Comparison of Lysyl-Transfer Ribonucleic Acid Species from Vegetative Cells and Spores of Bacillus subtilis by Methylated Albumin-Kieselguhr and Reversed-Phase Chromatography

Lysyl-transfer ribonucleic acid (tRNA) species from a spore-forming strain of Bacillus subtilis (168 trp2(-)) and an early blocked asporogenous mutant (spoA 12) were compared on reversed-phase and methylated albumin-kieselguhr columns. Lysyl-tRNA species from spores and the asporogenous mutant in stationary phase both exhibited altered chromatographic profiles compared to that of log-phase cells. The major peak in spore lysyl-tRNA species eluted later than that characteristic of vegetative cells, whereas the major peak of the lysyl-tRNA species from the asporogenous mutant in stationary phase eluted earlier. Although the early eluting lysyl-tRNA species was observable on methylated albumin columns, the late eluting peak was not detectable by that column technique. By using a shallower gradient on an RPC-2 column, the resolution of all lysyl-tRNA species increased. Several subspecies were revealed. The chromatographic comparisons clearly show that both the spore-forming strain and the asporogenous mutant undergo relative increases in different lysyl-tRNA species when grown to late stationary phase. No new species seem to be involved but rather altered amounts of minor species existing in log-phase cells. The experiments also demonstrate the usefulness of reversed-phase columns for such comparisons.     

Flow-microfluorometric analysis of Escherichia coli, Rhizobium meliloti, and Rhizobium japonicum at different stages of the growth cycle.

The applicability of flow-microfluorometry (FMF) to the study of bacterial samples was investigated on cultures of Rhizobium meliloti, Rhizobium japonicum, and Escherichia coli using fluorescent and light-scattering signals. This technique which analyzes individual bacterial cells in a population was used to monitor the relative change in nucleic acid content and cell size during the growth cycle of the three microorganisms which were known to have different growth rates. Early log-phase E. coli cells contained at least eightfold more nucleic acid and were significantly larger than the stationary-phase cells. Cultures of early log-phase R. meliloti cells contained three to four-fold more nucleic acid and were slightly larger than cells in the stationary phase. Rhizobium japonicum had very little change in either parameter. In general, the amount of change in both cell size and nucleic acid content upon initiation of log-phase growth was related to the overall growt rate of the organisms, with E. coli experiencing the greatest change and R. japonicum the least. Results obtained by FMF analysis, therefore, were consistent with observations reported by earlier workers. Cultures of R. meliloti also were used to demonstrate that the intensity of the fluorescent signals was sensitive to digestion by DNase and RNase and to prolonged storage and fixation. The potential use of FMF in the study of microorganisms is discussed.     

Update on treatment of vesiculitis in bulls

Four experiments were done to determine: (1) the effectiveness of early detection and treatment of vesiculitis in bulls; (2) whether antibiotic treatment at recommended dosages will result in adequate vesicular gland tissue concentrations of antibiotics to prevent in vitro bacterial growth; (3) whether intraglandular injection of antibiotics can be a successful alternative to systemic antibiotic treatment; and (4) the effectiveness of tilmicosin versus tulathromycin for treatment of clinical vesiculitis. In Experiment 1, there was a high rate of spontaneous remission from vesiculitis detected at 9-12 mo of age. Furthermore, there was no advantage for early antibiotic treatment versus no treatment for bulls of this age. In Experiment 2, bacteria on agar plates were exposed to fluid extracted from vesicular gland biopsies after antibiotic treatment of normal, healthy bulls. Although inadequate concentrations of antibiotics were achieved to inhibit bacterial growth when recommended dosages of various antibiotics were administered, doubling the antibiotic dosage increased in vitro bacterial growth inhibition. In Experiment 3, relatively nonirritating antibiotics were injected directly into the glands of bulls with clinical vesiculitis, demonstrating that intraglandular injections of antibiotic could be used as a successful alternative to systemic antibiotic treatment. Experiment 4 was a clinical field trial to compare the efficacy of tilmicosin versus tulathromide at recommended dosages for the treatment of clinical vesiculitis. Although the results favored tulathromycin, both antibiotics resulted in clinical cures of vesiculitis.     

