Polyunsaturated fatty acids in the psychrophilic bacterium Shewanella gelidimarina ACAM 456T: molecular species analysis of major phospholipids and biosynthesis of eicosapentaenoic acid.

The production of eicosapentaenoic acid [20:5omega3; EPA] from Shewanella gelidimarina (ACAM 456T) was investigated with respect to growth temperature and growth on sole carbon sources. The percentage and quantitative yield of EPA remained relatively constant at all growth temperatures within or below the optimal growth temperature region. At higher growth temperatures, these values decreased greatly. Growth on differing sole carbon sources also influenced the percentage and amount of EPA produced, with the fatty acid composition influenced by provision of potential acyl chain primers as sole carbon sources. The highest amounts of EPA occurred from growth on propionic acid and L-leucine respectively, while the highest percentage of EPA occurred from growth on L-proline. Monounsaturated fatty acid components and EPA were concentrated in phosphatidylglycerol (PG), while the proportion of branched-chain fatty acids was elevated in phosphatidylethanolamine (PE); the two major phospholipid classes. Specific associations of EPA with other acyl chains were identified within cellular phospholipid classes. The association of EPA with 17:1 and 18:0 acyl chains in phospholipid species was specific to PG, whereas the association of EPA with i13:0/13:0 and 14:0/i14:0 was specific to PE. Such acyl chain 'tailoring' is indicative of the important role of EPA in bacterial membrane adaptive responses. EPA was also a large component (22%) of a non-esterified fatty acid (NEFA) fraction within the total lipid extract of the bacterium. This may point toward a particular role of NEFA in polyunsaturated fatty acid (PUFA) metabolism. The formation of EPA was investigated by labelling with L-[U-14C]serine and sodium [1-14C]acetate. The accumulation of radiolabel within unsaturated intermediates (di-, tri- and tetraunsaturated fractions) was low, indicating a rapid formation and derivatisation of these components. Similar results were found for the unsaturated fatty acid fractions of both PE and PG using sodium [1-14C]acetate radiolabel. The regulation of triunsaturated fatty acid components may be a potential control site in PUFA biosynthesis.

Biosynthesis of furan fatty acids (F-acids) by a marine bacterium, Shewanella putrefaciens.

A mutant derived from Shewanella putrefaciens 8CS7-4 treated with N-methyl-N'-nitro-N-nitrosoguanidine was found to produce 15-20 mg of a furan fatty acid (F-acid), 10,13-epoxy-11-methyloctadeca-10,12-dienoic acid (F18), per liter of growth medium (10-15% of total fatty acids). Capillary gas chromatography-mass spectrometry and proton nuclear magnetic resonance analysis of the fatty acid methyl esters of the mutant revealed the presence of other F-acids, 8,11-epoxy-9-methylhexadeca-8,10-dienoic acid (F16), 6,9-epoxy-7-methyltetradeca-6,8-dienoic acid (F14), and methyl branched unsaturated fatty acids, 11-methyl-12E-octadecenoic acid (11-me-18:1) and 11-methyl-10E,12E-octadecadienoic acid (1-me-18:2). About 90% of F-acids were present in phospholipids, in which the F-acids were found to be exclusively linked at the sn-1 position. 11-me-18:1 and 11-me-18:2 were also detected in the sn-1 position. Firstly, 11-me-18:1 increased and reached a maximum at 12 h, and then decreased rapidly. Secondly, the 11-me-18:2 content reached a maximum at 24 h, when 11-me-18:1 was little detected, and then decreased. Finally, the amount of F18 began to increase after 20 h and reached a plateau at 36 h. These results suggest that 11-me-18:1 and 11-me-18:2 are precursors of F18.

Terminal oxidases of Azoarcus sp. BH72, a strictly respiratory diazotroph.

The nitrogen-fixing, grass-associated bacterium Azoarcus sp. BH72 was characterized with respect to its terminal oxidases. Inhibitory respiratory analysis revealed the presence of at least one cytochrome c oxidase and one quinol oxidase. The cytochrome c oxidase was preferably used by the cells under aerobic, whereas the quinol oxidase seemed to be dominant under microaerobic, nitrogen-fixing conditions. Differential spectroscopy and heme analysis of the membrane preparations indicated that the cytochrome c oxidase is probably of the cb type.

