Differences in aerosol survival between pathogenic and non-pathogenic strains of Legionella pneumophila serogroup 1

A strain of Legionella pneumophila serogroup 1 known to be virulent for guinea-pigs was found to be least stable at a relative humidity (r.h.) of 60% when stored as a small particle aerosol. Three L. pneumophila serogroup 1 strains of different virulence for guinea-pigs were then tested at a r.h. of 60% at 20°C. The most virulent strain was found to have the best survival and the avirulent strain was least stable. The strain of intermediate virulence did not survive as well as the virulent strain but was more stable than the avirulent strain. Strains of L. pneumophila serogroup epidemiologically associated with legionnaires' disease had better survival in small particle aerosols than strains which were not associated with disease. Subtyping with monoclonal antibodies also showed that the type more commonly associated with disease survived longer in aerosols than the other subtypes.     

Virulence conversion of Legionella pneumophila by conjugal transfer of chromosomal DNA

In this study, we examined whether virulence conversion occurs in Legionella pneumophila by conjugal transfer of chromosomal DNA. A virulent strain, K6, which has the genes for Kmr and LacZ+ transposed in the chromosome of strain Philadelphia-1, which belongs to serogroup 1, was used as one parent, and an avirulent strain, Chicago-2S, which is a spontaneous streptomycin-resistant derivative of strain Chicago-2 belonging to serogroup 6, was used as the other parent. Experiments in which K6 (approximately 2.6 x 10(9) CFU) and Chicago-2S (approximately 8.9 x 10(9) CFU) were mated typically yielded 10(3) Kmr Smr LacZ+ transconjugants. Thirty-two (about 2.8%) of 1,152 transconjugants belonging to serogroup 6 acquired the ability to grow intracellularly in Acanthamoeba castellanii and guinea pig macrophages. When guinea pigs were infected with sublethal doses of Legionella aerosols generated from one of these transconjugants (HM1011), they developed a severe pneumonia similar to that caused by donor strain K6. These results show that avirulent strain Chicago-2S changed into virulent strain HM1011 through conjugation with virulent strain K6. Furthermore, we showed that Legionella chromosomal virulence genes (icm-dot locus) were horizontally transferred by the conjugation system. The chromosomal conjugation system may play a role(s) in the evolution of L. pneumophila.     

Temperature-dependent expression of flagella in Legionella

Legionella pneumophila, the causative agent of Legionnaires' disease, was analysed by electron microscopy for production of surface structures. Crystalline surface (S-) layers and fimbriae were not detected, but monotrichous flagellation was seen. Polyclonal antibodies specific for the 47 kDa flagellin subunit of L. pneumophila Philadelphia I were used in Western blots to confirm the presence of flagella subunits in various L. pneumophila strains tested, but the antiserum also reacted with flagellin subunits of L. micdadei, L. hackelia [serogroup (SG) 1 and SG2] and L. longbeachae (SG2). Flagellation of Legionellae was shown to be temperature regulated. When the growth temperature of virulent and avirulent of strain L. pneumophila Philadelphia I was shifted from 30 degrees C to either 37 or 41 degrees C, a decrease in the percentage of flagellated bacteria within the population was observed.     

Gentamicin-Containing Peptone-Yeast Extract Medium for Cocultivation of Hartmannella vermiformis ATCC 50256 and Virulent Strains of Legionella pneumophila

We evaluated the use of peptone-yeast extract (PY) medium, different strains of Hartmannella vermiformis, and gentamicin in a coculture system to improve the discrimination of virulent and avirulent strains of Legionella pneumophila. H. vermiformis ATCC 50256 was unique among four strains of H. vermiformis, in that it multiplied equally well in Medium 1034 and PY medium (Medium 1034 without fetal calf serum, folic acid, hemin, and yeast nucleic acid and with a 50% reduction of peptone). However, both a virulent strain of L. pneumophila and its avirulent derivative strain multiplied in cocultures when PY medium was used. The multiplication of this avirulent strain was greatly reduced by incorporating gentamicin (1 (mu)g/ml) into the cocultivation system. Five virulent-avirulent sets of L. pneumophila strains were then tested for multiplication in cocultures with H. vermiformis ATCC 50256 and the gentamicin-containing PY medium. Only the virulent strains multiplied. The modified cocultivation system can discriminate between virulent and avirulent strains of L. pneumophila.     

