Anomalies in the enumeration of starved bacteria on culture media containing nalidic acid and tetracycline

Culturable counts of antibiotic resistant, genetically engineeredPseudomonas fluorescens were determined on antibiotic-containing plate count agar during starvation in water. Prior to starvation, colony counts obtained on all media separated into two groups. The mean of the colony counts on plate count agar with or without tetracycline (4.9 × 10⁶ ml⁻¹) was significantly higher than the mean colony counts on plate count agar containing either nalidixic acid or nalidixic acid plus tetraclycline (2.5×10⁶ ml⁻¹). After 20 days of starvation the highest mean colony counts continued to be obtained on plate count agar (7.2 × 10⁶ ml⁻¹) with slightly, but significantly, lower counts obtained on plate count agar containing either nalidixic acid (5.6 × 10⁶ ml⁻¹) or tetraclycline (1.5×10⁶ ml⁻¹). A combination of nalidixic acid and tetracycline in plate count agar, however, dramatically reduced colony counts (8.3 × 10² ml⁻¹) after this starvation period. The addition of catalase to plate count agar containing nalidixic acid and tetracycline negated the effect caused by this combination of antibiotics. When colony counts obtained over the entire 20 day incubation were considered, the addition of MgSO₄ to plate count agar containing nalidixic acid and tetracycline resulted in a significant increase in colony counts. Other combinations of antibiotics, nalidixic acid+carbenicillin, nalidixic acid+kanamycin, streptomycin+tetracycline, streptomycin+carbenicillin, rifampicin+tetracycline, rifampicin+carbenicillin, and rifampicin+kanamycin, did not inhibit colony formation of starved cells. Antibiotic resistant strains ofP. putida andEscherichia coli also displayed sensitivity to the combination of nalidixic acid and tetracycline in plate count agar after starvation.     

Transfer Region of a Bacteroides Conjugative Transposon Contains Regulatory as Well as Structural Genes

Conjugative transposons (CTns) are integrated elements that excise themselves from the chromosome to form a circular transfer intermediate that is transferred by conjugation to a recipient. In an earlier paper, the excision step was shown to be regulated by tetracycline and to be dependent on the regulatory gene, rteC. In this paper, we report that genes involved in conjugal transfer are also regulated by tetracycline but that regulation is more complex. Genes contained within a 20-kbp region that is sufficient for conjugal transfer were disrupted by single crossover integration events. Most of the disruptions abolished transfer of the CTn. None of them abolished excision. Antibodies to two of the proteins encoded in this region (TraG and TraN) were obtained and used to show that production of these proteins was dependent on tetracycline stimulation. Both TraG and TraN were membrane proteins. A surprising finding was that a disruption in the gene traQ increased transfer of CTnERL over 100-fold. Thus, TraQ may be a repressor protein that controls expression of transfer genes. If so, TraQ is not the only protein that controls expression of transfer genes because production of TraG and TraN in the traQ disruption mutant was still dependent on tetracycline stimulation.     

Incidence of resistance to ampicillin,chloramphenicol, kanamycin and tetracycline amongSalmonella species isolated in The Netherlands in 1972, 1973 and 1974

The resistance ofSalmonellae to drugs has been studied in the Netherlands since 1958. In 1972, 1973, and 1974 respectively, 14241, 13086, and 22927 strains were tested for resistance to ampicillin, chloramphenicol, kanamyci and tetracycline. From 1973 all strains were also tested for resistance to trimethoprim. In the period covered, the yearly incidence of resistance to at least one of the above drugs ranged from 39.2% to 45.6% of all strains obtained from various sources (humans, animals, animal products, sewage, etc.). A new finding in the period 1972 to 1974 was that many multiply resistant strains emerged inS. typhimurium and inS. dublin isolated from calves and cattle. In 1974, 64.4% of all strains ofS. typhimurium from these animals appeared to be resistant to ampicillin, tetracycline, chloramphenicol and kanamycin, and 25.5% of those ofS. dublin were found to be resistant to chloramphenicol and tetracycline. Of all strains ofSalmonellae examined in 1973 and 1974 respectively, 0.15 and 0.22% were resistant to trimethoprim, the main component of the twin-drug cotrimoxazol. Of the 142 strains ofS. typhi isolated in 1972 to 1974 two were resistant to tetracycline only, and one was resistant to all four antibiotics. The others had a normal susceptibility pattern.     