Silver bullets: A new lustre on an old antimicrobial agent

Silver was widely used in medicine to treat bacterial infections in the 19th and early 20th century, up until the discovery and development of the first modern antibiotics in the 1940s, which were markedly more effective. Since then, every new antibiotic introduced to the clinic has led to an associated development of drug resistance. Today, the threat of extensive bacterial resistance to antibiotics has reignited interest in alternative strategies to treat infectious diseases, with silver regaining well-deserved renewed attention. Silver ions are highly disruptive to bacterial integrity and biochemical function, with comparatively minimal toxicity to mammalian cells. This review focuses on the antimicrobial properties of silver and their use in synergistic combination therapy with traditional antibiotic drugs.     

Minimizing the release of proinflammatory and toxic bacterial products within the host: a promising approach to improve outcome in life-threatening infections

Various bacterial components (e.g., endotoxin, teichoic and lipoteichoic acids, peptidoglycans, DNA) induce or enhance inflammation by stimulating the innate immune system and/or are directly toxic in eukariotic cells (e.g., hemolysins). When antibiotics which inhibit bacterial protein synthesis kill bacteria, smaller quantities of proinflammatory or toxic compounds are released in vitro and in vivo than during killing of bacteria by beta-lactams and other cell-wall active drugs. In general, high antibiotic concentrations liberate lower quantities of bacterial proinflammatory or toxic compounds than concentrations close to the minimum inhibitory concentration. In animal models of Escherichia coli Pseudomonas aeruginosa and Staphylococcus aureus peritonitis/sepsis and of Streptococcus pneumoniae meningitis, a lower release of proinflammatory bacterial compounds was associated with a reduced mortality or neuronal injury. Pre-treatment with a bacterial protein synthesis inhibitor reduced the strong release of bacterial products usually observed during treatment with a beta-lactam antibiotic. Data available strongly encourage clinical trials comparing antibiotic regimens with different release of proinflammatory/toxic bacterial products. The benefit of the approach to reduce the liberation of bacterial products should be greatest in patients with a high bacterial load.     

ppGpp介导的抗生素胁迫应答机制研究进展

抗生素是由微生物在生长发育后期产生的次级代谢产物,具有杀死或抑制细菌生长的能力,因此被广泛应用于细菌感染的临床治疗。在长期的进化过程中,细菌采取多种方式应对环境中抗生素的威胁。除了广为人知的抗生素耐药性(resistance)之外,细菌还能对抗生素产生耐受性(tolerance)和持留性(persistence),严重影响抗生素的临床疗效。鸟苷四磷酸(guanosine tetraphosphate, ppGpp)和鸟苷五磷酸(guanosine pentaphosphate, pppGpp) (本文统称ppGpp)是细菌应对营养饥饿等不利环境时产生的"报警"信号分子,其能够在全局水平调控基因的表达,使细菌适应不利的环境。越来越多的研究表明,ppGpp与细菌应对抗生素胁迫密切相关。基于此,本文综述了细菌中ppGpp的合成与水解及其作用机制,并重点阐述了ppGpp介导抗生素胁迫应答的分子机制,以期为新型抗生素的开发提供新思路。     

Protein synthesis during transition and stationary phases under glucose limitation in Saccharomyces cerevisiae.

Metabolic changes have been investigated during continuous growth of yeast cells inoculated in glucose-containing medium until the cells entered the stationary phase in response to glucose exhaustion. Well in advance of glucose exhaustion, a transition phase was observed, characterized by a decrease in the growth rate and a progressive reduction of protein and RNA accumulation. Two-dimensional gel analysis of the proteins synthesized during this stage showed that the pattern of proteins remained similar to that of log-phase cells. When the cells entered the stationary phase, protein accumulation was 10% of that in log-phase cells, and incorporation of labeled RNA precursor was undetectable. Analysis of protein synthesis gave evidence that the synthesis of 95% of the proteins present in log-phase cells was arrested in stationary-phase cells. Among the 20 proteins whose synthesis continues throughout the stationary phase were identified actin, aldehyde dehydrogenase, enolase, hexokinase, glyceraldehyde-3-phosphate dehydrogenase, and five heat shock proteins. In addition, the synthesis of six new proteins was observed. The occurrence of these new proteins in stationary-phase cells is presumed to result from the release of carbon catabolite repression due to glucose exhaustion.     

