Low-temperature biodegradation of high amounts of phenol by Rhodococcus spp. and basidiomycetous yeasts

Four cold-adapted microbial strains able to degrade high amounts of phenol were isolated from hydrocarbon-contaminated alpine soils. Two of the strains were bacteria identified as Rhodococcus spp., and two strains were basidiomycetous yeasts. One of the yeasts was identified as Trichosporon dulcitum, while the second yeast strain belonged to the Urediniomycetes and probably represents a novel species. This strain was not able to grow at temperatures above 20 degrees C, while the other three strains were cold-tolerant and could grow at temperatures ranging from 1-25 degrees C (T. dulcitum) or 1-30 degrees C (rhodococci). The yeast strains were characterized by a substantially lower optimum temperature for growth and biodegradation compared to the bacteria. The urediniomycete strain degraded 5 mM phenol at 1 degrees C faster than the two bacteria at 10 degrees C. The optimum temperature for phenol degradation was 10 degrees C (novel yeast species), 20 degrees C (T. dulcitum), or 30 degrees C (rhodococci). Using fed-batch cultivation in mineral medium with phenol as the sole carbon source, high amounts of phenol were degraded at 10 degrees C. Both rhodococci degraded up to 12.5 mM phenol, while the two yeast strains even utilized as much as 15 mM phenol.     

Identification of different regions among strains of Yersinia pestis by suppression subtractive hybridization

Yersinia pestis, the causative agent of bubonic and pneumonic plague, has been classified into four biovars: Antiqua, Mediaevalis, Orientalis and Microtus. Although the entire genome sequences of three Y. pestis strains, CO92, KIM and 91001, of biovar Orientalis, Mediaevalis and Microtus, respectively, have been decoded, the genome sequence of the biovar Antiqua strain is unknown. In an initial effort to find Antiqua-specific sequences, suppression subtractive hybridization (SSH) was performed and four different regions (DFRs) were identified. Among the four DFRs, only DFR4 was specific to the tester (strain 49006, biovar Antiqua). PCR demonstrated that DFR4 was present only in 57 of 60 Antiqua strains from the Marmota baibacina-Spermophilus undulates plague focus in the Tianshan Mountains (focus B) and in three strains of Y. pseudotuberculosis (serotypes I and II), showing that not all Antiqua strains had DFR4. Five DFR profiles were identified based on the presence or absence of these four DFRs in 636 strains of Y. pestis from 10 plague foci in China.     

Natural transformation-based foreign DNA acquisition in a Ralstonia solanacearum mutS mutant

Mutator strains with defective methyl-mismatch repair (MMR) systems have been shown to play an important role in adaptation of bacterial populations to changing and stressful environments. In this report, we describe the impact of mutS::aacC3-IV inactivation on foreign DNA acquisition by natural transformation in the phytopathogenic bacterium Ralstonia solanacearum. A mutS mutant of R. solanacearum exhibited 33- to 60-fold greater spontaneous mutation frequencies, in accordance with a mutator phenotype. Transformation experiments indicated that intra- and interspecific DNA transfers increased up to 89-fold. To assess horizontal gene transfer (HGT) from genetically modified plants to R. solanacearum, fitness of the mutator was first evaluated in soil and plant environments. Competitiveness was not modified after 61 days in soil and 8 days in tomato, and the progress of plant decay symptoms was similar to that of the wild-type strain. Despite its survival in soil and in planta, and the powerful capacities of HGT, R. solanacearum was not genetically transformed by transgenic plant DNA in a wide range of in vitro and in planta tests.     

