Passage through alternative hosts changes the fitness of Fusarium graminearum and Fusarium pseudograminearum

Species of the necrotrophic fungal pathogen Fusarium that cause head blight and crown rot of cereals including wheat also infect a number of alternative host plants. This raises the prospect of more damaging pathogen strains originating and persisting as highly successful saprophytes on hosts other than wheat. The immediate impact on pathogenic (aggressiveness) and saprophytic (growth rate and fecundity) behaviour of six isolates with low, moderate or high initial aggressiveness was examined in two species of Fusarium after their passage through 10 alternative plant hosts. One passage through alternative hosts significantly reduced the pathogenic fitness of most isolates, but this change was not associated with a concomitant change in their overall saprophytic behaviour. The overall weak association between aggressiveness, fecundity and growth rate both before and after passage through the alternative hosts indicate that pathogenic and saprophytic fitness traits may be independently controlled in both Fusarium species. Thus, there was no trade-off between pathogenic and saprophytic fitness in these necrotrophic plant pathogens.     

Naphthylamidase Activity of Leptospira

Extracts of 18 serotypes of the genus Leptospira were found to possess naphthylamidase activity, and differences in the pathogenic and saprophytic strains were noted. The former exhibited a preference for the leucyl naphthylamide substrate, whereas the latter demonstrated greater hydrolysis of alanyl naphthylamide. With the leucyl naphthylamide as substrate, pathogenic strains showed 10 to 20 times higher naphthylamidase activity than saprophytic strains. Optimal temperature and pH for enzymatic hydrolysis also differed between pathogenic and saprophytic strains. Maximal enzymatic activities for pathogenic and saprophytic naphthylamidases were 41 and 37 C, respectively, at pH 8.0 to 8.5. The pH and temperature optima suggested that the leptospiral enzyme activity was not leucine aminopeptidase.     

Differentiation of Pathogenic and Saprophytic Leptospires I. Growth at Low Temperatures

The minimal growth temperature of the pathogenic leptospires is between 13 and 15 C. The saprophytic leptospires have a minimal growth temperature between 5 and 10 C, or approximately 5 C below that of the pathogens. The capability of the saprophytic leptospires to grow at temperatures below those which allow the growth of the pathogenic leptospires provides a simple method of discrimination. With an inoculum yielding approximately 8 x 10(7) cells per ml in the test medium and an incubation temperature of 13 C, the saprophytic leptospires were easily differentiated from the pathogenic leptospires. All 13 saprophytic leptospires tested grew in the 10% rabbit serum medium at 13 C, whereas none of the 20 pathogens grew during the 30-day incubation period.     

Characterization of microaerophilic bacteria isolated from the coastal waters of Tokyo Bay, Japan

Abstract A total of 84 strains of microaerophiles were isolated from the Edo River mouth, Tokyo Bay, Japan, and their morphological, physiological and biochemical properties and mole percent guanine-plus-cytosine contents (mol%) of DNAs extracted from them were examined. Eighty-eight to 100% of the strains, collected in summer, grew at 15–37°C but only 5% of the strains grew at 5°C. Seventy-seven to 100% of the strains, collected in winter, were able to grow at 10–25°C but 79% of the strains were unable to grow at 37°C. The shift in growth-range of bacteria strongly suggests that the microflora of estuarine microaerophiles changes seasonally. All the strains could grow at 50% seawater while 70 and 42% of the strains could not grow at 0 and 100% seawater, respectively.
On the other hand, 72 strains, out of the 84 strains, clustered into 9 phena using numerical taxonomy methods. The strains belonging to phena 1 through 9 were all Gram-negative, non-spore-forming rod-shaped organisms which were negative for susceptibility to vibrostatic agent O/129, H₂S production and acid from dulcitol, and the G + C contents of DNAs ranged from 29.8 to 56.6 mol%. However, almost all strains could not be identified and are suggested to be new species.     