Antimicrobial activity and spectrum investigation of eight broad-spectrum beta-lactam drugs: a 1997 surveillance trial in 102 medical centers in the United States. Cefepime Study Group.

Because antimicrobial agents become less effective after the emergence of resistance mechanisms in clinically prevalent pathogens, physicians must utilize local, regional, and national antimicrobial susceptibility surveillance data to assist in choices of appropriate agents. An investigation of the spectrum and potency of eight broad-spectrum beta-lactam drugs (cefepime, cefotaxime, ceftazidime, ceftriaxone, imipenem, piperacillin with or without tazobactam, and ticarcillin/clavulanic acid) was performed using a common protocol and method (Etest; AB BIODISK, Solna, Sweden) in 102 clinical microbiology laboratories in the United States. A total of 9777 strains of Gram-negative bacilli were tested from late 1996 through April 1997. Quality assurance measures using three control strains observed quality control failures in 13 laboratories (usually ticarcillin/clavulanic acid or piperacillin), but only 2% of results required deletion. A total of 33.4% of Enterobacter spp. (1977 strains) were either resistant or intermediately susceptible to ceftazidime. Only imipenem (99.6% susceptible) and cefepime (99.1%) remained highly active against strains of Enterobacter, as well as Citrobacter freundii, indole-positive Proteae, and Serratia spp. Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae were detected at rates of 10.3% and 23.8%, respectively. Although these were participant-selected strains, only imipenem and cefepime had broad-spectrum coverage (> or = 97.1%) against these extended-spectrum beta-lactamase phenotypes. A dominant number of these extended-spectrum beta-lactamase phenotypes were reported from medical centers in the Northeast, but a nationwide distribution was observed. Among the nonenteric Gram-negative bacilli (4057 strains), the rank order of susceptibility (percent inhibited at published breakpoint concentrations) was: imipenem (86.1%) > piperacillin/tazobactam (80.1%) > cefepime (77.1%) > ceftazidime = piperacillin (74.9%) > ticarcillin/clavulanic acid (61.6%) > cefotaxime (18.2%) > ceftriaxone (12.9%). The cephalosporins, cefepime and ceftazidime, had rates of resistance for the 3005 Pseudomonas aeruginosa isolates of 10.1% and 14.4%, respectively. For all Gram-negative strains tested, only two contemporary beta-lactam antimicrobials exhibited > 90% inhibition of strains, imipenem at 93.6% and cefepime at 90.2%. These drugs were superior to the other tested compounds (48.8-84.3%). Ticarcillin/clavulanic acid had the narrowest spectrum of activity (48.8% of isolates susceptible). These results indicate that carbapenems and a new fourth-generation cephalosporin, cefepime, possess usable in vitro potencies against current clinical strains of Gram-negative bacilli, many of which harbored resistance to other antimicrobial agents.

Structure and properties of novel inclusions in Shewanella putrefaciens.

Cytoplasmic inclusions surrounded by a bilayer membrane were seen in thin sections. negatively stained and freeze-fractured preparations of Shewanella putrefaciens. Cells harvested from the late exponential and early stationary phase showed a higher number of these vesicles than bacteria isolated from early exponential or late stationary phase. Chemical dyes for polyphosphate or poly-beta-hydroxybutyrate did not stain the material enclosed within these vesicles. Elemental analysis of the material indicated that the content was organic in nature and might be a protein. HPLC analysis of the material showed that it was probably not a carbon source, nor an electron acceptor used by S. putrefaciens.

Broad host range plasmids carrying the Escherichia coli lactose and galactose operons.

We have developed a number of broad-host-range plasmids that allow the expression of the Escherichia coli lac operon from any cloned promoter, and the creation of 'in phase' fusions between lacZ and other cloned genes. In a second series of constructions, the E. coli gal operon has been cloned into the broad-host-range vector and a plasmid carrying both the E. coli gal and lac genes is described. These plasmids have been transferred into Pseudomonas aeruginosa and Zymomonas mobilis and their effects on the utilisation of lactose and galactose have been investigated.

Thauera linaloolentis sp. nov. and Thauera terpenica sp. nov., isolated on oxygen-containing monoterpenes (linalool, menthol, and eucalyptol) nitrate.