Prevalence of tet gene and complete genome sequencing of tet gene-encoded plasmid (pAHH01) isolated from Aeromonas species in South Korea

Aims: To investigate the tetracycline resistance related to tet genes in Aeromonas isolates collected from water and diseased fish in South Korea. Methods and Results: A total of 34 Aeromonas strains were examined for their susceptibility to tetracycline using the minimum inhibitory concentration (MIC) assay, and the genetic determinants (tetA to E) were analysed. Among these strains, the tetA and tetE genes were predominant (tetA was found in six strains, and tetE was found in nine strains), and 15 strains were tetracycline‐resistant by the MIC assay. Additionally, the 8979‐bp plasmid that contains the tetE gene was fully sequenced. Conclusions: These data may be important with regard to the spread and persistence of tetracycline resistance genes in the bacterial populations that are present in aquaculture systems. Significance and Impact of the Study: Interestingly, no isolate has previously been shown to harbour three tet genes that are mediated by efflux systems, but the tetA, tetD and tetE genes were all isolated from one strain, which had the highest MIC value for tetracycline among the strains analysed in this study. We also investigated the full‐length plasmid that encoded the tetE gene from a tetracycline‐resistant strain.     

Tetracycline treatment targeting Wolbachia affects expression of an array of proteins in Brugia malayi parasite

Wolbachia is an intracellular endosymbiont of Brugia malayi parasite whose presence is essential for the survival of the parasite. Treatment of B. malayi‐infected jirds with tetracycline eliminates Wolbachia, which affects parasite survival and fitness. In the present study we have tried to identify parasite proteins that are affected when Wolbachia is targeted by tetracycline. For this Wolbachia depleted parasites (B. malayi) were obtained by tetracycline treatment of infected Mongolian jirds (Meriones unguiculatus) and their protein profile after 2‐DE separation was compared with that of untreated parasites harboring Wolbachia. Approximately 100 protein spots could be visualized followed by CBB staining of 2‐D gel and included for comparative analysis. Of these, 54 showed differential expressions, while two new protein spots emerged (of 90.3 and 64.4 kDa). These proteins were subjected to further analysis by MALDI‐TOF for their identification using Brugia coding sequence database composed of both genomic and EST sequences. Our study unravels two crucial findings: (i) the parasite or Wolbachia proteins, which disappeared/down‐regulated appear be essential for parasite survival and may be used as drug targets and (ii) tetracycline treatment interferes with the regulatory machinery vital for parasites cellular integrity and defense and thus could possibly be a molecular mechanism for the killing of filarial parasite. This is the first proteomic study substantiating the wolbachial genome integrity with its nematode host and providing functional genomic data of human lymphatic filarial parasite B. malayi.     

紫茎泽兰对四环素的吸收特性

[目的] 探究四环素在水和紫茎泽兰间的传递以及在紫茎泽兰体内的累积特征。[方法] 利用高效液相色谱检测紫茎泽兰幼苗在水培过程中对四环素的吸收及其在根茎叶中的积累。[结果] 在10~20 mg·L^-1四环素的处理浓度范围内,紫茎泽兰根、茎、叶均能吸收并积累四环素,且吸收累积量均随处理浓度和处理时间的增加而升高。当紫茎泽兰在20 mg·L^-1四环素的水培液处理20 d时,茎中的四环素累积量最高,为（59.34±3.86）mg·kg^-1;根中的次之,为（52.52±5.89）mg·kg^-1;而叶中的最低,为（23.19±4.17）mg·kg^-1。此外,紫茎泽兰茎的四环素富集系数最大,根的次之,叶的最小。[结论] 紫茎泽兰能够较好地从水培液中吸收并累积四环素,具有吸收净化四环素污染水源的潜力。     

Construction of a Chimeric Plasmid in Bacillus subtilis

Staphylococcal plasmids pTP4 (2.7 megadaltons encoding resistance to chloramphenicol) and pTP5 (2.6 megadaltons encoding resistance to tetracycline), which replicate and express resistance in B. subtilis, were found to cut by HindIII endonuclease respectively at a single site and three sites. A chimeric plasmid pTA1245 (4.1 megadaltons) was constructed from pTP4 and pTP5 by HindIII digestion and ligation with E. coli DNA ligase. pTA1245 expresses resistances to chloramphenicol and tetracycline in B. subtilis, and pTA1245 is amplified in the presence of tetracycline. A physical map of pTA1245 was constructed.     