A model of insect—pathogen dynamics in which a pathogenic bacterium can also reproduce saprophytically

We developed a model of the population dynamic interaction between an insect and a pathogenic bacterium motivated by study of Serratia entomophila, a commercially exploited pathogen of the New Zealand grass grub (Costelytra zealandica). The bacterium is able to reproduce saprophytically, though it competes for saprophytic substrates with non-pathogenic strains, which appear to be superior competitors, probably because they lack a plasmid that carries genes required for pathogenicity. The effect of saprophytism and competition on the invasion criterion (R₀), short-term dynamics and long-term dynamics are described. Saprophytism can reduce (possibly to zero) the host threshold at which the pathogen can invade, though this reduction is less when there is competition with non-pathogenic strains. In a model of short-term population dynamics designed to mimic the application of bacteria to a host epizootic, saprophytism enhances the reduction in host density, though again this is tempered by competition with non-pathogens. In the long term, a pathogen that can develop saprophytically can drive its host to extinction in the absence of competition with non-pathogens. When the latter are present, host extinction is prevented. The addition of saprophytic reproduction can stabilize an otherwise unstable host–pathogen model, but we were unable to find a stable equilibrium given the further addition of a wholly saprophytic bacterial strain. The model suggests that enhancing or selecting for saprophytic ability could be a way of improving biological control.     

The growth profile, thermotolerance and biofilm formation of Enterobacter sakazakii grown in infant formula milk

AIMS: To study the growth, thermotolerance and biofilm formation of the emergent pathogen Enterobacter sakazakii in infant formula milk (IFM). METHODS AND RESULTS: The temperature range, death kinetics and biofilm formation of E. sakazakii were determined using impedance microbiology and conventional methods. In IFM the organism grew as low as 6 degrees C and optimally at 37-43 degrees C. In faecal coliform tests, 23% of strains (n = 70) produced gas from lauryl sulphate broth (LSB) at 44 degrees C after 48 h incubation. Three strains failed to grow in LSB at any of the temperatures. The D-value of cells suspended in IFM was determined between 54 and 62 degrees C. The resultant z-value was 5.7 degrees C. The organism was able to adhere and grow on latex, polycarbonate, silicon and to a lesser extent stainless steel. CONCLUSIONS: Enterobacter sakazakii was able to grow at refrigeration temperatures and on infant-feeding equipment. The thermotolerance of the organism was similar to other Enterobacteriaceae and should be killed during standard pasteurization treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: Enterobacter sakazakii has been associated with infant meningitis through consumption of contaminated IFM. Enterobacter sakazakii is able to grow in IFM during storage at refrigeration temperatures and attach to infant-feeding equipment, which may become reservoirs of infection.     

Morphological, Biochemical, and Growth Characteristics of Pseudomonas cepacia from Distilled Water

Studies were conducted on three strains of Pseudomonas cepacia isolated and maintained in distilled water and on a laboratory-subcultured strain transferred to distilled water. Optimum growth rates and maximum population yields of the four strains in distilled water were obtained at 37 C, although high population levels (10(6)-10(7)/ml) were reached and maintained over extended incubation periods at temperatures from 18 C to 42 C. Two strains were able to grow in distilled water at temperatures ranging from 12 C to 48 C and to survive 48 h and 21 days at 50 C and 10 C, respectively. Cells from distilled water cultures inoculated into Trypticase soy broth showed an immediate two- to three-log drop at upper and lower temperature limits; survivors were able to initiate logarithmic growth. Results obtained in morphological, biochemical, and antibiotic tests affirmed the strain differences noted in growth studies.     