The monoterpenes menthol, linalool, and eucalyptol were recently used as sole electron donor and carbon source for the isolation of three denitrifying bacterial strains 21Mol, 47Lol, and 58Eu. The motile, mesophilic, Gram-negative rods had a strictly respiratory metabolism. Monoterpenes were completely mineralised to carbon dioxide, nitrate was reduced to dinitrogen. Strain 47Lol utilised aliphatic monoterpenes, strain 21Mol oxygenated monocyclic monoterpenes, and strain 58Eu the bicyclic eucalyptol and monocyclic monoterpene alkenes. The fatty acid composition of the strains indicated an allocation to the rRNA group III of pseudomonads. Comparative 16S rRNA gene sequence analyses revealed that the new isolates can be assigned as members of the genus Thauera within the beta subclass of Proteobacteria. DNA-DNA hybridisation studies indicated a relateness of 68.5% between strains 21Mol and 58Eu which shared 36.0% and 40.6% DNA similarity with strain 47Lol. The strains are described as new species belonging to the genus Thauera, strain 47Lol (DSM 12138T) as T. linaloolentis sp. nov. and strains 21Mol and 58Eu as T. terpenica sp. nov. with strain 58Eu (DSM 12139T) as type strain.

Modelling of microbial activity and prediction of shelf life for packed fresh fish.

Prediction of shelf life based on growth of specific spoilage organisms (SSO) in model substrates was studied. The effect of CO2 on the growth kinetics for Photobacterium phosphoreum and Shewanella putrefaciens was quantified and modelled. Results showed that microbial spoilage of packed cod stored with various concentrations of CO2 was accurately predicted from the effect of CO2 on P. phosphoreum grown in model substrates. The short shelf life extensions previously reported for packed cod therefore can be explained by the high CO2 resistance of this Gram negative organism. S. putrefaciens was very sensitive to CO2 and growth rates could not be related to the shelf life of packed cod. Growth curves without lag phases were found for all concentrations of CO2 and for both the microorganisms studied. For the fitting of these growth curves the log-transformed Logistic models were selected after comparison with the 'modified Gompertz' models and with the model of Baranyi et al. (1993). The effect of CO2 on mu max was well described by a 2 parameter square root model. Validation of kinetic models by comparison of shelf life predictions with shelf life determined by sensory evaluations in product experiments was preferred for comparison of microbial growth rates determined in product and model system experiments. Kinetic modelling was found to be valuable for both evaluation and prediction of microbial fish spoilage and an iterative approach for development of kinetic shelf life models was suggested.

Origin and spoilage potential of the microbiota dominating genus Psychrobacter in sterile rehydrated salt-cured and dried salt-cured cod (Gadus morhua).

Salt-cured and dried salt-cured cod rehydrated using sterile water and equipment have a short shelf life at 4 degrees C due to high bacterial counts. The microbiota develops off-odours which partly can be described as musty, causing sensory rejection within 7-10 days of chilled storage. The microbiota composition was studied in a total of 38 samples obtained from 10 different, both commercial and laboratory produced, salt-cured and dried salt-cured cod products. The dominating bacterium, representing at least 90% of the total viable count in all products studied, was identified as belonging to the genus Psychrobacter; a Gram-negative, oxidase- and catalase-positive, nonpigmented, halotolerant, psychrotolerant, facultative aerobe and nonmotile bacterium. The morphology of the bacterium resembles coccobacilli and the cells occur most often in pairs. The bacterium was able to hydrolyze lipids, but not proteins. It did not produce H(2)S or TMA and the spoilage in rehydrated salt-cured and dried salt-cured cod is therefor different from what is observed in fresh cod. However, samples inoculated with Psychrobacter immobilis gave the same musty odour as spoiled control samples but earlier in the storage period and of a stronger intensity. In a field experiment, carried out to investigate the origin of the dominating bacterium, it was found that the microbiota in both sterile rehydrated commercially produced and laboratory (aseptically) produced salt-cured cod was dominated by this same bacterium. The bacterium was also isolated from cod skin mucus immediately after capture. The bacterium survived NaCl concentrations up to 25% (w/v) NaCl, stating its ability to survive during the salt-curing process. The dominating bacterium in rehydrated salt-cured and dried salt-cured cod seems to mainly originate from the fresh fish itself and not from contamination during processing.

Effect of modified atmosphere packaging on the TVB/TMA-producing microflora of cod fillets.