Tetracyclines and Tetracycline Resistance in Agricultural Soils: Microcosm and Field Studies

The influence of the use of antibiotics on the prevalence of resistance genes in the environment is still poorly understood. We studied the diversity of tetracycline and sulfonamide resistance genes as influenced by fertilization with pig manure in soil microcosms and at two field locations. Manure contained a high diversity of resistance genes, regardless of whether it stemmed from a farm operation with low or regular use of antibiotics. In the microcosm soils, the influence of fertilization with manure was clearly shown by an increase in the number of resistance genes in the soil after manuring. Spiking of the tetracycline compounds to the microcosms had only little additional impact on the diversity of resistance genes. Overall, the tetracycline resistance genes tet(T), tet(W), and tet(Z) were ubiquitous in soil and pig slurries, whereas tet(Y), tet(S), tet(C), tet(Q), and tet(H) were introduced to the microcosm soil by manuring. The diversity of tetracycline and sulfonamide [sul(1), sul(2), and sul(3)] resistance genes on a Swiss pasture was very high even before slurry amendment, although manure from intensive farming had not been applied in the previous years. The additional effect of manuring was small, with the tetracycline and sulfonamide resistance diversity staying at high levels for the complete growth season. At an agricultural field site in Germany, the diversity of tetracycline and sulfonamide resistance genes was considerably lower, possibly reflecting regional differences in gene diversity. This study shows that there is a considerable pool of resistance genes in soils. Although it is not possible to conclude whether this diversity is caused by the global spread of resistance genes after 50 years of tetracycline use or is due to the natural background in soil resistance genes, it highlights a role that environmental reservoirs might play in resistance gene capture.     

Mixed continuous culture experiments with an antibiotic-producing streptomycete andEscherichia coli

Four strains ofStreptomyces aureofaciens capable of producing different amounts of tetracycline have been grown in continuous culture with either of two strains ofEscherichia coli orBacillus pumillus. Each of the bacteria had faster specific growth rates than any of the streptomycetes.E. coli NTCT 5993 had a higher affinity for sucrose than didS. aureofaciens SR 11. In mixed culture experiments, the bacteria displaced the streptomycete under the following conditions: (a) when the streptomycete produced no tetracycline; (b) in tetracycline-producing cultures after a fraction of the bacteria had died—the bacterial population that subsequently developed was resistant to the drug; and (c) when using tetracycline-resistant bacteria. Under nutrient conditions leading to high antibiotic levels, it was possible to kill all the bacteria, and the streptomycete survived after a transient fall in mycelium and tetracycline levels. No stable mixed population was ever seen. Once tetracycline-resistant bacteria had displaced the streptomycete, and tetracycline concentrations had fallen, a sensitive bacterial population reappeared. Competition between sensitive and resistantE. coli NCTC 5993 in the chemostat confirmed the selective advantage of the sensitive strain. Results were discussed in terms of the role of antibiotics in nature.     

Phenotypic and genotypic analysis of bacteria isolated from three municipal wastewater treatment plants on tetracycline‐amended and ciprofloxacin‐amended growth media

Aims: The goal of this study was to determine the antimicrobial susceptibility of bacteria isolated from three municipal wastewater treatment plants. Methods and Results: Numerous bacterial strains were isolated from three municipal wastewater treatment facilities on tetracycline‐ (n = 164) and ciprofloxacin‐amended (n = 65) growth media. These bacteria were then characterized with respect to their resistance to as many as 10 different antimicrobials, the presence of 14 common genes that encode resistance to tetracycline, the presence of integrons and/or the ability to transfer resistance via conjugation. All of the characterized strains exhibited some degree of multiple antimicrobial resistance, with nearly 50% demonstrating resistance to every antimicrobial that was tested. Genes encoding resistance to tetracycline were commonly detected among these strains, although intriguingly the frequency of detection was slightly higher for the bacteria isolated on ciprofloxacin‐amended growth media (62%) compared to the bacteria isolated on tetracycline‐amended growth media (53%). Class 1 integrons were also detected in 100% of the queried tetracycline‐resistant bacteria and almost half of the ciprofloxacin‐resistant strains. Conjugation experiments demonstrated that at least one of the tetracycline‐resistant bacteria was capable of lateral gene transfer. Conclusions: Our results demonstrate that multiple antimicrobial resistance is a common trait among tetracycline‐resistant and ciprofloxacin‐resistant bacteria in municipal wastewater. Significance and Impact of the Study: These organisms are potentially important in the proliferation of antimicrobial resistance because they appear to have acquired multiple genetic determinants that confer resistance and because they have the potential to laterally transfer these genetic determinants to strains of clinical importance.     