Staphylococci in Competition: III. Influence of pH and Salt on Staphylococcal Growth in Mixed Populations

Previous results showed definite repressive effects on the growth of staphylococci in mixed cultures due to the competitive growth of psychrophilic saprophytes. This study was continued, and the influence of other environmental factors, pH and salt, on the competition between staphylococci and saprophytes was investigated. Initial pH values varied from 5 to 9. At the extremes of the pH range, staphylococci failed to grow, while the saprophytes grew under all of the conditions tested. At pH 5, the growth curves for the saprophytes were markedly altered from those obtained at neutral pH. The lag phases were greatly lengthened at and below 20 C, but normal numbers of saprophytes were reached in the stationary phase. At pH 6 and 8, staphylococcal growth showed the same inhibition observed at pH 7, at and below 20 C; normal multiplication was observed above this temperature, but with accelerated death phases. Thus, pH did not primarily effect staphylococcal growth through its influence on saprophyte growth and competition, but rather directly affected the growth of Staphylococcus cultures. Salt concentrations from 3.5 to 9.5% were investigated for influence on staphylococcal growth in mixed populations. Above 3.5% salt, staphylococcal inhibition at and above 20 C was not as marked as in the controls, although normal numbers were never reached. The saprophytes were increasingly inhibited, and their lag phases materially lengthened as salt concentration was increased. Salt acted directly on the Staphylococcus population and also, by repressing saprophyte growth, decreased competition, which allowed the staphylococci to grow.     

Lactic acid bacteria biofilms and their ability to mitigate Escherichia coli O157:H7 surface colonization

Lactic acid bacteria (LAB) exert antagonistic activities against diverse microorganisms, including pathogens. In this work, we aimed to investigate the ability of LAB strains isolated from food to produce biofilms and to inhibit growth and surface colonization of Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 at 10°C. The ability of 100 isolated LAB to inhibit EHEC O157:H7 NCTC12900 growth was evaluated in agar diffusion assays. Thirty-seven LAB strains showed strong growth inhibitory effect on EHEC. The highest inhibitory activities corresponded to LAB strains belonging to Lactiplantibacillus plantarum, Pediococcus acidilactici and Pediococcus pentosaceus species. Eighteen out of the 37 strains that showed growth inhibitory effects on EHEC also had the ability to form biofilms on polystyrene surfaces at 10°C and 30°C. Pre-established biofilms on polystyrene of four of these LAB strains were able to reduce significantly surface colonization by EHEC at low temperature (10°C). Among these four strains, Lact. plantarum CRL 1075 not only inhibited EHEC but also was able to grow in the presence of the enteric pathogen. Therefore, this strain proved to be a good candidate for further technological studies oriented to its application in food-processing environments to mitigate undesirable surface contaminations of E. coli.     

Simultaneous detection and differentiation of pathogenic and nonpathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay

Leptospirosis, caused by pathogenic Leptospira, is one of the most important zoonoses in the world. Several molecular techniques have been developed for detection and differentiation between pathogenic and saprophytic Leptospira spp. The aim of this study was to develop a rapid and simple assay for specific detection and differentiation of pathogenic Leptospira spp. by multiplex real-time PCR (TaqMan) assay using primers and probes targeting Leptospira genus specific 16S ribosomal RNA gene, the pathogen specific lig A/B genes and nonpathogen Leptospira biflexa specific 23S ribosomal RNA gene. Sixteen reference strains of Leptospira spp. including pathogenic and nonpathogenic and ten other negative control bacterial strains were used in the study. While the 16S primers amplified target from both pathogenic and non-pathogenic leptospires, the ligA/B and the 23S primers amplified target DNA from pathogenic and non-pathogenic leptospires, respectively. The multiplex real-time PCR (TaqMan) assay detection limit, that is, the sensitivity was found approximately 1 x 10(2) cells/ml for ligA/B gene and 23S ribosomal RNA gene, and 10 cells/ml 16S ribosomal RNA. The reaction efficiencies were 83-105% with decision coefficients of more than 0.99 in all multiplex assays. The multiplex real-time PCR (TaqMan) assay yielded negative results with the ten other control bacteria. In conclusion, the developed multiplex real-time PCR (TaqMan) assay is highly useful for early diagnosis and differentiation between pathogenic and non-pathogenic leptospires in a reaction tube as having high sensitivity and specificity.     