Characteristics of the interaction of legionellae and Tetrahymena pyriformis

L. pneumophila avirulent strains have been shown to lose their capacity for multiplication in T. pyriformis, while the concentration of the virulent strain increases 1000-fold. The loss of the hemolytic activity of L. pneumophila virulent strain leads to the loss of its capacity for multiplication in infusorians.     

Interaction of virulent and non-virulent Rhodococcus equi human isolates with phagocytes, fibroblast- and epithelial-derived cells

Abstract Rhodococcus equi is a facultative, intracellular, Gram-positive coccobacillus, increasingly reported in pneumonia of AIDS-infected patients. We investigated killing resistance properties of human R. equi virulent and avirulent human strains. Avirulent β-lactam-susceptible strains had lower intracellular colony forming units after 45 min incubation in murine macrophages J774 and human monocyte-macrophage TPH-1 than those of virulent strains. Only virulent β-lactam-resistant strains persisted within macrophages for at least 18 min only. A β-lactam-resistant mutant was obtained from a β-lactam-susceptible strain after selection in a penicillin G-containing culture medium. This mutant strain, like the natural virulent strains, persisted within macrophages, harboured cell-associated appendages, produced phage-like particles and induced, after its intravenous inoculation, a chronic infection in BALB/c nude mice. Supernatant culture of virulent strains transferred partial macrophage-killing resistance properties to avirulent strains. The same supernatant was toxic for L-929, HeLa and Vero cell cultures. These supernatant effects were heat-inactivated, trypsin-inactivated and did not seem to be linked to phage-like particle presence. These data argue that virulence, β-lactam-resistance, and macrophage-killing resistance are associated in human R. equi isolates. Moreover, only virulent strains produced uncharacterized toxic factors.     

Differences in the respiratory burst of human polymorphonuclear leukocytes induced by virulent and avirulent Legionella pneumophila Serogroup 1

Two strains of Legionella pneumophila of different virulence were examined for their influence on the metabolic oxidative activity of human polymorphonuclear leukocytes. The leukocytes exhibited decreased rates of oxygen consumption and diminished chemiluminescence activity following phagocytosis of a virulent strain of L. pneumophila serogroup 1. In contrast, phagocytosis of its multipassaged derivative rendered avirulent, was accompanied by increased rates of both oxygen consumption and chemiluminescence activity. Although no differences were observed in oxygen uptake induced by the virulent legionellae compared to leukocytes at rest, statistically significant differences were observed in the chemiluminescence responses. These observations were not unexpected, since the luminol-enhanced chemiluminescence assay, is more sensitive than the oxygen uptake assay. In spite of decreased metabolic activity of PMN in the presence of virulent legionellae, electron microscope studies showed higher numbers of intracellular L. pneumophila than the avirulent subtype. Thus, virulent and avirulent L. pneumophila can be differentiated on the basis of oxygen consumption and chemiluminescence assays.     

Conjugational transfer of genes determining plant virulence in Erwinia amylovora.

A stable virulent donor strain (EA 178R1-99) of Erwinia amylovora can transfer, by conjugation during a 3-h mating period, the gene or genes which determine(s) plant virulence to avirulent recipient strains (EA178-M64S1 and EA178-M173S1) of Escherichia amylovora. The virulence of over 200 recombinant clones was tested; they all were as virulent on immature Bartlett pear fruits (and, in the smaller series of strains tested, also, on Pyracantha twigs) as was the parent donor strain. Although the avirulent recipeint strains are amino acid auxotrophs, addition of the required amino acids to the inocula in plant virulence trials does not of itself restore virulence. Two small series of prototrophic revertant clones were selected from the auxotrophic avirulent recipient strains; only nine of the 21 prototrophic revertant clones regained virulence, whereas the other 12 prototrophic revertant clones remained avirulent, again suggesting a lack of parallelism between nutritional status and virulence in this system. Preliminary interrupted mating trials, carried out at 15-min intervals over 3 h, show that ser is transferred during the first 15 min, that pro starts entering at about 75 min (and with a higher frequency later), and that lac (originating from an integrated Escherichia coli F'lac) enters toward the end of the 3-h mating period and at a reduced frequency compared to the other markers. The gene or genes which determine(s) plant virulence in this Escherichia amylovora donor strain appear(s) to be transferred readily and seemingly completely to recipient strains during the first 15 min of a 3-h mating period. Exposure of the virulent donor strain to acridine orange or ethidium bromide does not result in loss of virulence, suggesting (but, of course, not proving conclusively) that the determinant(s) of virulence in Escherichia amylovora might be chromosomal rather than extrachromosomal.     