Cod fillets (Gadus morhua) were packed under modified atmospheres, with four different gas compositions (60% CO2-10% O2-30% N2, 60% CO2-20% O2-20% N2, 60% CO2-30% O2-10% N2, 60% CO2-40% O2), and stored at 6 degrees C. Plate counts were carried out after 3, 4, 5, 6 and 7 days, to follow the growth of aerobic and anaerobic bacteria, lactic acid bacteria, H2S-producing bacteria and Enterobacteriaceae. The production of total volatile bases (TVB) and trimethylamine (TMA), and the changes in pH of the fillets were measured. Modified atmosphere packaging (MAP) had in general an inhibitory effect on the growth of the microflora but limited inhibition of the production of TVB and TMA. Despite the fact that increased oxygen proportions in the atmosphere contributed in a slightly lower production of TMA, all the samples had a TVB and TMA content high enough to be considered as spoiled after 4 days' storage at 6 degrees C. A total aerobic plate count at 25 degrees C of a 10(6) cfu/g, combined with the presence of only a 10(3) cfu/g of H2S-producing bacteria, which are normally considered as TMAO-reducing organisms in fish, cannot explain the strong increase in TMA. A high cell concentration of more than 10(8) cfu/g of Shewanella putrefaciens is required for production of a TMA level normally found in spoiled fish. This suggests that there could be another type of bacterium in fish, not involved in the spoilage of unpacked fish, which is resistant to 60% CO2, is not H2S-producing, and shows a high TMAO-reducing capacity. This bacterium could be Photobacterium phosphoreum.

Volatile components associated with bacterial spoilage of tropical prawns.

Analysis of headspace volatiles by gas chromatography/mass spectrometry from king (Penaeus plebejus), banana (P. merguiensis), tiger (P. esculentus/semisulcatus) and greasy (Metapenaeus bennettae) prawns stored in ice or ice slurry, which is effectively an environment of low oxygen tension, indicated the presence of amines at the early stages of storage (less than 8 days) irrespective of the nature of the storage media. Esters were more prevalent in prawns stored on ice (normal oxygen conditions) at the latter stages of storage (more than 8 days) and were only produced by Pseudomonas fragi, whereas sulphides and amines occurred whether the predominant spoilage organism was Ps. fragi or Shewanella putrefaciens. The free amino acid profiles of banana and king prawns were high in arginine (12-14%) and low in cysteine (0.1-0.17%) and methionine (0.1-0.2%). Filter sterilised raw banana prawn broth inoculated with a total of 15 cultures of Ps. fragi and S. putrefaciens and incubated for two weeks at 5 degrees C, showed the presence of 17 major compounds in the headspace volatiles analysed using gas chromatography/mass spectrometry (GC/MS). These were mainly amines, sulphides, ketones and esters. Principal Component Analysis of the results for the comparative levels of the volatiles produced by pure cultures, inoculated into sterile prawn broth, indicated three subgroupings of the organisms; I, Ps. fragi from a particular geographic location; II, S. putrefaciens from another geographic location; and III, a mixture of Ps. fragi and S. putrefaciens from different geographic locations. The sensory impression created by the cultures was strongly related to the chemical profile as determined by GC/MS. Organisms, even within the same subgrouping classified as identical by the usual tests, produced a different range of volatiles in the same uniform substrate.

Development of intestinal flora of human-flora-associated (HFA) mice in the intestine of their offspring.

Development of intestinal flora in newborn human-flora-associated (HFA) mice was compared with that in newborn conventional (CV) mice. Facultative anaerobes were detected from the first day after birth in both CV and HFA mice but anaerobes were not detected in the first week. Anaerobes rapidly increased from the 2nd week after birth and became predominant in newborn intestine. Most of the intestinal bacteria in adult CV and HFA mice were colonized in the intestine of CV and HFA mice, respectively, within 3 weeks after birth. The human intestinal flora established in the intestine of HFA mice finally reproduced without any remarkable change in composition in the intestine of newborn HFA mice. The development of intestinal flora in HFA mice was similar to that in CV mice but not that in human infants. These results indicated that human flora associated in HFA mice could be transferred from mothers to their offspring although HFA mice could not simulate the development of intestinal flora of the human infant.

Effect of nisin on two cultures of rumen ciliates.

