Virulence, viability and antibiotic sensitivity of the causative agent of plague growing on nutrient media at 28 and 37 degrees C

A comparative study of virulence, viability and antibiotic sensitivity of Y. pestis strains grown at 28 degrees C and 37 degrees C in yeast-casein medium, yeast medium with Hottinger's meat digest and yeast medium with protein hydrolysate obtained from sunflower seed groats has been made. These media have been found to be suitable for the prolonged cultivation of Y. pestis at 28 degrees C and 37 degrees C, for the determination of its sensitivity to antibiotics, as well as for the preservation of Y. pestis cultures.     

Survival of Bacteria on Metal Surfaces

Survivor curves were determined for Serratia marcescens, Sarcina lutea, Pasteurella tularensis, and P. pestis deposited from the airborne state onto metallic surfaces and subsequently stored at various humidities and temperatures. Cells of all species tested remained alive longest in a dry atmosphere, except that cells of S. marcescens survived best in a saturated atmosphere. Survival decreased most rapidly at the intermediate humidity level for three of the test organisms, yet P. tularensis died most rapidly in a saturated atmosphere. An increase in temperature decreased survival of P. pestis and P. tularensis.     

Isolation of Antigens of Pasteurella pestis I. Lipopolysaccharide-Protein Complex and R and S Antigens.

Larrabee, Allan R. (Fort Detrick, Frederick, Md.), John D. Marshall, and Dan Crozier. Isolation of antigens of Pasteurella pestis. I. Lipopolysaccharide-protein complex and R and S antigens. J. Bacteriol. 90:116-119. 1965.-Pasteurella pestis contains at least 18 different antigens, 2 of which will protect experimental animals from challenge infection. A specific polysaccharide isolated and described as a hapten was isolated as a complete antigen. Two additional antigens were isolated from P. pestis. The preparation of antisera directed against these three antigens and the content of protein, lipid, and carbohydrate of each preparation were studied. None of the preparations will protect mice from challenge infection with virulent P. pestis. A basis for naming the new antigens which does not conflict with previously published designations is presented.     

Pesticinogeny: a Characteristic Useful for Presumptive Identification and Isolation of Pasteurella pestis

Current methods of identifying Pasteurella pestis rely heavily on tests specific for detecting fraction I, the envelope antigen. Pesticin I, a bacteriocin inhibitory for P. pseudotuberculosis, has been demonstrated in nearly all tested strains isolated from human infections. The results of using this characteristic as an identifying trait for P. pestis were compared with results reported for detecting fraction I by fluorescent-antibody and antiserum-agar techniques. Data indicate that, although certain atypical strains of P. pestis fail to react in one system or the other, a combination of these tests provides positive identification in all cases. Detection of P. pestis in contaminated materials is greatly facilitated, and the simplicity of this test makes it a valuable tool in the study of plague infections and an important adjunct to methods currently in use. The use of the pesticin I assay is not intended to replace other accepted techniques, but rather to supplement them and increase the effectiveness of plague investigation.     

LcrD, a membrane-bound regulator of the Yersinia pestis low-calcium response.

Yersinia pestis, the etiologic agent of bubonic plague, contains a 75-kb virulence plasmid, called pCD1 in Y. pestis KIM. The low-Ca(2+)-response genes of Y. pestis regulate both bacterial growth and the expression of pCD1-encoded virulence determinants in response to temperature and the presence of Ca2+ or nucleotides. This study characterizes the nucleotide sequence and protein product of the lcrD locus. An lcrD mutant, in contrast to the parent Y. pestis, did not undergo growth restriction or induce strong expression of the V antigen when grown under conditions (37 degrees C, no Ca2+) expected to elicit maximal expression of pCD1 genes. DNA sequence analysis of the cloned lcrD locus showed a single open reading frame that could encode a protein with a molecular weight of 77,804 and a pI of 4.88. LcrD was identified as a 70-kDa inner membrane protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis. LcrD membrane topology was investigated by using lcrD-phoA translational fusions generated with the transposon TnphoA. The alkaline phosphatase activities of the resultant hybrid proteins were consistent with a model predicting eight amino-terminal transmembrane segments that anchor a large cytoplasmic carboxyl-terminal domain to the inner membrane.     