Chemical treatment of cereal seed in relation to plant vigour and control of soil fungi

Treating oat seeds not contaminated with any known pathogen of this crop with an organo-mercury preparation (Ceresan) increased seedling vigour only when they were grown in natural or simulated winter conditions with periods of frost. This beneficial effect on vigour was largely confined to seedlings and young plants. The fungicide protected the mesocotyls from contamination or invasion by certain soil-borne fungi normally regarded as saprophytes. Cylindrocarpon radicicola and Fusarium sambucinum were closely associated with discoloured mesocotyls bearing lesions and reduced vigour of untreated plants. Protection from such fungi enabled mesocotyls to grow thicker, and, when seedlings were grown in unsterilised soil and exposed to frost, reduced the number with lesions or discolouration. Thus the ability of the saprophytic soil-borne fungi to cause damage seems to be influenced by environmental conditions.     

Thermosensitive H1 plasmids determining citrate utilization.

Twelve thermosensitive H1 plasmids from strains of Salmonella typhi that had caused outbreaks of chloramphenicol-resistant typhoid fever in Vietnam, Thailand and India mediated citrate utilization (Cit+) in a prototrophic Escherichia coli K12 strain but not in the S. typhi strains from which they were derived. Four H1 plasmids from a similar outbreak in Mexico differed from the Far Eastern plasmids in not mediating citrate utlization but in mediating mercury resistance. H1 plasmids resembling the Far Eastern and the Mexican plasmids in regard to citrate utilization and mercury resistance were found in sewage in Britain. Citrate utilization was transferred to eight pathogenic strains of E. coli and to one strain each of Shigella flexneri and Shigella sonnei. Cultures of Cit+ bacteria grew more rapidly in citrate media at 28 degrees C than at 37 degrees C. Plasmid mutants that were more efficient at utilizing citrate were present in all such cultures--they grew equally well or better at 37 degrees C than at 28 degrees C. None of 222 strains of E. coli or Shigella that contained a variety of different plasmids were able to utilize citrate. This property was not transferred to the prototrophic E. coli K12 strain from Citrobacter (3 strains), Salmonella (39 strains), Proteus (44 strains), Klebsiella pneumoniae (33 strains) or Pseudomonas aeruginosa (44 strains).     

Comparative study of potential virulence factors in human pathogenic and saprophytic Trichoderma longibrachiatum strains

Potential virulence factors of 9 saprophytic and 12 clinical Trichoderma longibrachiatum strains were examined in the present study, in order to compare their capacity to cause infection in humans. All of the strains were able to grow at temperatures up to 40 degrees C and at pH values ranging from 2.0 to 9.0. Carbon and nitrogen source utilization experiments revealed that all of the strains were able to utilize a series of basic amino acids both as sole carbon and nitrogen sources. The MIC values of the tested antifungal drugs were found to be 0.016-8 microg/ml for amphotericin B, 64-256 microg/ml for fluconazole, 0.5-32 microg/ml for itraconazole and 0.008-1 microg/ml for ketoconazole in the case of the examined isolates. Metabolites of the strains inhibited the growth of different bacteria, furthermore, compounds produced by three clinical isolates reduced the motility of boar spermatozoa, indicating their toxicity to mammalian cells as well. On the whole, there were no significant differences in the examined features between strains derived from clinical or soil samples. The question, however, whether all environmental Trichoderma longibrachiatum strains have the capacity to cause infections or not, remains still unanswered.     