水稻黄单胞菌水稻致病变种的超氧化物歧化酶活性及诱导

以水稻黄单胞菌水稻致病变种(Xanthomonas oryzae pv.oryzae)的毒性菌株PXO99Ａ和无毒菌株PXO99A(pBUavrXa10F1),检测液体培养中菌体超氧化物歧化酶(SOD)活性变化,以说明SOD与菌株致病性的关系。结果表明,菌体SOD活性高峰出现于延迟期末,之后下降;毒性菌株SOD活性高于无毒菌株。两个菌株的SOD活性的胞内定位均以胞质为主,占总活性的70%以上;酶体周质中SOD活性占总活性的20%～30%。以50～800μmol/L外源O-2处理细菌培养物1?h,可诱导菌体中SOD活性的增加。其 中以200?μmol/L O-2处理SOD活性最高;12?h菌龄培养物的诱导效果优于24?h培养物;对毒性菌株SOD的诱导作用更为明显。外源O-2处理后细菌存活率明显降低,2 4?h培养物的存活率下降大于12?h培养物;毒性菌株存活率下降大于无毒菌株。     

Analysis of Legionella pneumophila serogroup 6 strains isolated from a hospital warm water supply over a three-year period by using genomic long-range mapping techniques and monoclonal antibodies.

Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown from warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NotI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 300-kb NotI fragment when a legiolysin (lly)-specific DNA probe was used. The NotI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six NotI cleavage types obtained by pulsed-field electrophoresis.     

Analysis of Legionella pneumophila serogroup 6 strains isolated from a hospital warm water supply over a three-year period by using genomic long-range mapping techniques and monoclonal antibodies.

Over a period of 3 years, Legionella pneumophila serogroup 6 strains were isolated from warm water outlets and dental units in the Dental Faculty and from the Surgery and Internal Medicine Clinics at the University of Dresden, Dresden, Germany. In the bacteriological unit of the above-mentioned facility, L. pneumophila serogroups 3 and 12 were grown from warm water specimens. The medical facilities are located in separate buildings connected with a ring pipe warm water system. All L. pneumophila serogroup 6 strains isolated from the warm water supply reacted with a serogroup-specific monoclonal antibody, but not with two other monoclonal antibodies which are subgroup specific, reacting with other serogroup 6 strains. The NotI genomic profiles obtained by pulsed-field gel electrophoresis of 25 serogroup 6 strains isolated from the Dental Faculty over a 3-year period, 1 isolate from the Internal Medicine Clinic, and 4 strains from the Surgery Clinic were identical. Furthermore, all these strains hybridized with a 300-kb NotI fragment when a legiolysin (lly)-specific DNA probe was used. The NotI pattern, however, differed from those of six serogroup 6 strains of other origins, one serogroup 12 strain from the bacteriological unit, and another six unrelated strains of serogroups other than serogroup 6. L. pneumophila serogroup 6 strains which can be divided into only two subgroups by the use of monoclonal antibodies are differentiated in at least six NotI cleavage types obtained by pulsed-field electrophoresis.     

