The utilization of morpholine as a sole nitrogen source by Gram-negative bacteria

The ability of Gram-negative bacteria to degrade morpholine when growing in pure culture is reported for the first time. Several bacterial strains were able to degrade morpholine and to utilize it as a sole nitrogen source but not as a sole carbon and energy source. The organisms studied were obtained from river water and activated sludge and could not be isolated directly on morpholine-containing media which always yielded growth of Gram-positive bacteria using morpholine as a carbon and energy source. The Gram-negative strains were isolated on the basis of their ability to grow on the structurally-related heterocyclc amines piperidine and pyrrolidine.     

The frequency and characteristics of highly thermophilic bacteria in cool soil environments

Following enrichment at 70 degrees C and 80 degrees C, five highly thermophilic aerobic eubacteria have been isolated from cool soil environments. These organisms show a temperature range for growth of 40-80 degrees C and have optimal and very high growth rates around 70 degrees C with generation times less than 30 min. All isolates are narrow rods, which stain Gram-negative, but have a Gram-positive cell wall structure and only one of five isolates is a spore former. All cultures contain a small proportion of previously unreported extremely long flexuous rods, which can be seen to divide eventually. Biochemical testing of five strains reveals a significant ability to utilize alkanes and some aromatic hydrocarbons. Using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) of 16S rDNA the five strains were differentiated into three categories, which paralleled the biochemical results. 16S rDNA sequences showed high similarity with thermophilic Bacillus species now reclassified as Geobacillus. These bacteria are present in high numbers in apparently all soils and the question is raised of how these organisms, which are apparently unable to grow at the temperatures experienced in these cool soils, are so prominent.     

Glucanase-producing organisms in human dental plaques.

Selective media were used to isolate a wide range of bacteria from sixty human dental plaques. Glucanase activities of the isolates were determined on dextran- and starch-containing media. All sixty samples of dental plaque yielded some colonies showing amylolytic and dextranolytic activities. The glucanase-producing organisms comprised 20% of the isolates. Of these 38% were Gram-positive rods, 27% Gram-positive cocci, 28% Gram-negative rods and 7% were Gram-negative cocci. The cultural groups most commonly represented among the glucanase-producing isolates were Actinomycetaceae, streptococci, haemophili and Gram-negative anaerobes. Species prominent among these isolates included Streptococcus sanguis, Streptococcus mitior, Actinomyces naeslundii, Actinomyces viscosus, Bacterionema matruchotii, Bifidobacterium sp. and Bacteroides sp. No isolates capable of degrading starch or dextran were identified as Streptococcus milleri, Rothia dentocariosa or Fusobacterium sp. This study has shown that a wide range of bacterial species commonly isolated from human dental plaques exhibit both amylolytic and dextranolytic activities. In order to understand glucan metabolism in human dental plaques further investigation of these catabolic activities is necessary.     

Studies in Gram Staining

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuchsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal viokt-stained organisms with alcoholic safranin (0.25%) for 15 scc will distinguish Gram-positive bacteria (viokt) from Gram-negative bacteria (pink).

Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily dccolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Studies in gram staining.

Gram-negative bacteria stained with crystal violet are decolorized by 95% alcohol within 2 min, whereas Gram-positive bacteria require at least 3 min treatment. Aqueous solutions of safranin, neutral red, and fuschsin replace crystal violet from stained Gram-positive bacteria more quickly than alcohol alone, and alcoholic solutions of these counterstains are in most cases still more effective. Treatment of crystal violet-stained organisms with alcoholic safranin (0.25%) for 15 sec will distinguish Gram-positive bacteria (violet) from Gram-negative bacteria (pink). Alcohol containing very low concentrations of iodine generally decolorizes crystal violet-stained Gram-positive bacteria more quickly than alcohol alone. Increasing concentrations of iodine in alcohol reduce the rate of decolorization of stained bacteria, but stained Gram-negative bacteria are still readily decolorized. The addition of 0.1% iodine to alcohol increases the rate of extraction of crystal violet by alcohol from Gram-negative organisms, but delays extraction of dye from Gram-positive organisms, and this applies when counterstain is also present. A two-solution modification of Gram staining is described in which crystal violet-stained bacteria are treated with an alcoholic solution of safranin, fuchsin, and iodine.     