Congo Red-Agar Plating Medium for Detecting Pigmentation in Pasteurella pestis

Ability to detect pigmented and nonpigmented Pasteurella pestis is essential in plague research, and is currently dependent on use of the synthetic hemin-agar of Jackson and Burrows. We have devised a new differential medium for this purpose, containing Congo red dye and common, commercially available laboratory media. The ease and simplicity of preparation make the Congo red-agar a practical routine laboratory tool in plague research. These findings, possibly indicating a common binding site for hematin and Congo red, should be useful in efforts to determine the chemical nature of a bacterial component associated with high virulence in P. pestis.     

Influence of growth temperature on the production of extracellular virulence factors and pathogenicity of environmental and human strains of Aeromonas hydrophila

The biochemical properties, virulence for mice and trout, and the extracellular virulence factors at 28° and 37°C of 11 environmental and nine human strains of Aeromonas hydrophila were compared. All the environmental isolates and four of the human group were virulent for trout at 3 x 10⁷ cfu, but only human strains were able to cause death or lesions in mice by the intramuscular route. Extracellular virulence factors such as haemolysins, cytotoxins and proteases were also investigated in supernatant fluids of cultures grown at 28°C and 37°C. The production of haemolysins, caseinases, elastases and growth yields of environmental strains decreased sharply during cultivation at 37°C but cytotoxins were produced to the same extent, or slightly less, than at 28°C. The human strains differed from the environmental strains in response to growth temperatures: protease activity decreased at 37°C, although growth yield was not affected, but more haemolysins and cytotoxins were produced by the virulent strains at this temperature than at 28°C. Sodium caseinate SDS-PAGE of culture supernatant fluids of selected human strains revealed that temperature selectively inhibited the production of certain proteases.     

Virulence and viability of Yersinia pestis 25 years after lyophilization.

When equal volumes of 6% lactose and a broth culture of Yersinia pestis were mixed before freezing, approximately 50% of the cells survived lyophilization and reconstitution on the following day. Concomitantly, the number of viable cells per 50% lethal dose increased from about 16 to 125 organisms. On subsequent storage of the lyophilized cells under vacuum in glass ampoules at 4 degrees C for 25 years, more than 25% of the cells remained viable. When stored cultures were assayed immediately after reconstitution, virulence for mice was significantly reduced (as many as 4,000 cells/50% lethal dose), but the virulence was fully restored when reconstituted cultures were held for 24 h at room temperature, or when a subculture was prepared in fresh medium.     

Specific Identification of Fraction I-Positive Pasteurella pestis Colonies on Antiserum-Agar Plates

A method is described for the use of antiplague serum in Blood Agar Base plating media to detect fraction I-positive Pasteurella pestis. The antiserum was produced conveniently and in large volume in rabbits by use of Cutter plague vaccine combined with Freund's complete adjuvant. P. pestis colonies were specifically identified within 48 hr after plating by the presence of a precipitin ring surrounding each colony. The basis of the test was shown to be a precipitin reaction between fraction I antigen released from P. pestis colonies after chloroform vapor treatment and fraction I antibody present in the antiserum-agar medium.     

THTA, a thermotolerance gene of Aspergillus fumigatus

Aspergillus fumigatus grows optimally from 37 to 42 degrees C but can grow at temperatures up to 55 degrees C. To study the genetic basis of thermotolerance and its role in virulence of A. fumigatus, temperature sensitive mutants were isolated. One of the mutants that grew at 42 degrees C but not at 48 degrees C was complemented and the gene, THTA, was identified. Deletion of THTA showed the same temperature sensitivity as the original mutant. THTA encodes a putative protein of 141 kDa with unknown function and the HA-tagged ThtAp accumulated to similar levels in cultures grown at either 37 or 48 degrees C. Southern blot analysis and database searches revealed the presence of THTA-related sequences in several other ascomycetous fungi. No difference in virulence was observed between the deltathtA and wild-type strains. Thus, THTA is essential for growth of A. fumigatus at high temperatures but does not contribute to the pathogenicity of the species.     

