Preliminary Studies of Some Physiological Properties of Torula jeanselmei

The morphological characteristics of pathogenic and saprophytic strains of Torula jeanselmei (Fungi Imperfecti) are indistinguishable. Some basic physiological properties of T. jeanselmei were investigated in the hope of separating the human pathogenic strain from common saprophytes in a clinical laboratory. Nitrogen and carbon assimilation tests were not useful for distinguishing the pathogen, since all strains, pathogenic and saprophytic, were able to assimilate nitrogen from ammonium, nitrate, and nitrite sources, and all were able to utilize glucose, fructose, galactose, cellobiose, lactose, glycerol, and starch. It appeared that temperature range for growth might be useful in the identification of the pathogen. All pathogenic strains grew well at 37 C but poorly at 10 C; in contrast, all saprophytes could grow at 10 C but were unable to grow at 37 C (with two exceptions).     

Comparison between two techniques for endometrial swab culture and between biopsy and culture in barren mares

The endometria of 77 barren mares was swabbed simultaneously using a swab guarded with a single cannula and distal, gelatin capsule (completely guarded swab - CGS) and a partially guarded swab (PGS) with an open cannula. Sheep blood (5%) agar plates were inoculated with each swab, while MacConkey's agar plates were inoculated with the swabs from 44 mares. The presence of bacterial or fungal growth was determined after 24 and 48 hours of aerobic incubation at 37 C. Organisms present were identified, counted, and categorized as saprophytic or pathogenic flora. The endometria of all mares were biopsied immediately following swabbing. Histologic evidence of inflammation in biopsy specimens was classified as (1) none, (2) slight, discrete, focal, and (3) slight or moderate, diffuse, widespread infiltration of inflammatory cells. The number of inflammatory cells migrating through the luminal epithelium was counted and averaged. There were significantly fewer CGS than PGS cultures that yielded growth at 24 and 48 hours of incubation after being streaked on blood agar and MacConkey's agar plates. There were fewer pathogenic bacterial or fungal colonies present at 48 hours of incubation on blood agar plates after being streaked with CGS as compared to PGS. There were no differences in the number of pathogenic bacterial or fungal colonies present at 24 hours of incubation on blood agar or at 24 and 48 hours of incubation on MacConkey's agar plates. There was no correlation between CGS or PGS culture of pathogens and severity of histologic inflammation. There was a positive correlation between culture of pathogens and number of inflammatory cells migrating through the luminal epithelium.     

Regulation of histidine biosynthetic enzymes in a mutant of Escherichia coli with an altered histidyl-tRNA synthetase

Summary A mutant of Escherichia coli was isolated that grew at a normal rate in minimal medium at 26°C, grew at a normal rate in minimal medium at 37°C only if exogenous histidine was supplied, and grew more slowly than normal at 42°C even in the presence of histidine. In very rich media the growth rate of the mutant was normal at 26°C and 30°C, but not at 37°C or 42°C. It may be described as a temperature-conditional histidine bradytroph with a decreased ceiling to its growth rate.The histidyl-tRNA synthetase of the mutant was found to be abnormal; in crude extracts the enzyme activity was less stable and had approximately a tenfold higher apparent K _Mfor histidine than normal.Under many growth conditions the histidine biosynthetic enzymes in the mutant were derepressed several hundred fold compared to the wild strain, even in the presence of exogenous genous histidine. In general, the degree of derepression in the mutant was proportional to the difference in growth rate between the mutant and normal strains; this relationship, however, did not hold below 30°C or above 37°C.The properties of the mutant could be related to the properties of its histidyl-tRNA synthetase by assuming that the enzyme participates both in protein synthesis and in histidine biosynthetic enzyme regulation and that at low temperature it functions relatively more effectively in protein synthesis than in repression, while at high temperature it functions relatively more effectively in repression.Abbreviations used tRNA transfer RNA - AICAR aminoimidazole carboxamide ribose-5-phosphate     

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.     