Specific detection of Xanthomonas axonopodis pv. dieffenbachiae in anthurium (Anthurium andreanum) tissues by nested PCR

Efficient control of Xanthomonas axonopodis pv. dieffenbachiae, the causal agent of anthurium bacterial blight, requires a sensitive and reliable diagnostic tool. A nested PCR test was developed from a sequence-characterized amplified region marker identified by randomly amplified polymorphic DNA PCR for the detection of X. axonopodis pv. dieffenbachiae. Serological and pathogenicity tests were performed concurrently with the nested PCR test with a large collection of X. axonopodis pv. dieffenbachiae strains that were isolated worldwide and are pathogenic to anthurium and/or other aroids. The internal primer pair directed amplification of the expected product (785 bp) for all 70 X. axonopodis pv. dieffenbachiae strains pathogenic to anthurium tested and for isolates originating from syngonium and not pathogenic to anthurium. This finding is consistent with previous studies which indicated that there is a high level of relatedness between strains from anthurium and strains from syngonium. Strains originating from the two host genera can be distinguished by restriction analysis of the amplification product. No amplification product was obtained with 98 strains of unrelated phytopathogenic bacteria or saprophytic bacteria from the anthurium phyllosphere, except for a weak signal obtained for one X. axonopodis pv. allii strain. Nevertheless, restriction enzyme analysis permitted the two pathovars to be distinguished. The detection threshold obtained with pure cultures or plant extracts (10(3) CFU ml(-1)) allowed detection of the pathogen from symptomless contaminated plants. This test could be a useful diagnostic tool for screening propagation stock plant material and for monitoring international movement of X. axonopodis pv. dieffenbachiae.     

Putative virulence characteristics of <Emphasis Type="Italic">Stenotrophomonas maltophilia</Emphasis>: a study on clinical isolates

Stenotrophomonas maltophilia is an important evolving pathogen, especially in patients with cystic fibrosis (CF), but its mechanism of pathogenesis is poorly understood. The purpose of this study was to investigate the presence of potential virulence determinants in five septicemic clinical isolates of S. maltophilia. When screened for EPS biosynthesis, all five strains produced colonies on two different growth media both at 30 and 37 °C. LPS could be extracted from all strains successfully and all were positive for both cell-free and cell-bound hemolysin production but failed to agglutinate 3% human RBCs. Variation in the ability to produce protease and phospholipase C was observed. In addition, all strains were unable to produce pyochelin but were able to produce ornibactin in the form of hydroxamate derivatives. It was also observed that all strains showed adherence to mouse tracheal epithelium.     

Recognition of pathogenic Yersinia enterocolitica by crystal violet binding and polymerase chain reaction

Cefsulodin-Irgasan-Novobiocin (CIN) agar is used for the selective isolation and enumeration of Yersinia enterocolitica from clinical specimens and food. The medium contains crystal violet and about 1 mmol l^-1 calcium and can be used for the phenotypic characterization of strains that carry a virulence plasmid. At 32°C, irrespective of pathogenicity, colonies are translucent with a pale pink centre surrounded by a transparent border ('bullseye'), while at 37°C pathogenic strains grow as calcium-dependent microcolonies which, because of crystal violet binding, are intensely coloured. These results were confirmed by the polymerase chain reaction with primers directed at the vir F gene, which is present only in pathogenic strains of Y. enterocolitica. Pathogenic strains of Y. enterocolitica can be recognized by growth at 37°C on Yersinia selective agar.     

The Influence of the Concomitant Microflora on Establishment and Dieback of Decay Fungi in Standing Timber

The microbiological aspects that designate development of decay in standing timber were assessed by determining the resistance to succession as well as the competitive saprophytic ability (kratovirulence, KV) of 40 species of wood destroying basidiomycetous fungi (WBF), proved to be members of 7 ecological categories. The categories (i) to (iv) comprise predominantly pathogenic WBF (pathogens) with high pathovirulence (PV) and correspondingly low KV properties that are dominant in the decay of standing timber. The categories (v) to (vii) include saprophytic WBF (saprophytes) with occasional or no PV and increasingly dominant KV properties which are native to slash and plant residues. On sterilely overgrown blocks of fertilized wood meal which had been exposed to airborne contaminants, most pathogens displayed the same degree of resistance to the insinuation of successive microbes as did the saprophytes. In the inoculation attempts of randomly contaminated wood meal, however, the low-kratovirulence, pathogens were unable to repress the multitude of the antagonistic resident microorganisms in order to establish themselves in this substrate. In contrast, most high-kratovirulence saprophytes reduced the number of, or completely eliminated, these antagonistic resident microorganisms in wood meal to create the preconditions for colonizing this substrate themselves for up to 1,200 days. In application of these results to the conditions in standing timber it is concluded that the wounded living tree involuntarily promotes the entry of a pathogenic WBF by keeping its natural antagonists down. The once established WBF can then protect its vegetative thallus itself from being overgrown by successive microbes.     