Common components of industrial metal-working fluids as sources of carbon for bacterial growth

Water-based metal-working fluids used in large-scale industrial operations consist of many components, but in the most commonly used formulations only three classes of components are present in high enough concentrations that they could, in principle, provide enough carbon to support the high bacterial densities (10 CFU/ml) often observed in contaminated factory fluids. These components are petroleum oil (1 to 5%), petroleum sulfonates (0.1 to 0.5%), and fatty acids (less than 0.1%, mainly linoleic and oleic acids supplied as tall oils). We isolated pure strains of predominating bacteria from contaminated reservoirs of two metal-working systems and randomly selected 12 strains which we tested in liquid culture for growth with each of the metal-working fluid components as the sole source of carbon. Of the 12 strains, 7 reached high density (10 CFU/ml from an initial inoculum of less than 2 x 10) in 24 h, and 1 strain did the same in 48 h with 0.05% oleic or linoleic acid as the carbon source. These same strains also grew on 1% naphthenic petroleum oil but required up to 72 h to reach densities near 10 CFU/ml. One strain grew slightly and the others not at all on the petroleum sulfonates. The four remaining strains did not grow on any of the components, even though they were among the predominating bacteria in the contaminated system. Of the seven strains that grew best on the fatty acids and on the naphthenic petroleum oil, five were tentatively identified as Acinetobacter species and two were identified as Pseudomonas species. Four of the bacteria that did not grow were tentatively identified as species of Pseudomonas, and one could not be identified.     

医院内尿路感染致病菌变迁及其耐药性监测分析

目的调查院内尿路感染致病菌分布及耐药性变化情况.方法对我院1998年1月-2000 年12月医院内尿路感染患者分离的细菌菌株、真菌菌株及细菌耐药性进行回顾性调查.结果 医院内尿路感染仍以G 菌为主(39.9%),其次为真菌(32.9%),G+菌(27.2%);G -菌以大肠埃希菌为主(41.7%),G+菌以D组链球菌为主(50.3%),真菌以白色念珠菌为主(44.7%),与19 98年分离的菌株相比,2000年G 菌所占比例有下降趋势,而真菌由25.7%上升至36.6%(P< 0.05),3年间主要病原菌对常用抗生素的耐药率基本呈上升趋势.结论院内尿路感染致病菌正在发生变迁 ,耐药性严重,临床应重视病原学检查,开展细菌耐药性监测,合理使用抗生素.     

Isolation and molecular characterization of a novel broad-host-range plasmid from Bordetella bronchiseptica with sequence similarities to plasmids from Gram-positive organisms

A 2.6 kb plasmid, named pBBR1, was isolated from Bordetella bronchiseptica S87. After insertion of an antibiotic resistance marker, this plasmid could be transferred into Escherichia coli, Bordetella pertussis, B. bronchiseptica, Vibrio cholerae, Rhizobium meliloti, and Pseudomonas putida by transformation or conjugation. Conjugation was possible only when the IncP group transfer functions were provided in trans. As shown by incompatibility testing, pBBR1 does not belong to the broad-host-range IncP, IncQ or IncW groups. DNA sequence analysis revealed two open reading frames: one was called Rep, involved in replication of the plasmid, and the other, called Mob, was involved in mobilization. Both the amino-terminal region of Mob and its promoter region show sequence similarities to Mob/Pre proteins from plasmids of Gram-positive bacteria. In spite of these sequence similarities, pBBR1 does not replicate via the rolling-circle mechanism commonly used by small Gram-positive plasmids. We therefore speculate that pBBR1 may combine a mobilization mechanism of Gram-positive organisms with a replication mechanism of Gram-negative organisms. Determination of the plasmid copy number in E. coli and B. pertussis indicated that pBBR1 has a rather high copy number, which, in conjunction with its small size and broad host range, renders it particularly interesting for studies of broad-host-range replicons and for the development of new cloning vectors for a wide range of Gram-negative bacteria.     

酸性土壤中耐铝细菌的筛选鉴定及其耐铝能力分析

以含有1mmol／LAl3＋的s—LB培养基作为筛选培养基,从酸性土壤中分离到13株耐铝的细菌菌株,选取其中6株进行形态学分析,结果观察到这些菌株的菌体均呈杆状,其中1株为革兰阳性反应,其余5株为革兰阴性反应。以细菌通用引物扩增这些菌株的16SrDNA并测序,将得到的序列与GenBank中的序列进行BLAST比对,利用MEGA4．0软件,按照Neighbor-joining法构建系统进化树,这6个菌株分别与Enterobacter endosymbiont,Serratia marcescens ,Pantoea agglomerans ,Enterobacter aerogenes .Bacillus subtilis 和 Enterobacter asburiae的亲缘关系最近。将这些菌株接种到加有2mmol／LAl3＋、pH4．5的s—LB固体培养基上培养时,它们都能生长,说明这些菌株具有较好的耐铝能力,这些菌株为进一步研究细菌的耐铝机制提供了极好的材料。     

