Confirmation of oxytetracycline, tetracycline and chlortetracycline residues in milk by particle beam liquid chromatography/mass spectrometry.

A method using particle beam liquid chromatography/mass spectrometry was developed for the confirmation of oxytetracycline, tetracycline and chlortetracycline residues in bovine milk. This method is one of the first to apply particle beam technology to the confirmation of animal drug residues in food products for regulatory purposes. The milk is centrifuged, using molecular weight cut-off filters to remove components of 25,000 daltons and above from the milk. The filtrate is passed through a C-18 sample preparation cartridge which retains the tetracyclines. After the columns are washed with water, the tetracyclines are eluted with 0.1 M oxalic acid in methanol and concentrated. The compounds are separated on a Novapak C-18 column with a methanol-oxalic acid-acetonitrile mobile phase. Negative chemical ionization with selective ion monitoring is used to identify the tetracyclines. The procedure was used to confirm the presence of each tetracycline at 100 ng ml-1 in fortified and incurred milk samples.     

Determination of flumequine and doxycycline in milk by a simple thin-layer chromatographic method

Tetracycline and quinolone antibiotics have for many years served as important classes of veterinary drugs. Two representatives of both classes: doxycycline from tetracyclines and flumequine from quinolones are often administered together. When the withdrawal periods are not obeyed, the antibiotic residues may be present in edible products, e.g., in meat, eggs or milk. In the present paper a simple thin-layer chromatography (TLC) screening method is established for determining these drugs in milk. Only two developments of the plate with concentrating zone are needed: one as a clean-up procedure, the other as a proper analysis. The spots were detected both by UV lamp with dual wavelength (254 and 366 nm) and by densitometry.     

Accumulation of Tetracyclines by Escherichia coli

The net accumulation of tetracyclines by Escherichia coli as a function of concentration was shown to be biphasic. At concentrations less than the bacteriostatic levels, the mode of uptake was not azide-sensitive and was considered to be physical adsorption on the cell surface. At concentrations above the minimal inhibitory level, a second, azide-sensitive, uptake component was functional in addition to the surface adsorption process. This second energy-requiring mode was judged to represent penetration of the cytoplasmic membrane by tetracycline molecules to their sites of inhibitory action. Each mode for a given tetracycline and culture is expressed algebraically by a characteristic Freundlich equation. Resistance in E. coli is shown to be a result of diminished transport of antibiotic. However, this resistance was due not to a reduction or loss of a transport mechanism but rather to a requirement for higher antibiotic concentrations before the second mode of uptake could become operative.     

Action of tetracyclines on the pseudotuberculosis microbe in vitro experiments and their effectiveness in experimental pseudotuberculosis in white mice]

227 out of 228 strains of pseudotuberculosis microbes studied in vitro proved to be sensitive to tetracyclines. The MIC of tetracycline and morphocycline ranged within 0.25--25 gamma/ml. The MIC of chlortetracycline and oxytetracycline was somewhat lower, i. e. 1--50 gamma/ml. When administered intramuscularly all the tetracyclines had pronounced therapeutic effect in experiments with albino mice infected with the antibiotic sensitive strains of J. pseudotuberculosis. Chlortetracycline proved to be the most active drug in treatment of albino mice per os.     

Genetic determinants of antibiotic resistance in gram negative bacteria

Modern data on spreading, structural and functional organization and evolution of the genetic determinants of antibiotic resistance in the gram-negative bacteria are reviewed. Some mechanisms for resistance to trimethoprime, sulphonamides, tetracyclines, chloramphenicol aminoglycosides, beta-lactam antibiotics controlled by the plasmid and chromosomal genes are presented. The problem of using the molecular DNA-probes containing the genetical determinants for antibiotic resistance in the practical work of clinical laboratories is discussed.     

污水处理厂中四环素和磺胺类抗生素抗性基因的分布、传播及去除

近年来,抗生素滥用造成的抗性基因(ARGs)污染问题引起了人们的关注.四环素及磺胺类抗生素由于价格低廉被广泛使用,大量残留的四环素和磺胺通过各种途径进入污水处理厂,并进一步导致ARGs的污染.为深入了解四环素和磺胺类ARGs的污染及治理现状,本研究对污水处理厂中四环素和磺胺类ARGs的分布情况及传播机制进行了综述,并重点讨论了不同污水处理工艺对ARGs的去除效果.在此基础上,从加大污水处理厂ARGs污染调查、改进污水处理工艺以及探讨ARGs传播机制等方面进行了展望.     