Enterotoxin production in relation to taxonomic grouping and source of isolation of Aeromonas species

A total of 19 of 20 (95%) strains of Aeromonas hydrophila biovar hydrophila and 16 of 17 (94%) strains of Aeromonas sobria isolated from a variety of clinical and environmental sources were found to be enterotoxin positive. Only 2 of 18 (11%) A. hydrophila biovar anaerogenes and 2 of 13 (15%) unidentified Aeromonas strains from a similar variety of sources produced enterotoxin. No association was apparent between the source of isolation, in particular diarrheal stools, and enterotoxigenicity; 41% of the isolates from diarrheal stools were enterotoxin negative. A strong correlation was noted between ability to produce enterotoxin and positive results in six characters: lysine decarboxylase and Voges-Proskauer reactions, production of gas from glucose, gluconate oxidation, xanthine hydrolysis, and hemolysis of human erythrocytes. In the majority of cases (35 of 39 strains), enterotoxigenicity was detected using cell-free filtrates of brain heart infusion broth cultures grown at 36 degrees C for 15; however, the other four positive isolates were detected after growth in the same broth at 30 degrees C or in Casamino Acids-yeast extract broth at 30 or 37 degrees C. It is recommended that for enterotoxin tests, strains should be grown in both media at both temperatures. The infant mouse test was found to be a simple and reliable method for detection of the enterotoxin. The toxin proved to be heat labile and not neutralized by cholera antitoxin.     

Methods for identification of flavobacteria.

Published reports disagree on the best features for detecting and distinguishing between Flavobacterium meningosepticum (biovar IIa) and Flavobacterium species CDC group IIb (biovar IIb; Flavobacterium indologenes). This report discloses that at least some of these disagreements may reflect the methods used. To detect production of indole, a modified Kovács reagent (not Ehrlich) and a buffered tryptophan medium were optimal, but not all strains of these two biovars produced indole. To distinguish the two biovars, hydrolysis of corn starch was preferable to that of soluble potato starch. Both biovars may hydrolyze DNA; the differentiation achieved varied with the methods used. Both biovars presented pigmented growth; only IIb, however, was obviously pigmented on a 2-day blood agar plate. Acidification of D-arabinose definitively distinguished these two biovars; several additional features were useful but not definitive.     

Within plant distribution of Potato Virus Y in hairy nightshade (Solanum sarrachoides): an inoculum source affecting PVY aphid transmission

Potato virus Y (PVY) is vectored by several potato-colonizing and non-colonizing aphid species in a non-persistent manner and has a wide host range. It occurs naturally in several plant families. Myzus persicae and Macrosiphum euphorbiae are the most efficient potato-colonizing aphid vectors of PVY. Rhopalosiphum padi, a cereal aphid that migrates in large numbers through potato fields during the middle of the growing season, does not colonize potato plants but can transmit PVY. Hairy nightshade, Solanum sarrachoides, a prevalent annual solanaceous weed in the Pacific Northwest (PNW) of the United States, is an alternative host for PVY and a preferred host for M. persicae and M. euphorbiae. Hence, hairy nightshade plants might play an important role as an inoculum source in the epidemiology of PVY. We looked at titre accumulation and distribution of PVY(O), PVY(N:O) and PVY(NTN) in S. sarrachoides and potato after aphid inoculation with M. persicae and studied the transmission of PVY(O) and PVY(NTN), by M. persicae, M. euphorbiae and R. padi from hairy nightshade to potato plants. Virus titre at different positions on the plant was similar in S. sarrachoides and potato plants with strains PVY(O) and PVY(N:O). Titres of PVY(NTN) were similar in S. sarrachoides and potato but differences in titre were observed at different positions within the plant depending on the plant phenology. Percentage transmission of PVY(NTN) by M. persicae and M. euphorbiae was twice as high (46 and 34%, respectively) from hairy nightshade to potato than from potato to potato (20 and 14%). Percentage transmission of PVY(O) by M. persicae and M. euphorbiae was not affected by the inoculum source. No effect of the inoculum source was observed in the transmission of either PVY strain by R. padi. These results show that hairy nightshade may be an equal or better virus reservoir than potato and thus, important in the epidemiology of PVY.     

Suitability of new chlamydia transport medium for transport of herpes simplex virus.

