Experimental validation of low virulence in field strains of Listeria monocytogenes

Several reports have described Listeria monocytogenes strains which were nonpathogenic or weakly pathogenic, but little is known about these low-virulence strains. We found that 9 field L. monocytogenes strains were hypovirulent and 17 were avirulent, based on the number of mice contaminated and the colonization of their spleens after subcutaneous inoculation. All these strains possessed the known virulence genes. We have now assessed the low virulence of these strains in other assays before determining how they differ from virulent strains. We have shown that the low-virulence strains exhibited a phenotypic stability and were not a mixture of virulent and avirulent bacteria. They did not recover virulence after many passages in mice and colonized the spleens of mice more poorly than virulent strains after i.v. inoculation. Their lethal capacities, determined by 50% lethal dose (LD(50)), were lower than those of virulent strains. Like Listeria innocua, 14 of 17 avirulent strains had no LD(50) and were eliminated by the lymph nodes after subcutaneous inoculation. The virulent, hypovirulent, and avirulent strains were always significantly different, whatever the tests of virulence used, confirming the importance of these low-virulence field strains in identifying the proteins involved in virulence.     

Protection and immune responses induced by attenuated Salmonella typhimurium UK-1 strains

We previously reported that Salmonella typhimurium SR-11 mutants with deletion mutations in the genes encoding adenylate cyclase (cya) and the cAMP receptor protein (crp) are avirulent and protective in mice. Salmonella typhimurium UK-1 is highly virulent for chicks (oral LD50 of 3x10(3) CFU) and mice (oral LD50 of 8.5x10(3) CFU) and is capable of lethal infections in pigs, calves and horses. We postulated that attenuated derivatives of this lethal strain would probably induce a higher level of protective immunity than achieved with attenuated derivatives of less virulent S. typhimurium strains such as SR11. To test this hypothesis, we have constructed S. typhimurium UK-1 Deltacya-12Deltacrp-11 mutant strain chi3985 and its virulence plasmid cured derivative chi4095 to investigate their avirulence and immunogenicity in mice. We found that the mutants are avirulent and able to induce protective immune responses in BALB/c mice. These mutant strains retained wild-type ability to colonize the gut associated lymphoid tissue but reach and persist in spleen and liver at a significantly lower level than the wild-type parent strain. Mice survived oral infection with >1x10(9) CFU of chi3985 (the equivalent to 10(5) 50% lethal doses of wild-type S. typhimurium UK-1) and were fully protected against challenge with 10(5)times the LD50 of the wild-type parent. Immunized mice developed a high level of serum IgG titre to Salmonella LPS and delayed-type hypersensitivity (DTH) response to S. typhimurium outer membrane proteins. Compared to the virulence plasmid-containing strain chi3985, the virulence plasmid cured DeltacyaDeltacrp mutant strain chi4095 was more attenuated and less protective, as some mice immunized with chi4095 died when challenged with the wild-type UK-1 strain. This work demonstrates that S. typhimurium UK-1 Deltacrp Deltacya mutant strain may be a potential live vaccine to induce protective immunity against Salmonella infection or to deliver foreign antigens to the immune system.     

Pseudomonas aeruginosa isolates from patients with cystic fibrosis: a class of serum-sensitive, nontypable strains deficient in lipopolysaccharide O side chains

Twenty-six Pseudomonas aeruginosa strains from patients with cystic fibrosis were typed by the Fisher immunotyping scheme. Only 6 strains were agglutinated by a single typing serum, whereas 15 strains were agglutinated with more than one serum and 5 were not agglutinated by any serum. Neither the polyagglutinable nor the nonagglutinable strains were typable by hemagglutination inhibition or immunodiffusion, suggesting that these polyagglutinable strains did not express multiple serotype antigens, but were instead being agglutinated by antibody to nonserotype determinants. Four typable isolates were resistant to pooled normal human serum, whereas the 12 polyagglutinable and nonagglutinable isolates studied were very sensitive to normal human serum. The outer membranes of 16 strains were isolated and characterized. The data suggested, in general, strong conservation of outer membrane protein patterns. Lipopolysaccharides (LPS) were purified by a new technique which allowed isolation of both rough and smooth LPS in high yields. Three of four typable, serum-resistant strains examined had amounts of smooth, O-antigen-containing LPS equivalent to our laboratory wild type, P. aeruginosa PAO1 strain H103. In contrast, 10 of 12 polyagglutinable or nonagglutinable, serum-sensitive strains had very little or no smooth, O-antigen-containing LPS, and the other two contained less smooth LPS than our wild-type strain H103. In agreement with this data, five independent, rough, LPS O-antigen-deficient mutants of strain H103 were nontypable and serum sensitive. We suggest that the LPS defects described here represent a significant new property of many P. aeruginosa strains associated with cystic fibrosis.     