Improved annotation of antibiotic resistance determinants reveals microbial resistomes cluster by ecology

Antibiotic resistance is a dire clinical problem with important ecological dimensions. While antibiotic resistance in human pathogens continues to rise at alarming rates, the impact of environmental resistance on human health is still unclear. To investigate the relationship between human-associated and environmental resistomes, we analyzed functional metagenomic selections for resistance against 18 clinically relevant antibiotics from soil and human gut microbiota as well as a set of multidrug-resistant cultured soil isolates. These analyses were enabled by Resfams, a new curated database of protein families and associated highly precise and accurate profile hidden Markov models, confirmed for antibiotic resistance function and organized by ontology. We demonstrate that the antibiotic resistance functions that give rise to the resistance profiles observed in environmental and human-associated microbial communities significantly differ between ecologies. Antibiotic resistance functions that most discriminate between ecologies provide resistance to β-lactams and tetracyclines, two of the most widely used classes of antibiotics in the clinic and agriculture. We also analyzed the antibiotic resistance gene composition of over 6000 sequenced microbial genomes, revealing significant enrichment of resistance functions by both ecology and phylogeny. Together, our results indicate that environmental and human-associated microbial communities harbor distinct resistance genes, suggesting that antibiotic resistance functions are largely constrained by ecology.     

Prevalence of Tetracyclines in Children's Teeth—Study II: Resurvey after Five Years

The capacity of the tetracyclines to discolour teeth has been widely recognized for over 10 years. This survey examined recent trends in tetracycline usage as shown by deposits of the antibiotic in primary molars extracted from 505 children aged from 3 to 5 years.It was found that 70% of the children had been given the antibiotic during their first three years of life, each having received on average 2·4 courses. This represented an increase of 12% in tetracycline usage in children of this age as compared with a similar series five years ago. There was no evidence of increasing preference by prescribers for oxytetracycline (the analogue least likely to cause dental discoloration). Staining of the permanent incisor teeth frequently occurs if tetracyclines are given to children under 6 years of age and it is predicted that some degree of staining will occur in about 23% of the children in this series.All things considered, there appears to be a strong case for stopping the manufacture of paediatric preparations of tetracycline except for those comprised of oxytetracycline for use in exceptional cases.     

The Lysine Acetylation Modification in the Porin Aha1 of Aeromonas hydrophila Regulates the Uptake of Multidrug Antibiotics

Protein lysine acetylation (Kac) modification plays important roles in diverse physiological functions. However, there is little evidence on the role of Kac modification in bacterial antibiotic resistance. Here, we compared the differential expressions of whole-cell proteins and Kac peptides in oxytetracycline sensitive and oxytetracycline resistance (OXY^R) strains of Aeromonas hydrophila using quantitative proteomics technologies. We observed a porin family protein Aha1 downregulated in the OXY^R strain, which may have an important role in the OXY resistance. Interestingly, seven of eight Kac peptides of Aha1 decreased abundance in OXY^R as well. Microbiologic assays showed that the K57R, K187R, and K197R Aha1 mutants significantly increased antibiotic resistance to OXY and reduced the intracellular OXY accumulation in OXY stress. Moreover, these Aha1 mutants displayed multidrug resistance features to tetracyclines and β-lactam antibiotics. The 3D model prediction showed that the Kac states of K57, K187, and K197 sites located at the extracellular pore vestibule of Aha1 may be involved in the uptake of specific types of antibiotics. Overall, our results indicate a novel antibiotic resistance mechanism mediated by Kac modification, which may provide a clue for the development of antibiotic therapy strategies.     