Structural and nonstructural protein genome regions of eastern equine encephalitis virus are determinants of interferon sensitivity and murine virulence

Eastern equine encephalitis virus (EEEV) causes sporadic epidemics of human and equine disease in North America, but South American strains have seldom been associated with human neurologic disease or mortality, despite serological evidence of infection. In mice, most North American and South American strains of EEEV produce neurologic disease that resembles that associated with human and equine infections. We identified a South American strain that is unable to replicate efficiently in the brain or cause fatal disease in mice yet produces 10-fold higher viremia than virulent EEEV strains. The avirulent South American strain was also sensitive to human interferon (IFN)-alpha, -beta, and -gamma, like most South American strains, in contrast to North American strains that were highly resistant. To identify genes associated with IFN sensitivity and virulence, infectious cDNA clones of a virulent North American strain and the avirulent South American strain were constructed. Two reciprocal chimeric viruses containing swapped structural and nonstructural protein gene regions of the North American and South American strains were also constructed and found to replicate efficiently in vitro. Both chimeras produced fatal disease in mice, similar to that caused by the virulent North American strain. Both chimeric viruses also exhibited intermediate sensitivity to human IFN-alpha, -beta, and -gamma compared to that of the North American and South American strains. Virulence 50% lethal dose assays and serial sacrifice experiments further demonstrated that both structural and nonstructural proteins are important contributors to neurovirulence and viral tissue tropism. Together, the results of this study emphasize the complex and important influences of structural and nonstructural protein gene regions on EEEV virulence.     

Multiplication of different Legionella species in Mono Mac 6 cells and in Acanthamoeba castellanii.

Survival and distribution of legionellae in the environment are assumed to be associated with their multiplication in amoebae, whereas the ability to multiply in macrophages is usually regarded to correspond to pathogenicity. Since most investigations focused on Legionella pneumophila serogroup 1, we examined the intracellular multiplication of different Legionella species in Mono Mac 6 cells, which express phenotypic and functional features of mature monocytes, and in Acanthamoeba castellanii, an environmental host of Legionella spp. According to the bacterial doubling time in Mono Mac 6 cells and in A. castellanii, seven clusters of legionellae could be defined which could be split further with regard to finer differences. L. longbeachae serogroup 1, L. jordanis, and L. anisa were not able to multiply in either A. castellanii or Mono Mac 6 cells and are members of the first cluster. L. dumoffi did not multiply in Mono Mac 6 cells but showed a delayed multiplication in A. castellanii 72 h after infection and is the only member of the second cluster. L. steigerwaltii, L. gormanii, L. pneumophila serogroup 6 ATCC 33215, L. bozemanii, and L. micdadei showed a stable bacterial count in Mono Mac 6 cells after infection but a decreasing count in amoebae. They can be regarded as members of the third cluster. As the only member of the fourth cluster, L. oakridgensis was able to multiply slight in Mono Mac 6 cells but was killed within amoebae. A strain of L. pneumophila serogroup 1 Philadelphia obtained after 30 passages on SMH agar and a strain of L. pneumophila serogroup 1 Philadelphia obtained after intraperitoneal growth in guinea pigs are members of the fifth cluster, which showed multiplication in Mono Mac 6 cells but a decrease of bacterial counts in A. castellanii. The sixth cluster is characterized by intracellular multiplication in both host cell systems and consists of several strains of L. pneumophila serogroup 1 Philadelphia, a strain of L. pneumophila serogroup 2, and a fresh clinical isolate of L. pneumophila serogroup 6. Members of the seventh cluster are a strain of agar-adapted L. pneumophila serogroup 1 Bellingham and a strain of L. pneumophila serogroup 1 Bellingham which was passaged fewer than three times on BCYE alpha agar after inoculation and intraperitoneal growth in guinea pigs. In comparison to members of the sixth cluster, both strains showed a slightly enhanced multiplication in Mono Mac 6 cells but a reduced multiplication in amoebae. From our investigations, we could demonstrate a correlation between prevalence of a given Legionella species and their intracellular multiplication in Mono Mac 6 cells. Multiplication of members of the genus Legionella in A. castellanii seems to be dependent on mechanisms different from those in monocytes.     

Isolation and characterization of a conjugative plasmid from Legionella pneumophila.