A new chlamydia transport medium (ChlamydiaPort; Scott Laboratories, Inc., Fiskeville, R.I.) was evaluated for its suitability as a transport medium for herpes simplex virus (HSV). Two laboratory HSV strains (McIntyre and 333) and two clinical isolates (AO218 and AO301) were suspended in ChlamydiaPort, ViraPort (Scott Laboratories), and cell culture medium and maintained at 2 and 22 degrees C. Samples were tested at various time intervals to determine surviving virus. The range of half-lives of the HSV strains held at 2 degrees C in ChlamydiaPort medium was from 3.5 to 10 days, while virus stability was greater in ViraPort and less in cell culture medium. These HSV strains held at 22 degrees C in ChlamydiaPort had half-lives from 1.5 to 6 days, which were significantly greater than the half-lives of the viruses held in either tissue culture medium or ViraPort. Clinical specimens were tested for virus by using the Selecticult-HSV (Scott Laboratories) system to determine the performance of the transport medium under field conditions. Clinical specimens maintained up to 5 days at ambient temperatures in ChlamydiaPort medium appeared suitable for diagnostic testing without detectable loss of positive specimens. In addition, there was a significant decrease in the average time required for diagnosis when compared with a standard transport system, Virocult (Microdiagnostics, Cleveland, Ohio). These results show that HSV infections can be successfully diagnosed in distant virology laboratories by shipping specimens in ChlamydiaPort transport medium at ambient temperatures.     

Molecular phylogenetics of multiple genes on Aspergillus section Fumigati isolated from clinical specimens in Japan.

A phylogenetic study based on sequence analysis of the beta-tubulin, hydrophobin and calmodulin genes was performed in 19 strains of Aspergillus fumigatus and related species isolated from clinical specimens in Japan. Correlations between detailed morphology and phylogeny were examined. Species in the section Fumigati were divided into five clades: clade I, typical strains of A. fumigatus; clade II, species including A. lentulus and A. fumisynnematus; clade III, species including A. fumigatiaffinis and A. novofumigatus, clade IV, atypical strains of A. fumigatus including A. viridinutans; and clade V, species including A. brevipes, A. duricaulis and A. unilateralis. Most of the examined strains from clinical specimens in Japan clustered together in clade I and exhibit globose conidia with lobate-reticulate ornamentation. Other strains from clinical specimens were divided into two clades (clades II and IV). The strains in clades II and the six strains in clade IV exhibit conidia with microtuberculate ornamentation, while A. viridinutans-complex in clades IV and the strains in clade V have conidia with lobate-reticulate ornamentation. The six strains are clearly distinguished from A. viridinutans-complex and are considered to be related to Neosartorya udagawae. The maximal growth temperatures of clades I, II, IV and V were above 50 degrees C, 45 degrees C, 42 degrees C and 42 degrees C, respectively. These data are useful for classification of species within the Aspergillus section Fumigati.     

Primary carrier sites of group B streptococci in pregnant women correlated with serotype distributions and maternal parity.

Perianal, perineal, vulval, and vaginal swabs from women attending an antenatal clinic were quantitatively cultured for group B streptococci using Islam's medium. The isolation rates from the four sites were very similar with an overall carriage rate of 21%. The findings suggest that the type II strains, a faecal flora, probably colonise the perianal site from a faecal source, and that the type III strains colonise primarily the genital site and then spread to the perineoperianal region. The type I strains did not conform to any pattern, suggesting a possible secondary involvement of these sites from another source. The types I, R, X, and non-typable isolates were almost exclusively isolated from primigravidae and second gravidae; the type III strains were conspicuously absent in 47 primigravidae. The primigravidae and second gravidae women consistently had high colony counts.     