The Clostridium perfringens Tet P determinant comprises two overlapping genes: tetA(P), which mediates active tetracycline efflux, and tetB(P), which is related to the ribosomal protection family of tetracycline-resistance determinants

The complete nucleotide sequence and mechanism of action of the tetracycline-resistance determinant Tet P, from Clostridium perfringens has been determined. Analysis of the 4.4 kb of sequence data revealed the presence of two open reading frames, designated as tetA(P) and tetB(P), The tetA(P) gene appears to encode a 420 amino acid protein (molecular weight 46079) with twelve transmembrane domains. This gene was shown to be responsible for the active efflux of tetracycline from resistant ceils. Although there was some amino acid sequence similarity between the putative TetA(P) protein and other tetracycline efflux proteins, analysis suggested that TetA(P) represented a different type of efflux protein. The tetB(P) gene would encode a putative 652 amino acid protein (molecular weight 72639) with significant sequence similarity to Tet(M)-like cytoplasmic proteins that specify a ribosomal-protection tetracycline-resistance mechanism. In both C. perfringens and Escherichia coli. tetB(P) encoded low-level resistance to tetracycline and minocycline whereas tetA(P) only conferred tetracycline resistance. The tetA(P) and tetB(P) genes appeared to be linked in an operon, which represented a novel genetic arrangement for tetracycline-resistance determinants. It is proposed that tetB(P) evolved from the conjugative transfer into C. perfringens of a fer (M)-like gene from another bacterium.     

Integration of Chloramphenicol Resistance Gene of an R Factor on Escherichia coli Chromosome

An unstable mutant R factor conferring only chloramphenicol (CM) resistance was obtained by spontaneous segregation. After storage in broth culture, a stable CM-resistant mutant was obtained and its CM-resistance could not be cured by treatment with acriflavine or transduced to a recombination-deficient strain of Escherichia coli K12. Recombinational analysis indicated that the cml gene governing CM resistance had been integrated into the E. coli chromosome and closely linked with met B locus. The cml gene was co-transduced with both met and arg markers by phage P1, and the linkage order was considered to be mtl-cml-met-arg-thi. When the strain carrying this chromosomal CM-resistance was infected with a transferable R (TC) factor capable of conferring tetracycline (TC) resistance, the CM-resistance became transferable by conjugation. This mechanism is considered to account for the formation of the recombinant R (TC.CM) factor.     

Effect of milk on antibacterial activity of tetracycline against Escherichia coli and Staphylococcus aureus isolated from bovine mastitis

The susceptibility of mastitis-causing Escherichia coli and Staphylococcus aureus to two commonly used antibiotics, tetracycline and penicillin G, was tested in raw milk and in Muller–Hinton (MH) broth by introducing a pH indicator, bromocresol purple, which was shown to be a simple, sensitive, and rapid method. The minimum inhibitory concentration (MIC) of penicillin G in milk was the same as those in MH broth, whereas the MIC of tetracycline in milk was 4 to 32 times that in MH. An irreversible binding between tetracycline and large molecules of milk, which might be due to a hydrophobic interaction, was demonstrated by a dialysis test, suggesting the observed impairing effect was due to the action of milk on the tetracycline being tested. Further investigation revealed that much of the reduction of tetracycline’s activity in milk was attributable to the milk protein casein, while other heat-sensitive components in milk also play some roles.     