Occurrence and some properties of aerobic psychrophilic soil bacteria

Summary The occurrence of aerobic psychrophilic bacteria in different types of soil was studied in regard to all the bacteria present. They constitute a considerable percentage of all the bacteria present in the soils investigated and represent a constant component of the soil microflora, irrespective of the season of the year. They are almost exclusively rod-like, half Gram-negative and half Gram-positive, non-sporulating organisms. It was found, however, that their reaction to Gram-straining varies and depends on the incubation temperature. The temperature of 35°C inhibited the growth of about 40 per cent of the selected psychrophilic strains and caused the development of involutionary forms in 37 per cent of the strains grown. The psychrophilic bacteria isolated from the soils mostly showed ability to decompose urea, and were concerned with ammonification of peptones and reduction of nitrates.Due attention was also given to the problem of adequate incubation temperature in the investigations on soil microorganisms.     

The theft of host heme by Gram-positive pathogenic bacteria

The element iron is essential for bacteria and plays a key role in the virulence and pathology of bacterial diseases. The largest reservoir of iron within the human body is in protoporphyrin IX, the compound commonly referred to as heme and bound by hemoglobin. For many years, the study of heme uptake in bacteria was restricted to Gram-negative organisms. However, recent studies have shed light on how bacteria containing a thick peptidoglycan, such as Gram-positive bacteria, acquire and transport heme. This review summarizes old and new research covering the acquisition, transport, and utilization of heme in Gram-positive bacterial pathogens.     

Aerobic Gram-Positive and Gram-Negative Bacteria Exhibit Differential Sensitivity to and Transformation of 2,4,6-Trinitrotoluene (TNT)

A systematic evaluation of the ability of different bacterial genera to transform 2,4,6-trinitrotoluene (TNT), and grow in its presence, was conducted. Aerobic Gram-negative organisms degraded TNT and evidenced net consumption of reduced metabolites when cultured in molasses medium. Some Gram-negative isolates transformed all the initial TNT to undetectable metabolites, with no adsorption of TNT or metabolites to cells. Growth and TNT transformation capacity of Gram-positive bacteria both exhibited 50% reductions in the presence of approximately 10 μg TNT ml⁻¹. Most non-sporeforming Gram-positive organisms incubated in molasses media amended with 80 μg TNT ml⁻¹ became unculturable, whereas all strains tested remained culturable when incubated in mineral media amended with 98 μg TNT ml⁻¹, indicating that TNT sensitivity is linked to metabolic activity. These results indicate that the microbial ecology of soil may be severely impacted by TNT contamination. Received: 2 December 1996 / Accepted: 3 February 1997     

Cadmium uptake by growing cells of gram-positive and gram-negative bacteria

The present study evaluates the effect of the cadmium (Cd2+) on the growth and protein synthesis of some Gram-positive (Staphylococcus aureus, Bacillus subtilis and Streptococcus faecium) and Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacteria and the cadmium uptake by the same micro-organisms. The Gram-negative bacteria tested were less sensitive to metal ions than the Gram-positive, and P. aeruginosa was the most resistant. The Gram-negative bacteria were also able to accumulate higher amounts of cadmium during growth than the Gram-positive bacteria. The maximum values of specific metal uptake (microgram of Cd2+ incorporated per mg of protein) were: 0.52 for S. aureus, 0.65 for S. faecium, 0.79 for B. subtilis, 2.79 for E. coli and 24.15 for P. aeruginosa, respectively. The differences in the ability to accumulate metal found between Gram-negative and Gram-positive bacteria seems to account for different mechanisms of metal resistance.     

Microorganisms isolated from the water phase of tropospheric clouds at the Puy de Dôme: major groups and growth abilities at low temperatures