Identification of bacteria isolated from diseased Neungee mushroom, Sarcodon aspratus

As the first step in an investigation of the problem with quality deterioration seen in the Neungee mushroom (Sarcodon aspratus) due to bacterial overgrowth during its storage, an attempt to isolate bacterial strains was made using infected gills of Sarcodon aspratus. Five bacterial strains were isolated; one phototrophic cyanobacterial species and four heterotrophic Gram negative rods. The four heterotrophic bacterial isolates (strains P, S, R, and MK1) were subjected to identification based on biochemical characteristics using the Biolog system, cellular fatty acid analysis using the MIDI system, cytology by scanning microscopy, and 16s rDNA sequence analysis. A slow grower, the P strain (ca. 0.7 microm x 1.5 microm), which forms pink colonies on Tryptic Soy agar (TSA) and glucose minimal salt medium containing thiamine (MT medium), belongs to genus Methylobacterium, and is likely M. radiotolerans. The methanol-utilizing capacity of the P strain was confirmed by growth on methanol-supplemented medium as a sole carbon source. Both the S and R strains (ca. 0.5 microm x 0.8 microm) produced smooth and slightly rough white colonies, respectively, on TSA, MT, and potato dextrose (PD) agar are members of the Burkholderia cepacia complex. Although both strains showed some differences from each other in colony morphology, nitrogen fixation capacity, and denitrification, they were considered to be Burkholderia stabilis because their 16s rDNA sequences showed 99.93% similarity with those of B. stabilis LMG 14294T (NCBI AF 148554). The MK1 strain, a rod-shaped bacterium with a tapered end (ca. 0.6 microm x 1.8 microm), produces a copious mucoid substance on MT and PD agar, but not on TSA. Despite extensive identification studies, the M strain is not currently identifiable, which suggests that it is a novel bacterium.     

Phenotypic and genotypic discrimination of Francisella tularensis ssp. holarctica clades

Francisella tularensis is the causative agent of tularemia, a zoonotic disease with a wide host range. F. tularensis ssp. holarctica (Fth) is of clinical relevance for European countries, including Germany. Whole genome sequencing methods, including canonical Single Nucleotide Polymorphism (canSNP) typing and whole genome SNP typing, have revealed that European Fth strains belong to a few monophyletic populations. The majority of German Fth isolates belong to two basal phylogenetic clades B.6 (biovar I) and B.12 (biovar II). Strains of B.6 and B.12 seem to differ in their pathogenicity, and it has been shown that strains of biovar II are resistant against erythromycin. In this study, we present data corroborating our previous data demonstrating that basal clade B.12 can be divided into clades B.71 and B.72. By applying phylogenetic whole genome analysis as well as proteome analysis, we could verify that strains of these two clades are distinct from one another. This was confirmed by measuring the intensity of backscatter light on bacteria grown in liquid media. Strains belonging to clades B.6, B.71 or B.72 showed clade-specific backscatter growth curves. Furthermore, we present the whole genome sequence of strain A-1341, as a reference genome of clade B.71, and whole proteomes comparison of Fth strains belonging to clades B.6, B.71 and B.72. Further research is necessary to investigate phenotypes and putative differences in pathogenicity of the investigated different clades of Fth to better understand the relationship between observed phenotypes, pathogenicity and distribution of Fth strains.     

Induction of tobacco chlorosis by certain cucumber mosaic virus satellite RNAs is specific to subgroup II helper strains

Two satellite (sat) RNAs of cucumber mosaic virus (CMV), B2- and WL3-sat RNAs, which induce systemic chlorosis on tobacco, were inoculated onto tobacco with a number of CMV strains. Systemic chlorosis was observed only when these satellite RNAs were associated with subgroup II CMV strains. Infection of tobacco with various pseudorecombinants of subgroup I and II CMV strains, together with WL3- or B2-sat RNA, suggests that chlorosis is associated with RNA 2 of subgroup II CMV strains. Chlorosis was not induced when B2- or WL3-sat RNAs were inoculated onto tobacco with tomato aspermy virus. In contrast, the induction of chlorosis on tomato by B1-sat RNA did not show any clear dependence on the subgroup of its CMV helper strain although chlorosis did tend to be more severe in association with subgroup II CMV strains.     

