Association between M. pneumoniae hemolysis, attachment, and pulmonary pathogenicity

Three different groups of hemolysis mutants were produced by treatment of the M. pneumoniae FH-P24 strain with N-methyl-N-nitro-nitrosoguanidine. The first group of mutants, strains P24-L1, L2, and L11, showed wide and clear hemolytic zones, and attaching ability to erythrocytes and to hamster lung cells were the same as the properties of the parent strain and produced significant microscopic lung lesions. Mutant P24-S1 showed non-hemolysis and non-hemadsorption, yet retained the attaching ability to lung cells and produced milder lung lesions. Mutant P24-S11 showed none of those activities, did not cause any lung lesion, and was never recovered from the lungs of hamsters. A close relationship between the hemolytic ability of M. pneumoniae and the histopathogenicity in the hamster lung is suggested in this study. The attaching ability of organisms seems to be an important factor at the initial stage of infection.     

Immunogenicity and protective effect of hemolysis mutants of Mycoplasma pneumoniae

Two attenuated strains of Mycoplasma pneumoniae, P24-S1 and P24-S11, were tested for their ability as a live vaccine to confer on hamsters immune resistance against challenge infection with a virulent strain of M. pneumoniae, FH-P24. Fifty percent protection was obtained by vaccination with the P24-S1 strain administered once or twice. In contrast, only 10% of the animals were protected by the P24-S11 vaccine even when it was given three times. Vaccination with the P24-S1 strain resulted in higher humoral and cellular immune responses than the P24-S11 did. These results suggest that the P24-S1 strain has the primary qualities a vaccine which may be used for protection against human mycoplasmal infection.     

Relationship between an 85 kDa protein and the protective effects of Mycoplasma pneumoniae.

In the immunoblot analysis, sera from patients infected with Mycoplasma pneumoniae reacted with the 168 kDa (P1) and the 85 kDa proteins of virulent strain FH-P24 and P24-S1 mutant strain but not with the 85 kDa protein of P24-S11. Sera of hamsters and BALB/c mice, which had been immunized with live vaccines, were tested. In FH-P24 immunized animals, 100% or 80%, and in P24-S1, 40% of hamsters and 60% of BALB/c mice, developed antibodies against the 85 kDa protein, but antibodies were not detected in sera of P24-S11 immunized animals. The correlation between the development of antibodies to 85 kDa protein in the sera of vaccinated animals and the effects of protection by living vaccines were suggested.     

Hemagglutinin-neuraminidase glycoprotein as a determinant of pathogenicity in mumps virus hamster encephalitis: analysis of mutants selected with monoclonal antibodies.

With the aid of monoclonal antibodies directed against a specific site on the hemagglutinin-neuraminidase surface glycoprotein, four mutants of the Kilham neurotropic strain of mumps virus were isolated. All four mutants had increased neuraminidase activity. Two mutants (M10 and M12) lost their hemagglutination capacity with human O erythrocytes but retained their ability to agglutinate guinea pig erythrocytes at 4 degrees C. A third mutant (M11) showed a change in the molecular weight of the hemagglutinin-neuraminidase glycoprotein. These three mutants (M10, M11, and M12) showed unaltered capacity to infect tissue cultures and to cause encephalitis in newborn hamsters. A fourth mutant (M13) retained its hemagglutination activity and capacity to infect Vero cell cultures but showed significantly lower neurovirulence in the suckling hamster brain than did the parental Kilham strain and the other three mutants. Both the number of infected neurons and the amount of infectious virus in the brain was reduced. On the other hand, there were no apparent differences in the occurrence of viral antigen in ependymal cells, indicating a selective change in affinity for neurons in the brain. These results suggest that certain changes in the hemagglutinin-neuraminidase glycoprotein may lead to an alteration of the neuropathogenicity of the Kilham strain of mumps virus.     

Role of Humoral Antibodies in Resistance to Mycoplasma pneumoniae Pneumonia in Hamsters

Golden Syrian hamsters adoptively immunized with hyperimmune Mycoplasma pneumoniae rabbit antiserum, immunoglobulin (Ig) M-rich (IgM) fraction, IgG-rich (IgG) fraction, antiserum absorbed with either killed M. pneumoniae or killed Staphylococcus aureus organisms, or antiserum treated with 2-mercaptoethanol (2-ME) were examined for resistance against aerosol infection with virulent M. pneumoniae. Significant resistance to the establishment of infection in the respiratory tract was shown in hamsters pretreated with the untreated antiserum, IgG fraction or 2-ME-treated antiserum, whereas animals pretreated with the IgM fraction and the antisera absorbed with M. pneumoniae or S. aureus organisms were not significantly resistant. Histopathologically, lung lesions were markedly suppressed in animals with high resistance, but were typically pneumonic in animals with low or no resistance. The efficacy of adoptively administered serum preparations was closely related to their antibody titers. The results indicate that humoral antibody plays an important role in protection against experimental M. pneumoniae pneumonia in hamsters, although the participation of the cell-mediated immune response was not determined.     