This work constitutes the first large report on aerobic cultivable microorganisms present in cloud water. Seven cloud-event samples were collected at the Puy de D?me summit, and cultivation was performed leading to the isolation of 71 bacterial, 42 fungal and 15 yeast strains. Most of the fungi isolated were of Cladosporium or Trametes affiliation, and yeasts were of Cryptococcus affiliation. Bacteria, identified on the basis of their 16S rRNA gene sequence, were found to belong to Actinobacteria, Firmicutes, Proteobacteria (Alpha, Beta and Gamma subclasses) and Bacteroidetes phyla, and mainly to the genera Pseudomonas, Sphingomonas, Staphylococcus, Streptomyces, and Arthrobacter. These strains appear to be closely related to some bacteria described from cold environments, water (sea and freshwater), soil or vegetation. Comparison of the distribution of Gram-negative vs. Gram-positive bacteria shows that the number of Gram-negative bacteria is greater in summer than in winter. Finally, a very important result of this study concerns the ability of half of the tested strains to grow at low temperatures (5 degrees C): most of these are Gram-negative bacteria, and a few are even shown to be psychrophiles. On the whole, these results give a good picture of the microbial content of cloud water in terms of classification, and suggest that a large proportion of bacteria present in clouds have the capacity to be metabolically active there. This is of special interest with respect to the potential role of these microorganisms in atmospheric chemistry.     

Comparison of cadmium biosorption by Gram-positive and Gram-negative bacteria from activated sludge

Summary Gram-positive and Gram-negative bacteria were isolated from activated sludge and used to evaluate differences in cadmium biosorption. Gram-positive bacteria exhibited approximately 20% more cadmium biosorption at 30°C and pH 6.6 than Gram-negatives. Biosorption was largely passive in both cases, although metabolic uptake appeared to occur to a higher extent with Gram-positive bacteria.     

Bladder cytopathic effects associated with Gram-negative bacterial genera

The interaction of bacteria belonging to several genera with the bladder epithelium was studied. An in vivo rat bladder model system was used to evaluate the cytopathic effects of members of eight representative genera of bacteria and six strains ofEscherichia coli on the bladder surface. Alterations were noted mainly with the Gram-negative bacteria (Escherichia, Klebsiella, Enterobacter, Proteus, Pseudomonas), although some strains were associated with more cytopathology than others. Foci of cytopathology consisted of swelling, exfoliated epithelial cells, strand formation, and ulceration. Aggregates of bacteria, epithelial cells, and debris were shed into the bladder lumen. In contrast, members of three genera of Gram-positive organisms (Lactobacillus, Streptococcus, Staphylococcus) caused little or no cytopathology. The study strongly suggests that the bladder responds in a relatively uniform manner to viable Gram-negative organisms, while the response to Gram-positive organisms is minimal.     

Synergistic action of Galleria mellonella anionic peptide 2 and lysozyme against Gram-negative bacteria

Lysozyme and antimicrobial peptides are key factors of the humoral immune response in insects. In the present work lysozyme and anionic defense peptide (GMAP2) were isolated from the hemolymph of the greater wax moth Galleria mellonella and their antibacterial activity was investigated. Adsorption of G. mellonella lysozyme on the cell surface of Gram-positive and Gram-negative bacteria was demonstrated using immunoblotting with anti-G. mellonella lysozyme antibodies. Lysozyme effectively inhibited the growth of selected Gram-positive bacteria, which was accompanied by serious alterations of the cell surface, as revealed by atomic force microscopy (AFM) imaging. G. mellonella lysozyme used in concentrations found in the hemolymph of naive and immunized larvae, perforated also the Escherichia coli cell membrane and the level of such perforation was considerably increased by GMAP2. GMAP2 used alone did not perforate E. coli cells nor influence lysozyme muramidase activity. However, the peptide induced a decrease in the turgor pressure of the bacterial cell. Moreover, in the samples of bacteria treated with a mixture of lysozyme and GMAP2 the sodium chloride crystals were found, suggesting disturbance of ion transport across the membrane leading to cell disruption. These results clearly indicated the synergistic action of G. mellonella lysozyme and anionic peptide 2 against Gram-negative bacteria. The reported results suggested that, thanks to immune factors constitutively present in hemolymph, G. mellonella larvae are to some extent protected against infection caused by Gram-negative bacteria.     

In vitro activity of ramoplanin and comparator drugs against anaerobic intestinal bacteria from the perspective of potential utility in pathology involving bowel flora