Staphylococci in Competition: V. Effect of Eggs, Eggs Plus Carbohydrates, and Lipids on Staphylococcal Growth

As is generally known, custards have been frequently involved in staphylococcal food poisonings and are regarded by some as an ideal culture medium. Previous studies showed that high carbohydrate concentrations repressed the growth of competing saprophytic species, allowing the growth of staphylococci in a mixture rather than stimulating the growth of the staphylococci. In an extension of those studies, the influence of the egg constituent of custard was investigated to determine its role in affecting the growth of pathogenic staphylococci in a competing mixture of psychrotrophic saprophytes. The normal competitive effect of the saprophytes on the growth of staphylococci was very slightly affected by the addition of 25% whole egg to the growth media. Approximately 9% egg yolk alone added to the medium resulted in a slight increase in the length of the lag period of the psychrotrophs and a slight increase in the number of staphylococci which grew. Addition of 25% whole egg plus 14.5% sucrose resulted in repression of saprophyte growth similar to that seen in high sucrose concentrations. Staphylococcal growth was more extensive in the presence of both whole egg and sucrose than in the presence of either ingredient alone. Incorporation of 4% corn oil in media was effective in repressing growth of the saprophytes at 37 C only. This allowed the staphylococci to dominate the population. At lower temperatures, staphylococci were unable to compete effectively. Buffering media of high carbohydrate content resulted in lengthened lag periods for the psychrotrophs and the appearance of very large staphylococcal populations.     

Isolation of Bacteria Which Can Grow at Both High pH and Low Temperature

Fifty bacterial strains able to grow at pH 10 and 0°C were isolated from soils, and growth characteristics of three selected strains were investigated. Strain 207, which showed the best growth rate of all the isolates at the conditions described above, could grow at a temperature of −5 to 39°C at pH 8.5. The optimum pH for this strain changed from 9.5 at 10°C to 9.0 at 20°C.     

Contribution of Complement System pathways to the killing of Leptospira spp.

The Complement System (CS) plays an important role in the immune response against leptospirosis and can be activated by the Alternative and Lectin Pathways (Innate Immunity) and by the Classical Pathway (Acquired Immunity). Here we analyzed a broad range of nonpathogenic and pathogenic Leptospira strains considering their interaction with each CS pathway. We determined bacterial survival rate and CS protein deposition in the presence of purified proteins, specific component depleted sera and NHS treated with the chelating agents EDTA (inhibits all three activation pathways) or EGTA (inhibits the Classical and Lectin Pathways). We suggest that the Lectin and the Alternative Pathways have an important role to eliminate saprophytic leptospires since i) approximately 50% survival of both saprophytic strains was observed in the presence of MBL-deficient serum; ii) approximately 50% survival of Leptospira biflexa Patoc I was observed in the presence of NHS – EGTA and iii) C1q-depleted serum caused significant bacterial lysis. In all serovars investigated the deposition of C5–C9 proteins on saprophytic Leptospira strains was more pronounced when compared to pathogenic species confirming previous studies in the literature. No difference on C3 deposition was observed between nonpathogenic and pathogenic strains. In conclusion, Leptospira strains interact to different degrees with CS proteins, especially those necessary to form MAC, indicating that some strains and specific ligands could favor the binding of certain CS proteins.