Antibiotic resistance of Lactobacillus strains]

One hundred and thirty six Lactobacillus strains isolated from poultry and 23 Lactobacillus strains isolated from long-living persons were tested for their antibiotic sensitivity. Occurrence of some type determinants of resistance to aminoglycoside antibiotics and tetracyclines in the Lactobacillus strains resistant to these antibiotics was studied. The majority of the strains from the both collections were resistant to aminoglycosides (73 and 79 per cent, respectively). The isolates from the poultry were characterized by multiple resistance. The isolates from the long-living persons were most frequently resistant to one of two antibiotics. All the tested Lactobacillus strains isolated from the long-living persons were sensitive to tetracyclines. The species composition of the isolates was different. The antibiotic-resistant strains were detected in all the species involved in the study. By hybridization of Lactobacillus colonies with the probes containing various genes of the resistance it was shown that in 14 per cent of the antibiotic-resistant strains belonging to Lactobacillus the antibiotic resistance was controlled by the genes homologous to resistance genes widely distributed in gramnegative organisms. This indicated a possible wide exchange and heterologous expression of the antibiotic resistance determinants between microorganisms of various taxonomic groups.     

Light-Induced tetracycline accumulation by rhodopseudomonas sphaeroides

Light has been used as a primary energy source in studies of tetracycline transport by Rhodopseudomonas sphaeroides. Accumulation of the antibiotic occurs in light, while efflux occurs in dark. Both fluorescence enhancement and radioisotopic tracing have been used to monitor transport. K_m's obtained from both techniques are similar. Light-induced accumulation of tetracyclines is inhibited by a variety of inhibitors, including antimycin A, N-ethylmaleimide, carbonylcyanide m-chloro-phenylhydrazone, and 2,4-dinitrophenol. A rapid efflux is observed after loading when cells are placed in the dark or treated with inhibitors.     

Analysis of multiple bacterial species and antibiotic classes reveals large variation in the association between seasonal antibiotic use and resistance

Understanding how antibiotic use drives resistance is crucial for guiding effective strategies to limit the spread of resistance, but the use–resistance relationship across pathogens and antibiotics remains unclear. We applied sinusoidal models to evaluate the seasonal use–resistance relationship across 3 species (Staphylococcus aureus, Escherichia coli, and Klebsiella pneumoniae) and 5 antibiotic classes (penicillins, macrolides, quinolones, tetracyclines, and nitrofurans) in Boston, Massachusetts. Outpatient use of all 5 classes and resistance in inpatient and outpatient isolates in 9 of 15 species–antibiotic combinations showed statistically significant amplitudes of seasonality (false discovery rate (FDR) < 0.05). While seasonal peaks in use varied by class, resistance in all 9 species–antibiotic combinations peaked in the winter and spring. The correlations between seasonal use and resistance thus varied widely, with resistance to all antibiotic classes being most positively correlated with use of the winter peaking classes (penicillins and macrolides). These findings challenge the simple model of antibiotic use independently selecting for resistance and suggest that stewardship strategies will not be equally effective across all species and antibiotics. Rather, seasonal selection for resistance across multiple antibiotic classes may be dominated by use of the most highly prescribed antibiotic classes, penicillins and macrolides.

A study of the relationship between antibiotic use and resistance in Boston, Massachusetts shows that antibiotic resistance in multiple pathogens peaks in the winter/spring and is most correlated with the use of penicillins and macrolides – the antibiotics with winter-peaking use.     

Permeation of tetracyclines through membranes of liposomes and Escherichia coli.

Uptake of tetracycline (tc), 2-tetracyclinonitrile (CN-tc), and 9-(N, N-dimethylglycylamido)-6-demethyl-6-deoxytetracycline (DMG-DMDOT) by liposomes containing Tet repressor (TetR) and by Escherichia coli cells overexpressing TetR was examined. TetR specifically binds to tetracyclines, enhances their fluorescence and thereby allows selective detection of tetracyclines that have crossed the membranes. Analysis of the diffusion of tc and DMG-DMDOT into liposomes yielded permeation coefficients of (2.4 +/- 0.6) x 10-9 cm.s-1 and (3.3 +/- 0.8) x 10-9 cm.s-1, respectively. Similar coefficients were obtained for uptake of these tetracyclines by E. coli, indicating that diffusion through the cytoplasmic membrane is the rate-limiting step. The permeation coefficients translate into half-equilibration times of approximately 35 +/- 15 min and explain how efflux pumps can mediate resistance against tetracyclines. Furthermore, diffusion of CN-tc into liposomes was at least 400-fold slower than that of tc, indicating that the carboxamide group at position C2 is required for efficient permeation of tc through lipid membranes and thereby explaining the lack of antibiotic activity of CN-tc.     