Susceptibility of <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> to antibiotics

A total of 74 Streptococcus thermophilus isolates collected between 1948 and 2005 from different environments were investigated to assess erythromycin, clindamycin, streptomycin, gentamicin, tetracycline and ampicillin susceptibility by means of microdilution, Etest and disk diffusion methods. For this purpose a new S. thermophilus Susceptibility test Medium (SSM) was developed. This medium allowed a better identification of strains with atypical tetracycline resistance. The recipe is a mixed formulation of Iso-Sensitest medium (90% v/v) and M17 medium (10% v/v) supplemented with lactose (0.5% w/v). The overall agreement of the techniques was good with exception of tetracycline, for which Etest provided lower MICs than the microdilution method. Most strains were susceptible to all the antibiotics tested while a few erythromycin, tetracycline and streptomycin resistant strains were detected.     

Incidence of resistance to chloramphenicol and tetracyclines among 13502Salmonella strains isolated in 1961

Summary The rate of resistance to tetracycline and chloramphenicol amongSalmonella strains isolated in the Netherlands in 1961 was found to be 3.96%, the corresponding figures for 1958/1959 and 1961 being 2.08 and 1.29 respectively. In this country the total number ofSalmonella types found to develop resistance to either tetracycline or chloramphenicol now amounts to 38. Almost 77% of all resistant strains isolated in 1961 were found among the human pathogenS.typhimurium. The relative frequency of resistance in this organism was 8.18%, as compared with 2.50% in 1958/1959 and 1.80% in 1960. In 1961 some cross infections caused by tetracycline resistant strains ofS.typhimurium were observed in man and on one occasion also in a herd of calves. A similar outbreak due to a tetracycline resistant strain ofS.bovis morbificans was seen in a hospital. As almost 87% of all antibiotic resistant strains found in 1961 originated from human patients, the resistance must be largely attributed to the therapeutic use of the drugs in question.     

Mass marking of Leuciscus idus larvae using Artemia salina as a vector of fluorescent dyes

A method for the mass marking of ide Leuciscus idus larvae by feeding them Artemia salina nauplii that were immersed in different solutions of alizarin red S, tetracycline hydrochloride and calcein was tested. The best quality marks were obtained after feeding fish for 4 days with nauplii that had been immersed in 200 mg l^?1 alizarin red S.     

盐酸四环素浓度和食物密度对萼花臂尾轮虫生活史特征的综合影响

近年来,抗生素的环境污染问题已引起人们的广泛关注,其在环境中的残留可对水生态系统的结构和功能产生重要影响。迄今,盐酸四环素浓度和食物密度对轮虫生活史特征的影响研究尚未见报道。以萼花臂尾轮虫(Brachionus calyciflorus)为受试动物,研究了在不同斜生栅藻(Scenedesmus obliquus)密度下,不同浓度的盐酸四环素(Tetracycline hydrochloride)对萼花臂尾轮虫生活史特征的影响。结果表明,食物密度和盐酸四环素浓度对轮虫的生命期望、净生殖率、世代时间、内禀增长率、平均寿命和后代混交率,以及食物密度和盐酸四环素浓度间交互作用对除了内禀增长率外的其他5个统计学参数均具有显著影响。在各食物密度下,轮虫的特定年龄繁殖率高峰值随着盐酸四环素浓度的增加呈现先增加后降低的趋势;盐酸四环素对轮虫生长和繁殖参数的影响呈现"低促高抑"的特点。3个食物密度下,高浓度的盐酸四环素显著提高了萼花臂尾轮虫的后代混交率,且在1.0×10~6个/mL藻密度下毒物浓度与轮虫后代混交率间具有显著的剂量-效应关系。食物密度的高低显著影响盐酸四环素对萼花臂尾轮虫的毒性效应。     

Arsenite-Induced Multiple Antibiotic Resistance Phenotype in Environmental Isolates of Yersinia enterocolitica

Minimal inhibitory concentrations (MICs) of five antibiotics namely amikacin, gentamicin, tetracycline, ciprofloxacin, and nitrofurantoin for pork isolates of Yersinia enterocolitica increased two- to eightfold after bacteria were grown in the presence of 5 mm arsenite. For Y. enterocolitica isolates obtained from wastewater (sewage effluents), an unequivocal increase in MICs was seen with amikacin and gentamicin. No change was discernible in the outer-membrane proteins after isolates were grown in the presence of arsenite. Received: 14 October 2000 / Accepted: 26 January 2001