Ralstonia paucula (Formerly CDC group IV c-2): unsuccessful strain differentiation with PCR-based methods, study of the 16S-23S spacer of the rRNA operon, and comparison with other Ralstonia species (R. eutropha, R. pickettii, R. gilardii, and R. solanacearum)

Ralstonia paucula (formerly CDC group IV c-2) can cause serious human infections. Confronted in 1995 with five cases of nosocomial bacteremia, we found that pulsed-field gel electrophoresis could not distinguish between the isolates and that randomly amplified polymorphic DNA analysis was poorly discriminatory. In this study, we used PCR-ribotyping and PCR-restriction fragment length polymorphism analysis of the spacer 16S-23S ribosomal DNA (rDNA); both methods were unable to differentiate R. paucula isolates. Eighteen strains belonging to other Ralstonia species (one R. eutropha strain, six R. pickettii strains, three R. solanacearum strains, and eight R. gilardii strains) were also tested by PCR-ribotyping, which failed to distinguish between the four species. The 16S-23S rDNA intergenic spacer of R. paucula contains the tRNA(Ile) and tRNA(Ala) genes, which are identical to genes described for R. pickettii and R. solanacearum.     

Relationship between cell surface composition, adherence, and virulence of Candida albicans.

A comparison was made of the adherence to acrylic and to human buccal epithelial cells of seven strains of Candida albicans isolated from active infections (I strains) and two strains obtained from asymptomatic carriers (C strains). After growth in defined medium containing a relatively low concentration (50 mM) of glucose as the carbon source, the adherence of I and C strains to either surface was similar and all strains were sensitive to spheroplast formation with Zymolyase 5000. Growth in medium containing a high concentration (500 mM) of sucrose or galactose enhanced the adherence of I strains up to 5- and 11-fold, respectively, and there were corresponding increases in resistance to spheroplast formation. Sucrose- or galactose-grown C strains showed only small increases in adherence and remained relatively sensitive to spheroplast formation. When inoculated intravenously into mice, I strains grown in 500 mM sucrose were up to five times more virulent than organisms grown in 50 mM glucose, while I strains grown in 500 mM galactose showed a 5- to 24-fold increase in virulence. Fifty percent lethal doses obtained for C strains were similar after growth on all three carbon sources. We conclude that I strains are able to modify their surface composition in response to high concentrations of certain sugars in the growth environment. Such modification can enhance both their ability to adhere to surfaces and their virulence. C strains lack this capability, or possess it to a lower degree, and may therefore have a lower pathogenic potential.     

The enigma of Yersinia enterocolitica biovar 1A

Yersinia enterocolitica, an important food- and water-borne enteropathogen causes acute diarrhea, terminal ileitis, and mesenteric lymphadenitis. It is represented by six biovars (1A, 1B, 2-5). The biovar 1A strains are generally regarded as avirulent as they lack pYV plasmid and major chromosomal virulence genes. Despite this, some biovar 1A strains produce disease symptoms indistinguishable from that produced by known pathogenic biovars (1B, 2-5). Suggested prospective studies to understand pathogenic potential of biovar 1A should focus on role of insecticidal toxins, urease, protease, superoxide dismutase, and host responses. These studies should also take into account the clonal groups of biovar 1A.     

Detection and differentiation of Plum pox virus using real-time multiplex PCR with SYBR Green and melting curve analysis: a rapid method for strain typing

A real-time multiplex PCR procedure with melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid and reliable identification of Plum pox virus (PPV) isolates of strains D and M. Members of the different strains were identified by their distinctive melting temperatures (T(m)s); 84.3-84.43 degrees C for D isolates, and 85.34-86.11 degrees C for M isolates. The associated amplicon sizes were 114 and 380 bp, respectively. The procedure was used for detection and identification of PPV in both herbaceous and woody hosts. The Tm for members of a particular strain was very similar, with a host effect that did not hinder strain identification. Universal primers included in the study detected all isolates of PPV tested, amplifying a 74 bp fragment. The Tm of this fragment varied from 80.12 to 81.52 degrees C and may have supplementary value for PPV identification. SYBR Green-based detection was compared to detection using a hybridization LUX fluorogenic primer. Better resolution of the melting peaks was observed with SYBR Green I, than with the LUX primers, hence strain identification with SYBR Green I was more reliable. This is a simple approach to PPV strain identification with the relatively inexpensive dye SYBR Green I, and eliminates any need for electrophoretic analysis of amplicons or RFLP patterns using ethidium bromide.     