The effect of nisin (in the form of Nisaplin) was determined using two species of rumen ciliate protozoa in vitro, on their co-culture bacterial population, and volatile fatty acid concentration. Nisaplin did not affect the in vitro growth of Entodinium caudatum at concentrations of 50-400 mg/L during short-term treatment (5 d). Long-term application (30 d) of Nisaplin (100 mg/L) significantly decreased growth of the Epidinium ecaudatum forma caudatum et ecaudatum but not growth of E. caudatum. Nisaplin moderately supported the growth of E. caudatum after omission of wheat gluten (source of amino acids for protozoan growth). An inhibition of Gram-positive facultative anaerobic bacterial population in the protozoan cultures (lactobacilli, enterococci, staphylococci and amylolytic streptococci) was observed during long-term Nisaplin treatment. The concentration of volatile fatty acids significantly increased during the long-term Nisaplin treatment of both cultures. The propionate concentration in the mixture of volatile fatty acids was nearly twice higher on the account of the decreased concentration (from 74 to 63%) of acetate.

Increased susceptibility to gingival colonization by specific HACEK microbes in children with congenital heart disease.

It is well established that infective endocarditis (IE) involving the HACEK (Hemophilus, Actinobacillus, Cardiobacter, Eikenella, Kingella) group of microbes occurs in patients with congenital heart defects (CHD) and in those with prosthetic grafts. Dental caries and gingival disease have been presumed to be the focus of microbial shedding. The purpose of this study was to determine if children with CHD had a more severe gingival inflammatory condition and harbored the HACEK group of microbes to a greater extent than normal children. Two groups of 12 age and sex matched children were selected for this study. The experimental group consisted of twelve children with CHD, 1-1/2 to 8 years of age. The control group consisted of 12 healthy children 2 to 8 years of age. Each child had a gingival index score recorded as described by Massler. Subgingival cultures were obtained. Gingival samples were cultured for HACEK microbes and total Streptococcus (spp) using standard techniques. Fisher's exact test was performed with significance defined at P < 0.05. Children with CHD had more severe gingival inflammatory index than the control group (P < 0.05). 8/12 CHD patient had Actinobacillus actinomycetemcomitans (A.a.) as compared with 2/12 controls (P < 0.05). Furthermore, all cyanotic CHD patients (4/4) had A.a. whereas, only 2/12 controls did (P < 0.05). 4/12 CHD patients harbored Eikenella corrodens (E.c.) compared to 1/12 controls (N.S.). There was no significant difference in colonization with E.c. or A.a. between cyanotic and acyanotic patients. No significant difference in total Streptococcus (spp) was found between the two groups. This study suggests that children with CHD have a more severe gingival inflammatory index and are colonized with specific HACEK microbes more so than normal children.

In vitro study of possible microbial indicators for drowning: Salinity and types of bacterioplankton proliferating in blood.

Numbers and types of bacterioplankton proliferating in blood samples mixed with water of various salinity levels were examined to determine the characteristics of species associated with salinity. Water samples (total n=88) were collected from the midstream of two rivers (freshwater; n=10; salinity <0.05%), from around their estuaries (areas of freshwater, n=20, salinity <0.05%; areas of brackish water, n=20, salinity <0.05-3.1%; areas of marine water beyond the mouths of the rivers, n=28, salinity 2.4-3.3%), and from the coast (areas of marine water; n=10; salinity 3.3-3.5%). Freshwater bacteria were identified in 41 of 42 blood samples mixed with water at ≤1.3% salinity, and the genus Aeromonas, which is universally distributed in freshwater environments, was predominant. Marine bacteria were identified in all of 46 blood samples mixed with water at ≥1.8% salinity, and most comprised the genera Vibrio and Photobacterium that are universally distributed in seawater environments. Aeromonas was undetectable in all blood samples mixed with brackish or sea water at ≥1.8% salinity although they are detectable even in seawater environments. Thus, the present results showed that bacterioplankton capable of proliferating in human blood reflects the salinity of water.

[Quinolone resistance in Gram negative rods in Iceland and association with antibiotic use]. / Onaemi fyrir kínólónum hjá Gram neikvaedum stöfum á Islandi og tengsl vid sýklalyfjanotkun.