Tetracyclines downregulate the production of LPS-induced cytokines and chemokines in THP-1 cells via ERK,p38, and nuclear factor-κB signaling pathways

Recent reports have shown that antibiotics such as macrolide, aminoglycoside, and tetracyclines have immunomodulatory effects in addition to essential antibiotic effects. These agents may have important effects on the regulation of cytokine and chemokine production. However, the precise mechanism is unknown. This time, we used Multi Plex to measure the production of cytokines and chemokines following tetracycline treatment of lipopolysaccharide (LPS)-induced THP-1 cells. The signaling pathways were investigated with Western blotting analysis. Three tetracyclines significantly suppressed the expression of cytokines and chemokines induced by LPS. Minocycline (50 μg/ml), tigecycline (50 μg/ml), or doxycycline (50 μg/ml) were added after treatment with LPS (10 μg/ml). Tumor necrosis factor-α was downregulated to 16%, 14%, and 8%, respectively, after 60 min compared to treatment with LPS without agents. Interleukin-8 was downregulated to 43%, 32%, and 26% at 60 min. Macrophage inflammatory protein (MIP)-1α was downregulated to 23%, 33%, and 16% at 120 min. MIP-1β was downregulated to 21%, 11%, and 2% at 120 min. Concerning about signaling pathways, the mechanisms of the three tetracyclines might not be the same. Although the three tetracyclines showed some differences in the time course, tetracyclines modulated phosphorylation of the NF-κB pathway, p38 and ERK1/2/MAPK pathways, resulting in inhibition of cytokine and chemokine production. In addition, SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly suppressed the expression of TNF-α and IL-8 in LPS-stimulated THP-1 cells. And further, the NF-κB inhibitor, BAY11-7082, almost completely suppressed LPS-induced these two cytokines production. Thus, more than one signaling pathway may be involved in tetracyclines downregulation of the expression of LPS-induced cytokines and chemokines in THP-1 cells. And among the three signaling pathways, NF-κB pathway might be the dominant pathway on tetracyclines modification the LPS-induced cytokines production in THP-1 cells. In general, minocycline and doxycycline suppressed the production of cytokines and chemokines in LPS-stimulated THP-1 cell lines via mainly the inhibition of phosphorylation of NF-κB pathways. Tigecycline has the same structure as the other tetracyclines, however, it showed the different properties of cytokine modulation in the experimental time course.     

Increase in tetracycline activity by using surface-active substances]

The antimicrobial effect of cationic and anionic surface-active substances, i.e. catamine AB and sulphonol NP-3 respectively was studied in vitro with respect to gramnegative bacteria. In non-bactericidal concentrations catamine AB significantly increased the efficacy of tetracyclines, while the anionic compound had no such effect. The increase in the tetracycline activity was due to the antibiotic increased absorption (14C-oxytetracycline as an example) on the treatment of gramnegative bacteria with catamine AB and staphylococci with catamine AB and sulphonol NP-3, which was mainly associated with impairement of the cell membrane permeability by the surface-active substances.     

A simple classification method for residual antibiotics using E. coli cells transformed by the calcium chloride method and drug resistance plasmid DNA

Using three different plasmid DNA codings for kanamycin (KM), chloramphenicol (CP), and ampicillin- (AMP) and tetracycline- (TC) resistance, four different competent Escherichia coli strains were transformed by the calcium chloride method to produce KM-, CP- and AMP- and TC-resistant strains. Evaluation of minimum inhibitory concentrations (MIC) of 22 antibiotics, showed KM-resistant E. coli to be cross resistant only to fradiomycin (FRM); CP-resistant E. coli, especially HB101 and JM109 strains, exhibited cross-resistance only to thiamphenicol (TP). On the other hand, AMP- and TC-resistant E. coli showed cross resistance to several penicillins, tetracyclines and erythromycin. E. coli ATCC-27166, the strain most sensitive to all drugs in this experiment, was employed for disc diffusion experiments and from the pattern of appearance of the inhibition zone, eight major antibiotics were divided into three groups depending on their activity against containing each of the three plasmids. Only gentamicin (GM) activity was not affected by any of the drug resistant strains. Assay techniques utilizing three resistant strains may be the technique for screening foods for antibiotic residues in the future.     