Lysis of erythrocytes byTrichomonas vaginalis

The in vitro hemolytic activity of 4 isolates ofTrichomonas vaginalis was investigated. Repetitive hemolysis assays of any one isolate showed cyclical fluctuations in hemolytic activity, varying over 24 hr of continuous culture. Maximal hemolytic activity was detected using trichomonads in the lag phase of the growth cycle. Investigations showed that hemolysis was a contact-dependent phenomenon and microscopic investigation of samples showed a significant correlation between hemolysis and attachment of erythrocytes to the trichomonad surface. Quantitative data from cytoadherence assays using [⁵¹Cr]-labeled erythrocytes were consistent with these observations. It is suggested that hemolytic activity is dependent upon adherence of red blood cells to the surface ofT. vaginalis.     

A tracheal culture model of respiratory tract infection withPseudomonas Aeruginosa

Summary The pathogenesis ofPseudomonas aeruginosa for the respiratory tract has been examined using hamster tracheal organ cultures. Tracheal rings prepared from male Syrian hamsters, strain LSH/LAK, were infected withP. aeruginosa for 4 h and processed at 4-h intervals for 24 h for examination by light- and electron microscopy. Tissue destruction was observed within 8 h after infection with 10⁸ colony-forming units (cfu)/ml and within 12 h after infection with 10⁴ or 10⁶ cfu/ml. Ciliated cells that contained abnormal subcellular organelles were expelled from the epithelium. By 20 h the epithelial borders were composed primarily of nonciliated cells. Transmission- and scanning electron microscopy revealed details of the cellular destruction and attachment ofP. aeruginosa to the ciliated epithelium.Pseudomonas aeruginosa causes a rapid destruction of the epithelium of hamster trachea in cultures. Hamster tracheal organ cultures have been shown to be useful in studying the pathogenesis ofP. aeruginosa for the respiratory tract. This work was supported by Grants G-430B and G-431B from the Cystic Fibrosis Foundation.     

Cell-envelope changes in mutants ofCitrobacter freundii with altered response to colicin A

Spontaneous colicin A-resistant and -tolerant mutants were isolated fromCitrobacter freundii and classified in five different groups on basis of their sensitivity to bacteriocin S6 produced byKlebsiella pneumoniae. One group of colicin A-resistant mutants was extremely sensitive to ampicillin and desoxycholate; one group of colicin-A-tolerant mutants was extremely sensitive to ampicillin, desoxycholate and EDTA. One of the bacteriocin-S6-insensitive mutants showed filament formation in liquid medium. The cell envelope of one representative strain of each group was isolated and fractionated in a cell-wall- and a cytoplasmic-membrane-enriched fraction. Polyacrylamide-gel electrophoresis of these fractions showed that both fractions differ from those of the wild-type strain in the relative amounts of some proteins. The differences in the cell envelope of all mutants concerned for the greater part the same proteins. Data obtained by phospholipid analysis of the cell envelope of the mutants showed no significant differences.     

Pathogenicity of axenically cultivated Entamoeba histolytica, strain 200:NIH, in the hamster.

Entamoeba histolytica strain 200:NIH cultured axenically for about 15 years produced lesions and abscesses in inbred adult hamsters following intrahepatic, intraperitoneal, and intradermal inoculation. These amebae, even in much higher doses, inoculated into the ceca of adult and young hamsters failed to produce lesions or to become established there. On the other hand, 200:NIH axenic amebae passaged through hamster liver (substrain 200:NIH-L) were able to become established and to produce lesions in the ceca of very young hamsters (1- and 3-week-old), the degree of pathogenicity appearing to be inversely proportional to age of the hamster.     