Production and Repair of Radiochemical Damage in Escherichia coli Deoxyribonucleic Acid; Its Modification by Culture Conditions and Relation to Survival

Late log-phase Escherichia coli B/r cells are 1.6 times more sensitive to killing by X rays than are stationary-phase cells when grown in Brain Heart Infusion (BHI) + glucose. The number of single-chain breaks formed per krad is the same for log- and stationary-phase cells. Stationary-phase cells show a somewhat greater ability to repair single-chain breaks (especially after high doses of X rays) than do log-phase cells. The rapidity and extent of postirradiation deoxyribonucleic acid (DNA) degradation are greater in log-phase cells than in stationary-phase cells. The enhanced viability exhibited by stationary-phase cells thus appears to correlate both with enhanced single-chain break repair and the reduced degradation of DNA. Cells grown to stationary phase in peptone medium (PO cells) are 3.4 times more sensitive to killing by X rays than cells grown to stationary phase in peptone medium supplemented with glucose and phosphate buffer (PG cells). The yield of single-strand breaks is the same for both types of cells (but the absolute yield is about two times higher than in the cells grown in BHI + glucose). The kinetics for the repair of single-chain breaks are the same for both types of cells for about 30 min. After this time period, further repair ceases in the PO cells but continues in the PG cells, provided that glucose is present in the medium. Postirradiation DNA degradation is both more rapid and more extensive in PO cells than in PG cells whether or not glucose is present in the postirradiation incubation medium. The survival of stationary-phase E. coli B/r grown in PO or PG medium is likewise unaffected by the presence of glucose in the plating medium, and thus correlates better with the lower DNA degradation seen in the PG cells than with the increased strand rejoining, since this latter process requires the presence of glucose.     

Escherichia coli produces linoleic acid during late stationary phase.

Escherichia coli produces linoleic acid in the late stationary phase. This was the case whether the cultures were grown aerobically or anaerobically on a supplemented glucose-salts medium. The linoleic acid was detected by thin-layer chromatography and was measured as the methyl ester by gas chromatography. The linoleic acid methyl ester was identified by its mass spectrum. Lipids extracted from late-stationary-phase cells generated thiobarbituric acid-reactive carbonyl products when incubated with a free radical initiator. In contrast, extracts from log-phase or early-stationary-phase cells failed to do so, in accordance with the presence of polyunsaturated fatty acid only in the stationary-phase cells.     

Antibiotic susceptibility of coagulase-negative staphylococci isolated from very low birth weight babies: comprehensive comparisons of bacteria at different stages of biofilm formation

Background

Coagulase-negative staphylococci are major causes of bloodstream infections in very low birth weight babies cared for in Neonatal Intensive Care Units. The virulence of these bacteria is mainly due to their ability to form biofilms on indwelling medical devices. Biofilm-related infections often fail to respond to antibiotic chemotherapy guided by conventional antibiotic susceptibility tests.

Methods

Coagulase-negative staphylococcal blood culture isolates were grown in different phases relevant to biofilm formation: planktonic cells at mid-log phase, planktonic cells at stationary phase, adherent monolayers and mature biofilms and their susceptibilities to conventional antibiotics were assessed. The effects of oxacillin, gentamicin, and vancomycin on preformed biofilms, at the highest achievable serum concentrations were examined. Epifluorescence microscopy and confocal laser scanning microscopy in combination with bacterial viability staining and polysaccharide staining were used to confirm the stimulatory effects of antibiotics on biofilms.