Efflux-mediated drug resistance in Gram-positive bacteria

Gram-positive bacteria express numerous membrane transporters that promote the efflux of various drugs, including many antibiotics, from the cell to the outer medium. Drug transporters can be specific to a particular drug, or can have broad specificity, as in so-called multidrug transporters. This broad specificity can be a consequence of the hydrophobic nature of transported molecules, as suggested by recent structural studies of soluble multidrug-binding proteins. Although the functions of drug transporters may involve both the protection of bacteria from outside toxins and the transport of natural metabolites, their clinical importance lies largely in providing Gram-positive pathogens with resistance to macrolides, tetracyclines and fluoroquinolones. A number of agents, discovered in recent years, that inhibit drug transporters can potentially be used to overcome efflux-associated antibiotic resistance.     

Survey of Shiga toxigenic Escherichia coli O157 and drug-resistant coliform bacteria from in-line milk filters on dairy farms in the Czech Republic

Aims: To determine the occurrence of Shiga toxin‐producing Escherichia coli (STEC) O157 and coliform bacteria isolates resistant to antimicrobial agents in dairy herds by examining milk filters and to analyse the influence of management factors and antibiotic use on antimicrobial resistance. Methods and Results: A total of 192 in‐line milk filters were sampled on 192 dairy farms in the Czech Republic. Information on feeding, husbandry, production, and antibiotic therapy were obtained by questionnaire. The milk filters were cultured for STEC O157 and coliform bacteria. All recovered isolates were examined for antimicrobial susceptibility and presence of antimicrobial‐resistance genes. STEC O157 was detected in four (2%) of the filters. Resistant nonpathogenic E. coli and coliform bacteria isolates with specific genes were detected in 44 (23%) of the filters. Conclusions: The study demonstrated a high prevalence of resistant coliform bacteria in milk filters obtained on Czech dairy farms. Significance and Impact of the Study: The occurrence of resistant coliform bacteria in milk filters was significantly higher among isolates from farms where antibiotic therapy against mastitis was employed during the dry period (P < 0·05).     

Acquired Genetic Mechanisms of a Multiresistant Bacterium Isolated from a Treatment Plant Receiving Wastewater from Antibiotic Production

The external environment, particularly wastewater treatment plants (WWTPs), where environmental bacteria meet human commensals and pathogens in large numbers, has been highlighted as a potential breeding ground for antibiotic resistance. We have isolated the extensively drug-resistant Ochrobactrum intermedium CCUG 57381 from an Indian WWTP receiving industrial wastewater from pharmaceutical production contaminated with high levels of quinolones. Antibiotic susceptibility testing against 47 antibiotics showed that the strain was 4 to >500 times more resistant to sulfonamides, quinolones, tetracyclines, macrolides, and the aminoglycoside streptomycin than the type strain O. intermedium LMG 3301^T. Whole-genome sequencing identified mutations in the Indian strain causing amino acid substitutions in the target enzymes of quinolones. We also characterized three acquired regions containing resistance genes to sulfonamides (sul1), tetracyclines [tet(G) and tetR], and chloramphenicol/florfenicol (floR). Furthermore, the Indian strain harbored acquired mechanisms for horizontal gene transfer, including a type I mating pair-forming system (MPF_I), a MOB_P relaxase, and insertion sequence transposons. Our results highlight that WWTPs serving antibiotic manufacturing may provide nearly ideal conditions for the recruitment of resistance genes into human commensal and pathogenic bacteria.     

1,12-substituted tetracyclines as antioxidant agents

Novel hydroxypyrazoline derivatives of tetracycline and minocycline have been synthesized through the reaction of these tetracyclines with hydrazine. The formation of a new chiral center at C12 is stereospecific to give 12S-12-hydroxy-1,12-pyrazolinotetracycline. A reaction mechanism for the formation of these novel tetracycline derivatives has been proposed. Hydroxypyrazolinotetracyclines exhibit no binding to Mg2+ and Zn2+, features that are required for antibiotic activity and matrix metalloproteinase (MMP) inhibitions, respectively. The modification toward their hydroxypyrazolino derivatives significantly improved the antioxidant activities of tetracycline and minocycline, as shown by three commonly used assays (DPPH, ABTS+, and superoxide scavenging). 12S-Hydroxy-1,12-pyrazolinominocycline is a promising tetracycline-based antioxidant devoid of antibiotic properties and MMP inhibitory activity, which could be beneficial in the treatment of complications related to oxidative stress.