Use of Antibiotics in the Preparation of Canine Kidney Tissue Culture Vaccines to Eliminate Leptospiral Infection Hazards

The potential leptospiral infection hazard in the use of vaccines prepared from canine kidney monolayer cultures was studied. Cell cultures were prepared from kidneys of dogs experimentally infected with Leptospira serotype canicola. Viable leptospires were found in kidney cell suspensions at the time of seeding, surviving trypsinization either at room temperature for approximately 2 hr or overnight at 4 C, even in the presence of antibiotics. In tissue cultures maintained without antibiotics, leptospires were cultured up to the time of involution of cells at 25 to 34 days of incubation. Cytopathogenic effects of leptospires on cultured kidney cells were not noted; neither was growth of leptospires remarkable. Generally, the leptospire culture titer decreased to 10^-4 or 10^-5 at the 4th hr or 1st day of incubation to 10^-1 or negative by the 30th or 34th day of incubation. The addition of either a combination of penicillin (100 units per ml) plus streptomycin (100 μg/ml) or polymyxin B (50 units per ml) plus dihydrostreptomycin (100 μg/ml) to seeding cell suspensions resulted in the elimination of viable leptospires by the 4th hr of incubation. From cell cultures treated with neomycin (100 μg/ml) or chloramphenicol (100 μg/ml), leptospires were recovered, respectively, after 24 and 48 hr, but not thereafter. It was apparent that antibiotics, particularly the combination of polymyxin B and dihydrostreptomycin, could be effectively used to eliminate leptospires in tissue culture. Other antibiotics with known antileptospiral activities probably would be effective also. If antibiotics are not used in canine kidney tissue culture employed for viral vaccine preparations, rigid testing for the presence of leptospires in donor dogs and tissue-culture vaccine is indicated.     

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.     

Oligonucleotides specific for pathogenic and saprophytic leptospira occurring in water

Sets of primers specific for both pathogenic (SPL) and saprophytic (SSL) Leptospira were designed from ribosomal 16S genes (rrs) available in databases. They were used as two sets of primer pairs for the PCR amplification of known pathogenic and saprophytic strains. It was possible to identify pathogenic strains by the use of SPL primers and saprophytic ones by SSL primers. Serovars from L. meyeri, of controversial pathogenicity status, confirmed the heterogeneity of the species representatives in this respect. Serovars ranarum, sofia and perameles were amplified by SPL and not SSL. Conversely, serovar semaranga was amplified by SSL and not SPL. In order to use SPL primers for the detection of pathogenic leptospires from a natural water environment, we set up an additional semi-nested PCR by employing a second internal primer which succeeded in detecting as few as 5 pathogenic leptospires per ml of water.     

Temperature and water activity minima for growth of spoilage moulds from meat

L owry , P.D. & G ill , C.O. 1984. Temperature and water activity minima for growth of spoilage moulds from meat. Journal of Applied Bacteriology 56 , 193–199.
Five species of fungi were isolated from mould spoilage on meat other than black spot. 'White spot' colonies yielded Chrysosporium pannorum or an Acremonium sp .; 'whiskers' colonies yielded Thamnidium elegans or Mucor racemosus , and blue-green colonies yielded Penicillium corylophilum. Chrysosporium pannorum was moderately xerotolerant with a minimum growth temperature of — 5C. The Acremonium sp. and P. corylophilum showed a similar level of xerotolerance but had a minimum growth temperature of — 2C. Mucor racemosus was no more xerotolerant than many spoilage bacteria and did not grow below - 1C, but grew rapidly at 3C and above. Thamnidium elegans grew at — 7C on supercooled medium and an intrinsic minimum growth temperature of — 10C was indicated. However, the low xerotolerance of this species precluded growth on frozen media below — 5C. It seems therefore that — 5C is the practical limiting temperature for mould growth on meat, and mould spoilage usually indicates that surfaces of freezer stored meats have approached and possibly exceeded 0C.     

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.     

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.     

Minimal Growth Temperature, Sodium Chloride Tolerance, pH Sensitivity, and Toxin Production of Marine and Terrestrial Strains of Clostridium botulinum Type C

Minimal growth temperatures of four marine and two terrestrial strains of Clostridium botulinum type C were determined in a laboratory culture medium, fortified egg meat medium (FEM), and in ground haddock. The inoculum equaled 2 × 10⁶ viable spores per tube with five-tube replicate sets. The spores were preheated in aqueous suspension at 71 C for 15 min prior to inoculation to reduce toxin carry-over. Similar results were obtained in both substrates. Both the marine and the terrestrial strains grew at 15.6 C, but only the terrestrial strains grew at 12.8 C. None of the strains grew at 10 C during prolonged incubation. The sodium chloride tolerance and the pH sensitivity of the marine and the terrestrial strains were determined at 30 C. The basal medium consisted of beef infusion broth. The inoculum level equaled 2 × 10⁶ unheated spores per replicate. Growth was inhibited at salt concentrations from 2.5 to 3.0%. The terrestrial strains were more pH-sensitive than the marine strains. Whereas the terrestrial strains failed to grow below pH 5.62, three of the marine strains grew at pH 5.10, but not at pH 4.96, during extended incubation. One marine strain grew at pH 5.25, but not below. FEM and proteose peptone-Trypticase-yeast extract-glucose medium permitted the production of high levels of botulinum toxin among four media tested. Toxin produced by the marine and terrestrial strains showed no increase in toxicity after incubation with trypsin.     