Susceptibility of intestinal bacteria to various antimicrobial agents in vitro, together with levels of those agents achieved in the gut, provides information on the likely impact of the agents on the intestinal flora. Orally administered drugs that are poorly absorbed may be useful for treatment of intestinal infections and for certain other situations in which intestinal bacteria may play a role. The antimicrobial activity of ramoplanin (MDL 62,198) against 928 strains of intestinal anaerobic bacteria was determined using the NCCLS-approved Wadsworth brucella laked-blood agar dilution method. The activity of ramoplanin was compared with that of ampicillin, bacitracin, metronidazole, trimethoprim/sulfamethoxazole (TMP/SMX), and vancomycin. The organisms tested included Bacteroides fragilis group (n=89), other Bacteroides species (n=16), other anaerobic Gram-negative rods (n=56) anaerobic cocci (n=114), Clostridium species (n=426), and non-sporeforming anaerobic Gram-positive rods (n=227). The overall MIC(90)s of ramoplanin, ampicillin, bacitracin, metronidazole, and vancomycin were 256, 32, 128, 16, and 128 mcg/ml, respectively. Ramoplanin was almost always highly active vs. Gram-positive organisms and relatively poor in activity against Gram-negative organisms, particularly Bacteroides, Bilophila, Prevotella, and Veillonella. Vancomycin was quite similar to ramoplanin in its activity. Ampicillin was relatively poor in activity vs. organisms that often produce beta-lactamase, including most of the Gram-negative rods as well as Clostridium bolteae and C. clostridioforme. Bacitracin was relatively poor in activity against most anaerobic Gram-negative rods, but better vs. most Gram-positive organisms. Metronidazole was very active against all groups other than bifidobacteria and some strains of other types of non-sporeforming Gram-positive bacilli. TMP/SMX was very poorly active, with an MIC(90) of >2048 mcg/ml.     

Regulatory mechanisms differ in UMP kinases from gram-negative and gram-positive bacteria

In this work, we examined the regulation by GTP and UTP of the UMP kinases from eight bacterial species. The enzyme from Gram-positive organisms exhibited cooperative kinetics with ATP as substrate. GTP decreased this cooperativity and increased the affinity for ATP. UTP had the opposite effect, as it decreased the enzyme affinity for ATP. The nucleotide analogs 5-bromo-UTP and 5-iodo-UTP were 5-10 times stronger inhibitors than the parent compound. On the other hand, UMP kinases from the Gram-negative organisms did not show cooperativity in substrate binding and catalysis. Activation by GTP resulted mainly from the reversal of inhibition caused by excess UMP, and inhibition by UTP was accompanied by a strong increase in the apparent K(m) for UMP. Altogether, these results indicate that, depending on the bacteria considered, GTP and UTP interact with different enzyme recognition sites. In Gram-positive bacteria, GTP and UTP bind to a single site or largely overlapping sites, shifting the T R equilibrium to either the R or T form, a scenario corresponding to almost all regulatory proteins, commonly called K systems. In Gram-negative organisms, the GTP-binding site corresponds to the unique allosteric site of the Gram-positive bacteria. In contrast, UTP interacts cooperatively with a site that overlaps the catalytic center, i.e. the UMP-binding site and part of the ATP-binding site. These characteristics make UTP an original regulator of UMP kinases from Gram-negative organisms, beyond the common scheme of allosteric control.     

Imipenem in the treatment of patients with severe surgical infection]

The clinical efficacy and safety of intravenously administered imipenem/cilastatin in the treatment of 45 patients with severe bacterial septicemia due to intra-abdominal abscesses, respiratory and urinary tract as well as skin, soft tissue and bone infections was studied in the prospective and open trial. The in vitro antimicrobial activity of imipenem has been assessed on the basis of 909 bacterial strains isolated from patients treated and non-treated with imipenem/cilastatin. Among them were 526 Gram-negative, 370 Gram-positive aerobic bacteria and 13 Gram-negative anaerobic bacteria (Bacteroides sp.). Pathogen susceptibility to imipenem was determined with a disc-diffusion technique using Merck, Sharp Dohme sensitive discs containing 10 mcg of imipenem. Highly sensitive to imipenem were 96.8% of Gram-negative 82.7% of Gram-positive aerobic bacteria and 100% of Bacteroides sp. All patients, in whom evident foci of infection e.g. intra-abdominal abscesses were discovered, were operated on. The dosage of imipenem/cilastatin ranged from 1.5 to 2.0 g/24 h. Clinical cure and bacteriological elimination was achieved in 39 (86.7%) of patients while 6 (13.3%) showed marked clinical improvement. Before and during therapy, aerobic and anaerobic cultures were taken from accessible sites. All specimens were worked up using conventional bacteriological techniques. Before during and after therapy, samples for hematology, biochemistry and urinanalysis were obtained. Adverse clinical effects were noted in 2 (4.4%) patients. One had nausea and vomiting which were probably related to rapid infusion and disappeared after increasing the administration time, and one had transient diarrhea. In conclusion, imipenem/cilastatin was a well tolerated and effective drug in the treatment of life-threatening surgical infections.