The conjugative properties of an indigenous 85 MDa plasmid (designated pCH1) from Legionella pneumophila were studied. To determine if pCH1 was transmissible by conjugation, mating experiments were performed between legionellae that harboured pCH1 and several plasmid-less recipients. Plasmid transfer was monitored by colony hybridization, using a cloned 21.0 kb SalI restriction fragment from pCH1 as a probe. The results from these experiments showed that pCH1 could be conjugatively transferred into several strains of L. pneumophila serogroup 1 but not into strain Bloomington-2 (serogroup 3) or Escherichia coli. Southern hybridization experiments in which pCH1 DNA was used as a probe showed that pCH1 does not share homology with other indigenous L. pneumophila plasmids. There was no detectable DNA homology between pCH1 and L. pneumophila chromosomal DNA. Additional mating experiments revealed that pCH1 was unable to mobilize the L. pneumophila chromosome. The conjugative transfer of pCH1 into plasmid-less avirulent or virulent serogroup 1 strains did not alter the intracellular growth characteristics of these strains in U937 cells, a human-monocyte-like cell line, or in the amoeba Hartmannella vermiformis. These results suggest that pCH1 does not contribute to the ability of L. pneumophila to enter or grow within eukaryotic cells.     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

鸭疫里默氏杆菌外膜蛋白生物学特性研究

血清2型鸭疫里默氏杆菌强毒菌株体外传200代获得了无毒力无免疫原性菌株,采用超声波裂解和超速离心法提取二株菌的外膜蛋白, 以比较分析鸭疫里默氏杆菌外膜蛋白的生物学特性。电镜观察细菌超微结构显示传代菌株外膜膜密度降低, 外膜泡的数量明显减少, 细胞质不均匀、内有空泡产生;免疫印迹结果表明二株菌的外膜蛋白免疫原性多肽存在明显区别;原代菌株的外膜蛋白仅与2型RA抗体出现特异性凝集, 而传代菌株的外膜蛋白与 1、2、10与11型RA抗体均出现凝集;二株菌的外膜蛋白均可诱导雏鸭产生抗体, 但原代菌株外膜蛋白诱导雏鸭产生抗体滴度显著高于200代次菌株;原代菌株外膜蛋白免疫鸭对同源RA菌株的攻击可产生100%的免疫保护, 而传代菌株外膜蛋白免疫鸭对同源RA菌株的攻击不产生免疫保护。序列分析显示两者的外膜蛋白A同源性达到99.9%。结果表明强毒菌株的外膜蛋白为良好的亚单位疫苗候选, 体外连续传代对RA外膜蛋白生物学特性影响显著。     

A note on the temperature tolerance of Legionella

A strain of Legionella pneumophila serogroup 1 isolated from the environment had a decimal reduction time in water at 50 degrees C (D50) of 111 min, a D54 of 27 min and a D58 of 6 min. There was little loss of viability at 46 degrees C. Other environmental organisms, a Pseudomonas sp., a Micrococcus sp. and a coliform survived less well at these temperatures. A species of Sarcina had a survival time greater than the L. pneumophila at all the temperatures tested. Other strains of legionellas were tested at 50 degrees C and decimal reduction times calculated. These ranged from 80 min for another strain of L. pneumophia serogroup 1 to 216 min for L. bozemannii . Legionella micdadei did not survive well at 50 degrees C.     

A note on the survival of Legionella pneumophila in stagnant tap water

Tap water, from an experimental hot water system, containing a known virulent strain of Legionella pneumophila was stored in screw-capped bottles for 14 months. Viable counts showed survival of L. pneumophila and at least three other bacterial species. This reinforces the view that L. pneumophila can survive in stagnant water foatively long periods of time.     

A comparative study of virulent and avirulent strains of Chromobacterium violaceum

A clinical isolate and a soil isolate of Chromobacterium violaceum were compared to determine differences in virulence-related characteristics. Purified lipopolysaccharide (endotoxin) from the virulent, clinical strain was more reactive than that from the avirulent soil strain as determined by the Limulus amebocyte lysate assay. There were no differences in hemolysin or cyanide production between the two strains. The virulent strain was more resistant to phagocytosis and intracellular killing by human polymorphonucleocytes. The clinical strain showed a superoxide dismutase activity 30% higher and a catalase activity fivefold higher than the activities of the soil-isolated strain. The clinical strain also was capable of producing approximately twice as much hydrogen peroxide during growth as compared with the soil isolate. This study suggests that virulence of C. violaceum may be, at least in part, associated with endotoxin, and some protection of the virulent, clinical strain from phagocytic attack is afforded by elevated levels of superoxide dismutase and catalase.