TEMPORAL ORGANIZATION OF FEEDING IN SYRIAN HAMSTERS WITH A GENETICALLY ALTERED CIRCADIAN PERIOD

The variation in spontaneous meal patterning was studied in three genotypes (tau +/+, tau +/?and tau ?/?) of the Syrian hamster with an altered circadian period. Feeding activity was monitored continuously in 13 individuals from each genotype in constant dim light conditions. All three genotypes had on average six feeding episodes during the circadian cycle (about 20h in homozygous tau mutants and 22h in heterozygotes compared with 24h in wild-type hamsters). Thus, homozygous tau mutant hamsters had significantly more feeding episodes per 24h than wild types, and heterozygotes were intermediate. The average duration of feeding bouts (FBs) was indistinguishable (around 30 minutes) among the three genotypes, whereas the intermeal (IM) intervals were significantly shorter for homozygote tau mutant hamsters (99 minutes), intermediate for heterozygotes (116 minutes), and the longest for wild-type hamsters (148 minutes). Thus, the meal-to-meal duration was on average 25% shorter in homozygous tau mutants (16% in heterozygous) than in wild-type hamsters. The reduction of the circadian period has a pronounced effect on short-term feeding rhythms and meal frequency in hamsters carrying the tau mutation. (Chronobiology International, 18(4), 657–664, 2001)     

Cariogenic virulence of Streptococcus mutans AHT mutants with altered in vitro adherence abilities in hamsters

Plaque-forming ability and cariogenic virulence of Streptococcus mutans AHT mutants having varying in vitro adherence abilities were studied in hamsters reared on a high sucrose diet over a period of 64 days. The parent wild-type strain produced heavy plaque and extensive carious lesions on molars. However, mutants M1, M2, and M13, which have lost the ability to form adhesive deposits on glass when grown in sucrose broth, and mutant M9, which adheres firmly to glass but in an extremely small amount, could not be recovered from the mouths of animals even after repeated inoculations at weekly intervals throughout the experimental period. Mutant M35, which produces a large amount of loosely adhering deposits in vitro, became established and persisted on molars, but its recovery was extremely low. None of the mutants, including M35, significantly produced plaque and caries in hamsters. Possible causes of this inability to form plaque and the avirulence of mutants are discussed.     

Development of orthogonal T7 expression system in Klebsiella pneumoniae

Most expression systems are tailored for model organisms rather than nonmodel organisms. However, heterologous gene expression in model organisms constrains the innate advantages of original strain carrying gene of interest. In this study, T7 expression system was developed in nonmodel bacterium Klebsiella pneumoniae for production of chemicals. First, we engineered a recombinant K. pneumoniae strain harboring two vectors. One vector was used to express T7 RNA polymerase (T7 RNAP) which would drive the expression of egfp in the other vector. This recombinant strain demonstrated 15.73-fold of fluorescence relative to wild-type K. pneumoniae and showed similar level of fluorescence to recombinant Escherichia coli overexpressing egfp. When egfp was replaced by puuC, an endogenous aldehyde dehydrogenase catalyzing 3-hydroxypropionic acid (3-HP) biosynthesis in K. pneumoniae, the recombinant strain coexpressing T7 RNAP and PuuC showed high-level PuuC expression. In shake-flask cultivation, this recombinant strain produced 1.72 g/L 3-HP in 24 hr, which was 3.24 times that of wild-type K. pneumoniae (0.53 g/L). To mitigate plasmid burden, the vector expressing T7 RNAP was eliminated, but the T7 RNAP expression cassette was integrated into K. pneumoniae genome. The resulting strain harboring only PuuC expression vector produced 2.44 g/L 3-HP in 24 hr under shake-flask conditions, which was 1.46 times that of the strain harboring both T7 RNAP and PuuC expression vectors. In bioreactor cultivation, this strain generated 67.59 g/L 3-HP and did not show significantly halted growth. Overall, these results indicate that the engineered T7 expression system functioned efficiently in K. pneumoniae. This study provides a paradigm for the development of T7 expression system in prokaryotes.     

Animal model of Mycoplasma fermentans respiratory infection

Background

Mycoplasma fermentans has been associated with respiratory, genitourinary tract infections and rheumatoid diseases but its role as pathogen is controversial. The purpose of this study was to probe that Mycoplasma fermentans is able to produce respiratory tract infection and migrate to several organs on an experimental infection model in hamsters. One hundred and twenty six hamsters were divided in six groups (A-F) of 21 hamsters each. Animals of groups A, B, C were intratracheally injected with one of the mycoplasma strains: Mycoplasma fermentans P 140 (wild strain), Mycoplasma fermentans PG 18 (type strain) or Mycoplasma pneumoniae Eaton strain. Groups D, E, F were the negative, media, and sham controls. Fragments of trachea, lungs, kidney, heart, brain and spleen were cultured and used for the histopathological study. U frequency test was used to compare recovery of mycoplasmas from organs.