Results

Most coagulase-negative staphylococcal clinical isolates were resistant to penicillin G (100%), gentamicin (83.3%) and oxacillin (91.7%) and susceptible to vancomycin (100%), ciprofloxacin (100%), and rifampicin (79.2%). Bacteria grown as adherent monolayers showed similar susceptibilities to their planktonic counterparts at mid-log phase. Isolates in a biofilm growth mode were more resistant to antibiotics than both planktonic cultures at mid-log phase and adherent monolayers; however they were equally resistant or less resistant than planktonic cells at stationary phase. Moreover, for some cell-wall active antibiotics, concentrations higher than conventional MICs were required to prevent the establishment of planktonic cultures from biofilms. Finally, the biofilm-growth of two S. capitis isolates could be enhanced by oxacillin at the highest achievable serum concentration.

Conclusion

We conclude that the resistance of coagulase-negative staphylococci to multiple antibiotics initially remain similar when the bacteria shift from a planktonic growth mode into an early attached mode, then increase significantly as the adherent mode further develops. Furthermore, preformed biofilms of some CoNS are enhanced by oxacillin in a dose-dependent manner.     

Combating bacteria and drug resistance by inhibiting mechanisms of persistence and adaptation

Antibiotics have revolutionized the treatment of infectious disease but have also rapidly selected for the emergence of resistant pathogens. Traditional methods of antibiotic discovery have failed to keep pace with the evolution of this resistance, which suggests that new strategies to combat bacterial infections may be required. An improved understanding of bacterial stress responses and evolution suggests that in some circumstances, the ability of bacteria to survive antibiotic therapy either by transiently tolerating antibiotics or by evolving resistance requires specific biochemical processes that may themselves be subject to intervention. Inhibiting these processes may prolong the efficacy of current antibiotics and provide an alternative to escalating the current arms race between antibiotics and bacterial resistance. Though these approaches are not clinically validated and will certainly face their own set of challenges, their potential to protect our ever-shrinking arsenal of antibiotics merits their investigation. This Review summarizes the early efforts toward this goal.     

Nonenzymatic turnover of an Erwinia carotovora quorum-sensing signaling molecule.

The production of virulence factors and carbapenem antibiotic in the phytopathogen Erwinia carotovora is under the control of quorum sensing. The quorum-sensing signaling molecule, N-(3-oxohexanoyl)-L-homoserine lactone (OHHL), accumulates in log-phase culture supernatants of E. carotovora but diminishes in concentration during the stationary phase. In this study, we show that the diminution in OHHL was not due to sequestration of the ligand by the cells, although some partitioning did occur. Rather, it was caused by degradation of the molecule. The rate of stationary-phase degradation of OHHL was as rapid as the rate of log-phase accumulation of the ligand, but it was nonenzymatic and led to a decrease in the expression of selected genes known to be under the control of quorum sensing. The degradation of OHHL was dependent on the pH of the supernatant, which increased as the growth curve progressed in cultures grown in Luria-Bertani medium from pH 7 to approximately 8.5. OHHL became unstable over a narrow pH range (pH 7 to 8). Instability was increased at high temperatures even at neutral pH but could be prevented at the growth temperature (30 degrees C) by buffering the samples at pH 6.8. These results may provide a rationale for the observation that an early response of plants which are under attack by Erwinia is to activate a proton pump which alkalizes the site of infection to a pH of >8.2.     

Luciferase inactivation in the luminous marine bacterium Vibrio harveyi.