Levels of fraction I and VW antigens in cultures of the plague microbe grown on yeast-casein, yeast-Hottinger broth and yeast-sunflower oil cake media

The content of fraction 1 and VW-antigens in Y. pestis cultures grown in different media (yeast-casein medium, yeast medium with Hottinger digest, and yeast medium with sunflower-seed protein) was studied over the course of their growth by means of the antibody neutralization and microprecipitation in agar tests. The media under study were not inferior to the casein sulfuric hydrolysate-based medium used for control in their capacity for ensuring the synthesis of VW-antigens. The maximum accumulation of fraction 1 was observed in yeast medium with sunflower-seed protein. In all media the maximum content of fraction 1 was registered on day 3 of cultivation, and the maximum accumulation of VW-antigens on days 8-9 of incubation at 37 degrees C. The data obtained in this study make it possible to regard fraction 1 and VW-antigens as the secondary metabolites of Y. pestis.     

Strain specific variation of outer membrane proteins of wild Yersinia pestis strains subjected to different growth temperatures

Three Yersinia pestis strains isolated from humans and one laboratory strain (EV76) were grown in rich media at 28 degrees C and 37 degrees C and their outer membrane protein composition compared by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). Several proteins with molecular weights ranging from 34 kDa to 71 kDa were observed to change in relative abundance in samples grown at different temperatures. At least seven Y. pestis outer membrane proteins showed a temperature-dependent and strain-specific behaviour. Some differences between the outer membrane proteins of full-pathogenic wild isolates and the EV76 strain could also be detected and the relevance of this finding on the use of laboratory strains as a reference to the study of Y. pestis biological properties is discussed.     

Typing of Yersinia pestis isolates from the state of Ceará, Brazil

AIMS: To investigate whether modifications in Yersinia pestis isolates from three plague foci from the state of Ceará, Brazil, had occurred over the years as a consequence of genetic adaptation to the environment. METHODS AND RESULTS: The isolates were studied with respect to susceptibility to antimicrobial drugs, plasmid and protein profiling, pigmentation on Congo red-agar plates, and the presence of some pathogenicity genes using PCR. Most of the expected virulence markers were detected in the cultures examined. There was no evidence of any alteration that could be associated with their origin (patients, rodents and fleas) or period of isolation (1971-1997). CONCLUSIONS, SIGNIFICANCE AND IMPACT OF THE STUDY: Phenotypic or genotypic changes were not detected in the cultures examined. However, the results obtained will serve as a reference to follow the evolution of Y. pestis in these foci.     

Growth of Male-Specific Bacteriophage in Pasteurella Harboring F-Genotes Derived from Escherichia coli

When either the F' lac or the F'Cm plasmid was transferred from Escherichia coli into Pasteurella pseudotuberculosis, the P. pseudotuberculosis (F') strains isolated formed plaques with both ribonucleic acid (RNA)-containing and deoxyribonucleic acid-containing male-specific phages. In contrast, strains of P. pestis harboring E. coli (F') plasmids did not form plaques with male-specific phages, although such strains permitted limited multiplication of phage MS2. The adsorption and burst size of MS2 were approximately the same in both species of Pasteurella, but the per cent of adsorbed MS2 that produced infective centers was much lower in P. pestis than it was in P. pseudotuberculosis. By use of a sib-selection technique of P. pestis (F') cells, we isolated a single clone that could form MS2 plaques. (32)P-labeled MS2 adsorbed equally to and its RNA penetrated equally into both the typical MS2-nonpermissive P. pestis cells and the MS2-permissive P. pestis cells. No host modification occurred after growth of MS2 in Pasteurella. Our data suggest that typical strains of P. pestis inhibit the intracellular development of phage MS2.     

Gene Transfer in Pasteurella pestis Harboring the F′Cm Plasmid of Escherichia coli

A strain of Pasteurella pestis, harboring the F'Cm plasmid from Escherichia coli, was able to donate its chromosome to auxotrophic recipient strains of P. pestis. The frequency of gene transfer in P. pestis was approximately 10(-6) per donor cell, 100 times less efficient than gene transfer in Pasteurella pseudotuberculosis, but efficient enough to determine entry times for the markers histidine, threonine, and tryptophan and to show linkage to the markers arginine and pigmentation. An attempt to extend the conjugation system to different serotypes of P. pseudotuberculosis and to Yersinia enterocolitica did not succeed.     