Results

Mycoplasmas were detected by culture and PCR. The three mycoplasma strains induced an interstitial pneumonia; they also migrated to several organs and persisted there for at least 50 days. Mycoplasma fermentans P 140 induced a more severe damage in lungs than Mycoplasma fermentans PG 18. Mycoplasma pneumoniae produced severe damage in lungs and renal damage.

Conclusions

Mycoplasma fermentans induced a respiratory tract infection and persisted in different organs for several weeks in hamsters. This finding may help to explain the ability of Mycoplasma fermentans to induce pneumonia and chronic infectious diseases in humans.     

Isolation and transportation ofTreponema pertenue in golden hamsters

Ten golden hamsters of the CB/Ss LAK strain were flown to Africa, and six of them were inoculated with lesion exudate from yaws patients in an attempt to isolate newer strains ofTreponema pertenue. Three of the six hamsters developed yaws lesions from which treponemes were isolated. Two isolates have been successfully maintained in laboratory animals; these have been designated CDC isolates numbers 1 and 2. Since older stock strains may be altered through repeated animal passage, these fresh isolates will be useful in antigenic and immunological analyses of the pathogenic treponemes. This strain of golden hamster is susceptible to yaws infection, easy to care for, and shipped safely by air; therefore, they are a practical means of transportingT. pertenue.     

Cytotoxic Effect of Hemolytic Culture Supernatant from Enterococcus faecalis on Mouse Polymorphonuclear Neutrophils and Macrophages

We reconfirmed that the LD₅₀S of hemolytic Enterococcus faecalis strains were significantly less than those of nonhemolytic E. faecalis strains in normal mice. Hemolysin produced by E. faecalis lysed human, horse, rabbit, and mouse erythrocytes, but not cow and sheep erythrocytes. Sphingomyelin comprises a part of the lipid composition of the erythrocyte membrane of all mammalian species tested. But phosphatidylcholine exists only in human, horse, rabbit, and mouse. These two lipids inhibited lysis of horse erythrocytes by hemolytic E. faecalis. Phosphatidylcholine is probably the binding component on the membrane of erythrocytes for E. faecalis hemolysin. The hemolytic culture supernatant lysed not only erythrocytes but also mouse polymorphonuclear neutrophils (PMNs) and macrophages.     

Prevention of Clostridium difficile -induced ileocecitis with Bacteriophage

A bacteriophage specific for Clostridium difficile was examined for its ability to prevent ileocecitis in a hamster model. This species- and strain-specific bacteriophage was isolated from a lysogenic strain of C. difficile . Hamsters were maintained in sterile isolation cages to prevent the acquisition of C. difficile from the environment. Bicarbonate neutralization of gastric acidity was necessary for bacteriophage survival in the hamster's gastrointestinal tract. Bacteriophage recovery from the hamster cecum was 2×10⁴plaque forming units/mL of cecal contents 24 h after orogastric challenge with 10⁸plaque forming units/mL of bacteriophage. However, there was no bacteriophage recovery 48 h post challenge, indicating dissipation of bacteriophage from the hamster intestinal tract within this time frame. Twenty-four hours after being challenged with clindamycin, one group of hamsters was challenged with C. difficile followed by a single dose of bacteriophage (10⁸plaque forming units/mL). Two additional groups of hamsters received phage doses immediately after C. difficile challenge and subsequently thereafter every 8 h up to 48 and 72 h, respectively. The gastric acidity was neutralized with bicarbonate buffer preceding every bacteriophage treatment. Control animals that received only clindamycin and C. difficile died within 96 h after challenge while the majority of bacteriophage treated hamsters survived. Two weeks after stopping bacteriophage treatment, the surviving hamsters were re-challenged with clindamycin and C. difficile . All the hamsters died within 96 h indicating susceptibility of the surviving hamsters to C. difficile disease in the absence of bacteriophage treatment.     