Electrophoretic study of the outer membrane proteins of leptospirae

The outer membranes of pathogenic and saprophytic leptospires have been isolated. The spectrum of outer membrane proteins in three saprophytic and one pathogenic Leptospira strains has been studied by means of electrophoresis in polyacrylamide gel. In Leptospira strains VGNKI-6 (pathogenic) and G-80 (saprophytic) identical proteins, as well as proteins similar in their Rf value, have been detected. The possibility of using strain G-80 for the development of leptospiral vaccine against serovars having common surface antigens with this strain has been suggested.     

Staphylococci in Competition: I. Growth of Naturally Occurring Mixed Populations in Precooked Frozen Foods during Defrost

In chicken pot pies, it was not possible to promote the growth of appreciable numbers of staphylococci under any condition of defrost. The pies spoiled under the same conditions, however. In macaroni and cheese dinners, tremendous numbers of saprophytic bacteria developed, but only after extended incubation at room temperature in which even spoilage was carried to an extreme. Under these conditions, staphylococci also multiplied vigorously. At the extreme temperature of 37 C, rank spoilage of such an advanced state as to render the product completely inedible was reached in less than 24 hr. Staphylococci grew very well under these extreme conditions.     

Suitability of poor medium in counting total viable airborne bacteria

The purpose of this study was to test the effect of incubation temperature and culture medium on viable counts of airborne bacteria. The incubation temperature had different effect on indoor and outdoor air bacteria. Indoor air bacteria grew as well at 20°C as 37°C, but less at 10°C. Outdoor air bacteria grew equally well at 10°C and 20°C, but less at 37°C. Both indoor and outdoor air bacteria grew differently on poor and rich media. The counts of both indoor and outdoor air bacteria were higher on poor R2A medium (low nutrient concentration) than on rich TYG and blood media (high nutrient concentration). The results indicate that a poor medium incubated at 20°C is adequate for counting viable airborne bacteria.     

Seasonal selection in a freshwater heterotrophic bacterial community

The objective of this study was to determine if a seasonal selection could be demonstrated in the heterotrophic component of a freshwater bacterial community. Surface samples were taken at approximately monthly intervals covering an annual seasonal cycle, and counts were made of the numbers of bacteria capable of growing at each of 10 incubation temperatures from 0° to 45°C at 5°C intervals. Evidence for seasonal selection was provided by a 6°C shift in the mean temperature of the counts from the summer sample to the winter sample. The selection was even more evident when the number of organisms capable of growing at 10°C and those capable of growing at 35°C were compared over the seasonal cycle. The counts at these two incubation temperatures varied inversely to each other. Although a negligible number of organisms from a representative summer sample grew at 10°C, 18% of the organisms from a representative winter sample grew at this temperature. The data of this study indicate that, although seasonal selection does occur, the magnitude of that selection is not great enough to permit the growth of bacteria during the coldest month to approach the levels of growth observed during the summer months. However, the selection appears to be adequate to permit significant activity during the spring and fall transition months.     

Enteric Bacterial Growth Rates in River Water

Enteric bacteria, including stocked strains of pathogenic species and organisms naturally present in the stream, were capable of growth in a chemostat with autoclaved river water taken 750 m below a sewage outfall. Maximal specific growth rates for all organisms occurred at 30 C, whereas culture generation times ranged between 33.3 and 116 hr. Of the six laboratory strains of enteric species used, Escherichia coli and Enterobacter aerogenes grew at generation times of 34.5 and 33.3 hr, respectively, while the remaining Proteus, Arizona, Salmonella, and Shigella spp. reproduced at a rate two to three times slower than the coliforms. Little or no growth occurred in the water at incubation temperatures of 20 and 5 C, and death was observed for Salmonella senftenberg at 20 and 5 C and for E. aerogenes and Proteus rettgeri at 5 C. When enteric bacteria naturally present in the river water were employed in similar experiments, coliform bacteria demonstrated a generation time of approximately 116 hr, whereas fecal coliforms failed to grow. Growth of the bacteria from the river demonstrated a periodicity of approximately 100 hr, which suggests that much of the growth of these organisms in the chemostat may be on the glass surfaces. This phenomenon, however, was not observed with any of the stocked enteric species. Neither the stock cultures nor the aquatic strains were capable of growth in autoclaved river water taken above the sewage outfall at the three temperatures tested.     