OBJECTIVE: Fluoroquinolones are bacteriocidal drugs that are widely used to treat severe urinary and respiratory tract infections. Studies show that resistance to fluoroquinolones is continuously increasing both in Europe and the United States. The purpose of this study was to measure the frequency of fluoroquinolone resistance in the most prevalent Gram negative rods and look at the correlation with fluoroquinolone use over the last 8 years. MATERIALS AND METHODS: All strains of Escherichia coli, Klebsiella sp., Proteus sp. and Pseudomonas aeruginosa identified from clinical specimens at the Department of Clinical Microbiology at the Landspitali University Hospital (LUH) during the time period 1.11.2006 to 31.1.2007. Antibiotic susceptibility testing was performed by disc diffusion tests and all strains were tested for ciprofloxacin susceptibility. Antibiotic resistance data for the last years were collected from the reports of the Department of Clinical Microbiology, but ciprofloxacin susceptibility was usually only tested for specimens from hospitalised patients and when there was resistance to two or more antimicrobial agents. Data on antibiotic use/sales was obtained from the State Epidemiologist at the Directorate of Health. RESULTS: Of the 1861 strains tested, 104 fluoroquinolone resistant strains were identified during the study period, including 91 E. coli (87%), 8 Klebsiella sp. (8%) and 5 P. aeruginosa (5%). No fluoroquinolone resistant Proteus sp. was identified. There was a significant positive correlation between fluoroquinolone use and the frequency of resistant strains of E. coli and Enterobacteriaceae. The frequency of resistant E. coli strains was 6% and it differed significantly between age groups (p >0.001) and sex, 6% for females and 11% for males (p = 0.015). The ratio of fluoroquinolone resistant E. coli was highest in the LUH and homes for the elderly. CONCLUSION: The frequency of fluoroquinolone resistance is increasing fast in Iceland but is still one of the lowest compared to the other European countries. The frequency is highest in the oldest age groups where the use of the quinolones is the greatest and there was a significant correlation between the quinolone use and the frequency of resistance in E. coli and Enterobacteriaceae. The results highlight the importance of prudent fluoroquinolone use and the need to monitor fluoroquinolone use and resistance.

Prospects for a bio-based succinate industry.

Bio-based succinate is receiving increasing attention as a potential intermediary feedstock for replacing a large petrochemical-based bulk chemical market. The prospective economical and environmental benefits of a bio-based succinate industry have motivated research and development of succinate-producing organisms. Bio-based succinate is still faced with the challenge of becoming cost competitive against petrochemical-based alternatives. High succinate concentrations must be produced at high rates, with little or no by-products to most efficiently use substrates and to simplify purification procedures. Herein are described the current prospects for a bio-based succinate industry, with emphasis on specific bacteria that show the greatest promise for industrial succinate production. The succinate-producing characteristics and the metabolic pathway used by each bacterial species are described, and the advantages and disadvantages of each bacterial system are discussed.

Root canal microbiota of dogs' teeth with periapical lesions induced by two different methods.

OBJECTIVE: The microbial composition was investigated in root canals of dogs' teeth with periapical lesions induced by 2 different methods: open versus sealed canals. STUDY DESIGN: Teeth from Group I (n = 16) were left open for a week, then sealed with composite resin for 120 days. The teeth from Group II (n = 16) were left open for the same period. Microbiological samples from the root canals were collected and processed by the anaerobic technique for identification and counting of microorganisms after establishment of periapical reactions. RESULTS: Seventy-four cultivable isolates were recovered in sealed canals (Group I). Strict anaerobes accounted for 64.9% of all species isolated, and gram-negative microorganisms accounted for 55.4%. Microbial genera most frequently isolated were Prevotella, Fusobacterium, Peptostreptococcus, Streptococcus, Enterococcus, Clostridium, and Porphyromonas. Statistical analysis by Pearson chi-square or Fisher's test revealed positive association between sealed teeth and strict anaerobes (P < .05). In open canals (Group II), from a total of 58 cultivable isolates, 19% were strict anaerobes and 81% facultative anaerobes, with predominance of gram-positive species (75.8%). Genera most frequently isolated were Streptococcus, Propionibacterium, Staphylococcus, Neisseria, and Prevotella. CONCLUSION: Strict anaerobes were most frequently found in sealed teeth rather than in the teeth with canals left exposed to the oral cavity for 4 months. Therefore, the method that induced periapical inflammatory lesions by intentional oral exposure, followed by tooth sealing, produced root canal microbiota similar to the same found in humans.

Diverse endophytic bacteria isolated from a leguminous tree Conzattia multiflora grown in Mexico.

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10(4)-10(6) individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.