Species specificity affects the choice of S9 preparations for use in the hamster embryo cell transformation system

Summary Before their use as a source of carcinogen-activating enzymes in the hamster embryo cell transformation assay, liver, kidney, lung, and small intestine S9 fractions from Syrian golden hamsters and Sprague-Dawley rats were evaluated for toxicity to hamster embryo target cells. Sprague-Dawley rat liver and kidney S9 were highly toxic to the hamster embryo cells (90 to 100%). When retested at lower concentrations these tissue fractions were still quite toxic (up to 75%). In contrast, hamster liver and kidney S9 were considerably less toxic (14 to 25%). The S9 preparations were also evaluated for their ability to metabolizeN-2-acetylaminofluorene to 2-aminofluorene andN-hydroxy-acetylamino-fluorene, products that transform hamster embryo cells. Large amounts ofN-hydroxy-acetylaminofluorene were formed in the presence of preparations from hamster liver and small intestine, whereas kidney and lung S9 fractions were considerably less active. No detectable levels ofN-hydroxy-acetylaminofluorene were formed after incubation ofN-2-acetylaminofluorene with any of the rat S9 preparations. High levels of deacetylase activity were found in hamster liver and small intestine S9 fractions, at least eightfold higher than those obtained from equivalent rat preparations. Hamster kidney and lung S9 fractions showed low levels of deacetylase activity. There was no detectable activity in equivalent preparations from rats. When tested withN-2-acetylaminofluorene in the hamster embryo cell clonal transformation system, transformed colonies were obtained with hamster liver S9, with and without an external NADPH-generating system. This work was supported by Contract N01-CO-75380 with the National Cancer Institute, NIH, Bethesda, MD 20205.     

Diversity and exploration of bioactive marine actinomycetes in the Bay of Bengal of the Puducherry coast of India

The present study was designed to investigate the Puducherry coast of the Bay of Bengal, India for the diversity of bioactive actinomycetes. A total of 50 actinomycete strains were isolated from the marine sediments and most of the strains were belongs to Streptomyces. These strains were identified by means of morphological physiological, biochemical and cultural characteristics. The isolates were subjected to shake flask fermentation and the secondary metabolites were extracted with ethyl acetate and screened for cytotoxicity, hemolytic activity and antimicrobial activity against selected bacterial and fungal pathogens. The cytotoxic activity was evaluated using HeLa cell lines by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, a tetrazole (MTT) assay, hemolytic activity on mouse erythrocytes and the antifungal activity was evaluated by MTT cytotoxic assay against Aspergillus niger, Aspergillus fumigatus and Candida albicans. The antibacterial activity was studied against Staphylococcus aureus, Bacillus subtilis, Escherichia coli and Klebsiella pneumoniae. The cytotoxicity and antimicrobial activity of secondary metabolite was found to be concentration dependent and nearly 24% of isolates showed significant antimicrobial, hemolytic and cytotoxic activity. The results of our study indicate the diversity and bioactive potential of marine actinomycetes isolated in the Puducherry coast.     

Expression of hemagglutination is dependent on environmental factors and bacterial concentration ofEscherichia coli andKlebsiella pneumoniae

Hemagglutination of human type A and guinea pig erythrocytes showed that both mannose-sensitive and mannose-resistant adhesive mechanisms (pili) were detectable in broth cultures ofEscherichia coli while only a mannose-sensitive mechanism (type 1 pili) was noted withKlebsiella pneumoniae. The degree of hemagglutination was shown to be related to differences in erythrocytes, bacterial concentration, and growth media constituents.Escherichia coli grown in human urine produced both types of hemagglutinins, whileK. pneumoniae cultured in urine continued to express only a mannose-sensitive hemagglutinin.     

Acquired immunity to Mycoplasma pneumoniae. Pneumonia in hamsters

An inactivated Mycoplasma pneumoniae vaccine was prepared from a culture in a liquid medium supplemented with water extract of egg yolk. Vaccinated Syrian hamsters were exposed to virulent M. pneumoniae aerosol and were examined for the retention of mycoplasmas and for histopathological changes in the respiratory tracts. When a vaccine prepared with strain FH was administered intramuscularly or by inhalation in aerosol, no significant resistance was shown with respect to mycoplasma proliferation. An increased resistance, however, was observed when an aluminium phosphate-adsorbed vaccine, and when a plain vaccine (although to a lesser degree) prepared with hamster 24-passaged strain FH, was administered intramuscularly. Histopathologically, lung lesions were markedly suppressed in groups showing high resistance. A correlation between the serum antibody titer and the resistance to infection was observed. Hamsters which received a hyperimmune rabbit antiserum intracordally showed a high resistance to M. pneumoniae infection. The suppression of histopathological changes also coincided with high complement-fixing antibody titers of either actively or passively immunized hamster serum. The results suggest that humoral immunity plays an important role in resistance to M. pneumoniae pneumonia in hamsters.