperature 548C) was developed
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in vitro activities of Spectinomycin and comparator agents against pasteurella multocida and mannheimia haemolytica from respiratory tract infections of cattle
Journal of Antimicrobial Chemotherapy, 2004Co-Authors: Stefan Schwarz, Corinna Kehrenberg, Sarah A Salmon, Jeffrey L. WattsAbstract:OBJECTIVES: Prior to the renewal of Spectinomycin licensing for veterinary uses in Germany, 154 Pasteurella multocida and 148 Mannheimia haemolytica strains from respiratory tract infections in cattle were investigated for their MICs of Spectinomycin and other antimicrobial agents. The data obtained should serve as a baseline from which to judge the future development of resistance. Moreover, the in vitro activity of Spectinomycin in comparison with other antimicrobials should be assessed. METHODS: MIC determination for all 302 strains was performed by the broth dilution method and evaluated according to NCCLS standards. MIC(50) and MIC(90) values were calculated. Strains resistant to Spectinomycin were subjected to PCR assays for genes known to mediate Spectinomycin resistance in Gram-negative and Gram-positive bacteria. RESULTS: With the exception of resistance to sulfamethoxazole in P. multocida and M. haemolytica, and resistance to ampicillin in M. haemolytica, an overall low level of resistance was detected. A total of 93.5% of the P. multocida and 98.6% of the M. haemolytica strains were susceptible to Spectinomycin, with MIC(90)s of 32 mg/L. PCR analysis showed that none of the Spectinomycin-resistant strains carried any of the aadA gene subtypes, nor the genes spc or aad(9). CONCLUSIONS: Prior to the renewal of Spectinomycin, only a small number of Spectinomycin-resistant strains was detected among bovine P. multocida and M. haemolytica. The genes responsible for Spectinomycin resistance in these strains seemed to be different from those so far known to occur in other Gram-negative and Gram-positive bacteria.
Pal Maliga - One of the best experts on this subject based on the ideXlab platform.
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Spectinomycin resistance mutations in the rrn16 gene are new plastid markers in medicago sativa
Theoretical and Applied Genetics, 2012Co-Authors: Brigitta Dudas, Barnabas Jenes, Gyorgy B Kiss, Pal MaligaAbstract:We report here the isolation of Spectinomycin-resistant mutants in cultured cells of Medicago sativa line RegenSY-T2. Spectinomycin induces bleaching of cultured alfalfa cells due to inhibition of protein synthesis on the prokaryotic type 70S plastid ribosomes. Spontaneous mutants resistant to Spectinomycin bleaching were identified by their ability to form green shoots on plant regeneration medium containing selective Spectinomycin concentrations in the range of 25–50 mg/l. Sequencing of the plastid rrn16 gene revealed that Spectinomycin resistance is due to mutations in a conserved stem structure of the 16S rRNA. Resistant plants transferred to the greenhouse developed normally and produced Spectinomycin-resistant seed progeny. In light of their absence in soybean, a related leguminous plant, the isolation of Spectinomycin-resistant mutants in M. sativa was unexpected. The new mutations are useful for the study of plastid inheritance, as demonstrated by detection of predominantly paternal plastid inheritance in the RegenSY-T2 × Szapko57 cross, and can be used as selective markers in plastid transformation vectors to obtain cisgenic plants.
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a guide to choosing vectors for transformation of the plastid genome of higher plants
Plant Physiology, 2007Co-Authors: Kerry Lutz, Arun K Azhagiri, Tarinee Tungsuchathuang, Pal MaligaAbstract:Plastid transformation, originally developed in tobacco (Nicotiana tabacum), has recently been extended to a number of crop species enabling in vivo probing of plastid function and biotechnological applications. In this article we report new plastid vectors that enable insertion of transgenes in the inverted repeat region of the plastome between the trnV and 3′rps12 or trnI and trnA genes. Efficient recovery of transplastomic clones is ensured by selection for Spectinomycin (aadA) or kanamycin (neo) resistance genes. Expression of marker genes can be verified using commercial antibodies that detect the accumulation of neomycin phosphotranseferase II, the neo gene product, or the C-terminal c-myc tag of aminoglycoside-3″-adenylytransferase, encoded by the aadA gene. Aminoglycoside-3″-adenylytransferase, the Spectinomycin inactivating enzyme, is translationally fused with green fluorescent protein in two vectors so that transplastomic clones can be selected by Spectinomycin resistance and visually identified by fluorescence in ultraviolet light. The marker genes in the new vectors are flanked by target sites for Cre or Int, the P1 and phiC31 phage site-specific recombinases. When uniform transformation of all plastid genomes is obtained, the marker genes can be excised by Cre or Int expressed from a nuclear gene. Choice of expression signals for the gene of interest, complications caused by the presence of plastid DNA sequences recognized by Cre, and loss of transgenes by homologous recombination via duplicated sequences are also discussed to facilitate a rational choice from among the existing vectors and to aid with new target-specific vector designs.
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high frequency plastid transformation in tobacco by selection for a chimeric aada gene
Proceedings of the National Academy of Sciences of the United States of America, 1993Co-Authors: Zora Svab, Pal MaligaAbstract:We report here a 100-fold increased frequency of plastid transformation in tobacco by selection for a chimeric aadA gene encoding aminoglycoside 3"-adenylyltransferase, as compared with that obtained with mutant 16S rRNA genes. Expression of aadA confers resistance to Spectinomycin and streptomycin. In transforming plasmid pZS197, a chimeric aadA is cloned between rbcL and open reading frame ORF512 plastid gene sequences. Selection was for Spectinomycin resistance after biolistic delivery of pZS197 DNA into leaf cells. DNA gel-blot analysis confirmed incorporation of the chimeric aadA gene into the plastid genome by two homologous recombination events via the flanking plastid gene sequences. The chimeric gene became homoplasmic in the recipient cells and is uniformly transmitted to the maternal seed progeny. The ability to transform routinely plastids of land plants opens the way to manipulate the process of photosynthesis and to incorporate novel genes into the plastid genome of crops.
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mutation proximal to the trna binding region of the nicotiana plastid 16s rrna confers resistance to Spectinomycin
Molecular Genetics and Genomics, 1991Co-Authors: Zora Svab, Pal MaligaAbstract:Nicotiana tabacum lines carrying maternally inherited resistance to Spectinomycin were obtained by selection for green callus in cultures bleached by Spectinomycin. Two levels of resistance was found. SPC1 and SPC2 seedlings are resistant to high levels (500 μg/ml), SPC23 seedlings are resistant to low levels (50 μg/ml) of Spectinomycin. Lines SPC2 and SPC23 are derivatives of the SR1 streptomycin-resistant plastome mutant. Spectinomycin resistance is due to mutations in the plastid 16S ribosomal RNA: SPC1, an A to C change at position 1138; SPC2, a C to U change at position 1139; SPC23, a G to A change at position 1333. Mutations similar to those in the SPC1 and SPC2 lines have been previously described, and disrupt a conserved 16S ribosomal RNA stem structure. The mutation in the SPC23 line is the first reported case of a mutation close to the region of the 16S rRNA involved in the formation of the initiation complex. The new mutants provide markers for selecting plastid transformants.