Stable Reagent for the Detection of Antibody to the Specific Fraction I Antigen of Yersinia pestis

A stable hemagglutinating antigen for detection of fraction I (FR-I) antibody of Yersinia pestis (Pasteurella pestis) is described. The antigen was prepared by sensitizing tanned, pyruvaldehyde-treated sheep erythrocytes (PAT SRBC) with FR-I antigen. Preliminary standardization by titration of each lot of FR-I was required to minimize the effect of molecular heterogeneity of specific FR-I antigen and to eliminate nonspecific reactions caused by the presence of a minor antigenic contaminant. In tests with sera from rabbits, dogs, and humans, FR-I PAT SRBC were as reactive as the previously employed standard antigen, FR-I-sensitized tanned erythrocytes. Fluid suspensions of FR-I PAT SRBC stored at 4 C for 3 months, or lyophilized preparations stored at ambient temperature for 6 months, showed no loss in antigenic activity.     

The Interactive Effects of Phytochrome, Nitrate and Thiourea on the Germination Response to Alternating Temperatures in Seeds of Ranunculus sceleratus L.: A Quantal Approach

Probert, R. J., Gajjar, K. H. and Haslam, I. K. 1987. The interactiveeffects of phytochrome, nitrate and thiourea on the germinationresponse to alternating temperatures in seeds of Ranunculussceleratus L.: A quantal approach.—J. exp. Bot. 38: 1012–1025. The interactive effects of phytochrome, potassium nitrate andthiourea on the germination response to alternating temperaturesin achenes (seeds) of Ranunculus sceleratus L. were studied.Using thermogradient bars, high levels of germination were recordedover a broad range of alternating temperatures providing seedsreceived daily irradiations. Reduced germination in temperaturecycles with a relatively long warm phase was related to thelevel of the active form of phytochrome (Pfr). Dose-responseexperiments to red light (R) and temperature shifts showed thatthe actions of Pfr and alternating temperatures were interdependent.Maximum germination was recorded when intermittent pulses ofR were combined with daily 4 h temperature shifts from 16°Cto 26°C. Whilst probit analysis showed that potassium nitrateand thiourea both increased population sensitivity to temperatureshifts, thiourea was a more potent stimulant. Although the effectof both chemicals was dependent on phytochrome photo-equilibriumthe threshold level of Pfr required for thiourea action wasclearly much lower than that required for nitrate action. Thioureapotentiated a response to daily temperature shifts even whenPfr was at a low, normally inhibitory level. These results indicatedifferent mechanisms of action for potassium nitrate and thioureain relation to phytochrome controlled seed germination. Key words: Phytochrome, nitrate, thiourea, alternating temperatures, germination     

Investigation into the role of the serine protease HtrA in Yersinia pestis pathogenesis

The HtrA stress response protein has been shown to play a role in the virulence of a number of pathogens. For some organisms, htrA mutants are attenuated in the animal model and can be used as live vaccines. A Yersinia pestis htrA orthologue was identified, cloned and sequenced, showing 86% and 87% similarity to Escherichia coli and Salmonella typhimurium HtrAs. An isogenic Y. pestis htrA mutant was constructed using a reverse genetics approach. In contrast to the wild-type strain, the mutant failed to grow at an elevated temperature of 39 degrees C, but showed only a small increase in sensitivity to oxidative stress and was only partially attenuated in the animal model. However, the mutant exhibited a different protein expression profile to that of the wild-type strain when grown at 28 degrees C to simulate growth in the flea.     

Lysogenic Conversion of Pasteurella by Escherichia coli Bacteriophage P1 CM

Bacteriophage P1 CM can convert Pasteurella pestis or P. pseudotuberculosis to chloramphenicol resistance and phage restriction, but no viable phage was induced from converted Pasteurella strains.