Growth of Salmonella typhimurium in Skim Milk Concentrates

The influence of various levels of skim milk solids and temperature on the duration of lag phase, growth rate, and extent of growth of Salmonella typhimurium was investigated. The effect on growth of salmonellae (and a strain of Escherichia coli) of reduced pressure at a constant solids level and under conditions simulating vacuum condensation of skim milk was also studied. S. typhimurium grew when inoculated into skim milk solutions ranging from 10 to 60% solids and over a temperature range of 23 to 44 C. At 10 to 12 C, growth was evident only in the 10% skim milk. As the total solids level was increased or incubation temperature was deviated from the optimum, or both, there was an increase in the lag phase and generation time of salmonellae. A lower cell population also resulted. The generation time at 37 C of S. typhimurium incubated at atmospheric pressure was approximately one-half that in skim milk concentrates held under reduced pressure. In addition, a slightly longer lag phase and lower cell yield characterized the growth under reduced pressure. Concentration of skim milk had little or no effect on viability of salmonellae or E. coli when the vapor temperature in the vacuum pan was below the maximum growth temperature for salmonellae. Increasing the vapor temperature to 48 C caused a two-log reduction in viable organisms during the concentrating period (65 min).     

Fitness of three Fusarium pathogens of wheat

Crown rot and head blight of wheat are caused by the same Fusarium species. To better understand their biology, this study has compared 30 isolates of the three dominant species using 13 pathogenic and saprophytic fitness measures including aggressiveness for the two diseases, saprophytic growth and fecundity and deoxynivalenol (DON) production from saprophytic colonization of grain and straw. Pathogenic fitness was generally linked to DON production in infected tissue. The superior crown rot fitness of Fusarium pseudograminearum was linked to high DON production in the stem base tissue, while Fusarium culmorum and Fusarium graminearum had superior head blight fitness with high DON production in grains. Within each species, some isolates had similar aggressiveness for both diseases but differed in DON production in infected tissue to indicate that more than one mechanism controlled aggressiveness. All three species produced more DON when infecting living host tissue compared with saprophytic colonization of grain or straw, but there were significant links between these saprophytic fitness components and aggressiveness. As necrotrophic pathogens spend a part of their life cycle on dead organic matter, saprophytic fitness is an important component of their overall fitness. Any management strategy must target weaknesses in both pathogenic fitness and saprophytic fitness.     

The usefulness of semi‐solid medium in the isolation of highly virulent Leptospira strains from wild rats in an urban area of Fukuoka,Japan

Leptospirosis is a worldwide zoonosis. The importance of urban leptospirosis is recognized in Japan: urban rats carry pathogenic leptospires and people acquire these pathogens through contact with surface water or soil contaminated by the urine of the infected animals. To determine the current Leptospira carriage rate in urban rats, 29 wild rats were trapped in the central area of Fukuoka and strains isolated from their kidneys and urine analyzed. When semi‐solid Korthof's medium containing 0.1% agar was used for isolation, 72.2% and 30.8% of the kidney and urine cultures, respectively, were found to be Leptospira‐positive. The isolates belonged to Leptospira interrogans, and were classified into two groups (serogroups Pomona and Icterohaemorrhagiae) based on the results of gyrB sequence analysis and microscopic agglutination testing (MAT). Strains belonging to serogroup Icterohemorrhagiae grew well in liquid medium. On the other hand, serogroup Pomona isolates multiplied very little in liquid medium, but did grow in a semi‐solid medium. Although strains belonging to serogroup Pomona have not been recognized as native to Japan, this strain may be widely distributed in urban rats. Representative strains from each group were found to be highly pathogenic to hamsters. Our findings should serve as a warning that it is still possible to become infected with leptospires from wild rats living in inner cities of Japan. Furthermore, the use of semi‐solid medium for culture will improve the isolation rate of leptospires from the kidneys of wild rats.