Luciferase was rapidly inactivated in stationary-phase cultures of the wild type of the luminous marine bacterium Vibrio harveyi, but was stable in stationary-phase cultures of mutants of V. harveyi that are nonluminous without exogenous aldehyde, termed the aldehyde-deficient mutants. The inactivation in the wild type was halted by cell lysis and was slowed or stopped by O2 deprivation or by addition of KCN and NaF or of chloramphenicol. If KCN and NaF or chloramphenicol were added to a culture before the onset of luciferase inactivation, then luciferase inactivation did not occur. However, if these inhibitors were added after the onset of luciferase inactivation, then luciferase inactivation continued for about 2 to 3 h before the inactivation process stopped. The onset of luciferase inactivation in early stationary-phase cultures of wild-type cell coincided with a slight drop in the intracellular adenosine 5'-triphosphate (ATP) level from a relatively constant log-phase value of 20 pmol of ATP per microgram of soluble cell protein. Addition of KCN and NaF to a culture shortly after this drop in ATP caused a rapid decrease in the ATP level to about 4 pmol of ATP per microgram whereas chloramphenicol added at this same time caused a transient increase in ATP level to about 25 pmol/microgram. The aldehyde-deficient mutant (M17) showed a relatively constant log-phase ATP level identical with that of the wild-type cells, but rather than decreasing in early stationary phase, the ATP level increased to a value twice that in log-phase cells. We suggest that the inactivation of luciferase is dependent on the synthesis of some factor which is produced during stationary phase and is itself unstable, and whose synthesis is blocked by chloramphenicol or cyanide plus fluoride.     

The role of thiol redox systems in the resistance of <Emphasis Type="Italic">Escherichia coli</Emphasis> in the stationary phase

The effect of glutathione (gshA), thioredoxin (trxA), and thioredoxin reductase (trxB) mutations on the adaptation of Escherichia coli to prolonged glucose starvation was investigated. The trxB mutation had the worst consequences for the stationary-phase cells. These bacteria exhibited decreased survival, increased sensitivity to oxidants, and decreased expression of the katE and sulA genes. As the stationary phase proceeded, the physiological resistance to antibiotics increased in all the strains tested; however, the thiol redox system mutants were far less able to develop antibiotic resistance than the parent strain cells. During the stationary phase, a considerable shift was observed in the redox status of intra- and extracellular glutathione toward the oxidative values. These results indicate that the thiol redox systems play an important role in the adaptation of Escherichia coli to prolonged starvation and antibiotic resistance.     

When the Most Potent Combination of Antibiotics Selects for the Greatest Bacterial Load: The Smile-Frown Transition

Conventional wisdom holds that the best way to treat infection with antibiotics is to ‘hit early and hit hard’. A favoured strategy is to deploy two antibiotics that produce a stronger effect in combination than if either drug were used alone. But are such synergistic combinations necessarily optimal? We combine mathematical modelling, evolution experiments, whole genome sequencing and genetic manipulation of a resistance mechanism to demonstrate that deploying synergistic antibiotics can, in practice, be the worst strategy if bacterial clearance is not achieved after the first treatment phase. As treatment proceeds, it is only to be expected that the strength of antibiotic synergy will diminish as the frequency of drug-resistant bacteria increases. Indeed, antibiotic efficacy decays exponentially in our five-day evolution experiments. However, as the theory of competitive release predicts, drug-resistant bacteria replicate fastest when their drug-susceptible competitors are eliminated by overly-aggressive treatment. Here, synergy exerts such strong selection for resistance that an antagonism consistently emerges by day 1 and the initially most aggressive treatment produces the greatest bacterial load, a fortiori greater than if just one drug were given. Whole genome sequencing reveals that such rapid evolution is the result of the amplification of a genomic region containing four drug-resistance mechanisms, including the acrAB efflux operon. When this operon is deleted in genetically manipulated mutants and the evolution experiment repeated, antagonism fails to emerge in five days and antibiotic synergy is maintained for longer. We therefore conclude that unless super-inhibitory doses are achieved and maintained until the pathogen is successfully cleared, synergistic antibiotics can have the opposite effect to that intended by helping to increase pathogen load where, and when, the drugs are found at sub-inhibitory concentrations.     