One case each of recurrent meningitis and hemoperitoneum infection with Ralstonia mannitolilytica.

Two clinical cases of infection with Ralstonia mannitolilytica are described: a recurrent meningitis on an implanted intraventricular catheter and an infected hemoperitoneum as a complication of a cholangiocarcinoma. The strains were first misidentified as Pseudomonas fluorescens and Burkholderia cepacia. Further testing lead to the identification as Ralstonia pickettii biovar 3/"thomasii," which was recently shown to represent a separate species, R. mannitolilytica (List editor N. Weiss, Int. J. Syst. Evol. Microbiol. 51:795-796, 2001), originally described as R. mannitolytica (De Baere et al., Int. J. Syst. Evol. Microbiol. 51:547-558, 2001). R. mannitolilytica can be distinguished from all described Ralstonia species by its acidification of D-arabitol and mannitol and by its lack of nitrate reduction and of alkalinization of tartrate. In order to determine the true prevalence of infections with this species, colistin-resistant "P. fluorescens" strains and strains growing on B. cepacia selective medium deserve further attention.     

Synthesis of alpha-amylase by Aspergillus oryzae in solid-state fermentation

Spent Brewing Grains (SBG) was evaluated for its efficacy to be used as sole carbon source for the synthesis of alpha-amylase in solid-state fermentation using a fungal strain of Aspergillus oryzae NRRL 6270. Enzyme production was superior when the culture grew on mesophilic temperatures and best yields were at 25 degrees C. At 30 degrees C, yields were almost comparable. Maximum production of alpha-amylase [6870 U/g dry substrate (gds)] was obtained when SSF was carried out at 30 degrees C for 96 h using SBG medium, which had initial moisture of 70% and was inoculated using a spore suspension containing 1 x 10(7) spores/ml. Supplementation of SBG with external carbon sources such as mono-, di and polysaccharides caused repression in enzyme synthesis by the fungal culture.     

Interaction of Escherichia coli with Different Fimbriae and Polymorphonuclear Leukocytes

The effects of Escherichia coli strains with various fimbriae on bacteria-polymorphonuclear leukocyte (PMN) interactions were studied. Strains of E. coli were cultivated at 37 degrees C to express and at 18 degrees C to suppress the formation of fimbriae. The presence of fimbriae was confirmed by electron microscopic studies and hemagglutination and salt aggregation tests. Fimbriated E. coli strains were more readily PMN associated than the nonfimbriated strains in the absence of opsonins, confirming the results of previous studies. However, the PMN chemiluminescence (CL) induced by the various strains in the absence of serum opsonins depended on the type of fimbriae they expressed. Strains with type 1 fimbriae expressing mannose-sensitive hemagglutination induced 5 to 15 times more CL than the same strains grown at 18 degrees C, i.e., not expressing this type of fimbriae. For strains showing mannose-resistant hemagglutination, the differences between fimbriated and nonfimbriated variants of the same strains grown at 37 and 18 degrees C, respectively, were less pronounced. Analysis of enterotoxigenic strains expressing colonization factor antigen I (CFA/I) fimbriae showed that these induced only 25 to 33% of the CL induced by the same E. coli strains not expressing CFA/I, whereas enterotoxigenic strains expressing CFA/II fimbriae induced 100 to 200% of the CL induced by the nonfimbriated variants. Although less CL was induced by bacteria with CFA/I fimbriae than by nonfimbriated variants, this situation was reversed when the microorganisms were opsonized. Thus, CFA/I fimbriae, while enhancing adhesion to cells, induce less activation of PMN-killing mechanisms in a serum-free environment. These findings may be relevant for the virulence in certain body sites, since CFA/I fimbriae, while facilitating adhesiveness, may protect the bacteria from PMN killing. Our findings indicate that PMN interactions with fimbriated E. coli in the host defense may be complex. Certain fimbriae may indeed be advantageous to the bacteria, enabling them to interact with PMNs without activating the bactericidal oxidative metabolism.