High-throughput microfluidic system for long-term bacterial colony monitoring and antibiotic testing in zero-flow environments

In this study, a high-throughput microfluidic system is presented. The system is comprised of seven parallel channels. Each channel contains 32 square-shaped microchambers. After simulation studies on samples loaded into the microchambers, and the solute exchange between the microchambers and channels, the long-term culture of Escherichia coli (E. coli) HB101 in the microchambers is realized. Using the principle that L-arabinose (L-Ara) can induce recombinant E. coli HB101 pGLO to synthesize green fluorescent protein (GFP), the real-time analysis of GFP expression in different initial bacterial densities is performed. The results demonstrate that higher initial loading densities of the bacterial colony cause bacterial cell to enter log-phase proliferation sooner. High or low initial loading densities of the bacterial cell suspension induce the same maximum growth rates during the log-phase. Quantitative on-chip analysis of tetracycline and erythromycin inhibition on bacterial cell growth is also conducted. Bacterial morphology changes during antibiotic treatment are observed. The results show that tetracycline and erythromycin exhibit different inhibition activities in E. coli cells. Concentrations of 3 μg/mL tetracycline can facilitate the formation of long filamentous bacteria with the average length of more than 50 μm. This study provides an on-chip framework for bacteriological research in a high-throughput manner and the development of recombinant bacteria-based biosensors for the detection of specific substances.     

Combinations of antibiotics and nonantibiotic drugs enhance antimicrobial efficacy

Combinations of antibiotics are commonly used in medicine to broaden antimicrobial spectrum and generate synergistic effects. Alternatively, combination of nonantibiotic drugs with antibiotics offers an opportunity to sample a previously untapped expanse of bioactive chemical space. We screened a collection of drugs to identify compounds that augment the activity of the antibiotic minocycline. Unexpected synergistic drug combinations exhibited in vitro and in vivo activity against bacterial pathogens, including multidrug-resistant isolates.     

Protein tyrosine phosphatase activity inLeishmania donovani

L. Donovani promastigotes were grown to late-log and 3-day stationary phase to determine the level of protein tyrosine phosphatase activity in crude extracts and in fractions following gel filtration column chromatography. Over 90% of the activity was soluble in a low salt extraction buffer in both phases of growth. Several peaks of activity were resolved following gel filtration of the crude extracts indicating that multiple tyrosine phosphatases are present in these cells. Tyrosine phosphatase activity was lower in 3-day stationary than in late log-phase cells and a reduction in the major peak of activity, eluting in a gel fraction corresponding to an M _r of approximately 168kDa, was observed.In vivo tyrosine phosphorylation was revealed by Western blot analysis. The degree of phosphorylation of at least two proteins differed in cells obtained from late log phase cultures as compared with 3-day stationary phase cultures. These observations indicate that changes in the balance between tyrosine phosphorylation and dephosphorylation occur with increasing culture age.Abbreviations MBP myelin basic protein - PMSF phenyl-methanesulfonylfluoride - PTP protein tyrosine phosphatase - RCML reduced, carboxyamidomethylated, maleylated lysozyme - YINAS Tyr-Ile-Asn-Ala-Ser     

The Effect of Ochre Suppression on Meiosis and Ascospore Formation in Saccharomyces

The effect of altered tyrosyl-tRNAs on the developmental process of sporulation was examined. Mutations in eight independent loci resulting in tyrosine-inserting nonsense suppressor were tested for their effects on sporulation. Different levels of inhibition were found ranging from SUP3-omicron, which caused the greatest reduction of sporulation (7-17% of wild type), to SUP11-omicron which caused no reduction in sporulation. Since the SUP3-omicron mutation exhibited the greatest effect, it was studied in detail. Although SUP3-omicron is a dominant nonsense suppressor, its effect on sporulation is recessive. Expression of the sporulation deficiency is dependent upon the stage of transfer from glucose growth medium (i.e., log, early stationary, etc.) to sporulation medium. SUP3-omicron/SUP3-omicron diploid cells transferred from log or early stationary phase are capable of sporulation, whereas cells transferred after early stationary phase (i.e., after adaptation to respiration) exhibit poor sporulative ability. Sporulation events were examined under restrictive conditions to observe those events completed by SUP3-omicron/SUP3-omicron diploids. The early events of sporulation occur in these cells. Later events are completed by progressively fewer cells. Premeiotic DNA synthesis occurred in approximately 40% of the cells, nuclear segregation occurred in 20%, and finally, only 2% formed asci. The fact that fewer late-sporulation events occur under restrictive conditions can be explained by increased efficiency of suppression.