Generation of Yersinia pestis attenuated strains by signature-tagged mutagenesis in search of novel vaccine candidates

In a search for novel attenuated vaccine candidates for use against Yersinia pestis, the causative agent of plague, a signature-tagged mutagenesis strategy was used and optimized for a subcutaneously infected mouse model. A library of tagged mutants of the virulent Y. pestis Kimberley53 strain was generated. Screening of 300 mutants through two consecutive cycles resulted in selection of 16 mutant strains that were undetectable in spleens 48 h postinfection. Each of these mutants was evaluated in vivo by assays for competition against the wild-type strain and for virulence following inoculation of 100 CFU (equivalent to 100 50% lethal doses [LD50] of the wild type). A wide spectrum of attenuation was obtained, ranging from avirulent mutants exhibiting competition indices of 10(-5) to 10(-7) to virulent mutants exhibiting a delay in the mean time to death or mutants indistinguishable from the wild type in the two assays. Characterization of the phenotypes and genotypes of the selected mutants led to identification of virulence-associated genes coding for factors involved in global bacterial physiology (e.g., purH, purK, dnaE, and greA) or for hypothetical polypeptides, as well as for the virulence regulator gene lcrF. One of the avirulent mutant strains (LD50, >10(7) CFU) was found to be disrupted in the pcm locus, which is presumably involved in the bacterial response to environmental stress. This Kimberley53pcm mutant was superior to the EV76 live vaccine strain because it induced 10- to 100-fold-higher antibody titers to the protective V and F1 antigens and because it conferred efficacious protective immunity.     

Studies on virulence of Shigella flexneri.

The antigenic structure and some biological properties were compared in virulent S. flexneri strains of differences in: 1) antigenic composition; 2) biochemical activity; 3) sensitivity to a set of Shigella phages and 4) susceptibility to phagocytosis. The only difference found concerned the LD50 for mice; it was 10 to 100 times larger for avirulent than for virulent strains of S. flexneri. The toxic products of the strains were also compared. The lipopolisaccharide and free endotoxin purified from virulent and avirulent variants behaved similarly when tested for: LD50, pyrogenicity and the local Shwartzman reaction. A striking difference was demonstrated in the ultrastructure of lipopolisaccharide and free endotoxin isolated from virulent and avirulent variant of S. flexneri 3a.     

Virulence of different Pseudomonas species in a burned mouse model: tissue colonization by Pseudomonas cepacia.

The virulence of Pseudomonas aeruginosa and other pseudomonads was examined in a burned mouse model. P. aeruginosa M-2 was highly virulent causing 100% mortality by 38 h with an injection of 10(2) CFU by either a subcutaneous or intraperitoneal route. Subcutaneous injection of 10(2) CFU revealed rapid multiplication of the bacteria at the burn wound with 10(8) CFU/g detectable in the burned skin by 28 h postinjection, 10(5) CFU/g of liver, and 10(3) CFU/ml of blood. Non-P. aeruginosa clinical isolates were markedly less virulent; an injection of greater than or equal to 10(7) CFU caused less than or equal to 60% lethality. P. cepacia SMH colonized the burned skin of thermally injured mice, persisting at levels of 10(7) to 10(8) CFU/g of burned skin after an initial injection of 10(5) CFU. P. cepacia persisted in the burn wound for at least 3 weeks. No organ invasion was detectable throughout this period. Studies with an additional clinical isolate of P. cepacia yielded similar results. An injection of a 10(2) CFU dose revealed that the level of persistence is dose dependent. Results suggest that the tenacious persistence of P. cepacia in the burn wound may provide a model for the study of persistent colonization and infection in a compromised host.     

Resistance to Both Complement Activation and Phagocytosis in Type 3 Pneumococci Is Mediated by the Binding of Complement Regulatory Protein Factor H

To study the role of surface-associated proteins in the virulence of Streptococcus pneumoniae, we used two serotype 3 strains, ATCC 6303 and WU2, and two PspA-negative mutants of WU2, an encapsulated one, JY1123 (Caps(+)/PspA(-)), and an unencapsulated one, DW3.8 (Caps(-)/PspA(-)). ATCC 6303 and WU2 were highly virulent in mice, while the virulence of JY1123 was slightly decreased (50% lethal doses [LD(50)s], 24, 6, and 147 CFU/mouse, respectively); DW3.8 was avirulent (LD(50), 2 x 10(8) CFU). In vitro, ATCC 6303, WU2, and JY1123 (Caps(+)/PspA(-)) strongly resisted complement activation and complement-dependent opsonophagocytosis, whereas DW3.8 (Caps(-)/PspA(-)) was easily phagocytized in fresh serum. Trypsin treatment of ATCC 6303, WU2, and JY1123 (Caps(+)/PspA(-)) resulted in enhanced complement activation and complement-dependent opsonophagocytosis. Trypsin had no deleterious effect on the polysaccharide capsule. In addition, trypsin pretreatment of ATCC 6303 strongly reduced virulence upon intraperitoneal challenge in mice. This indicated that surface proteins play a role in the resistance to complement activation and opsonophagocytosis and contribute to the virulence of type 3 pneumococci. In subsequent experiments, we could show that the modulation of complement activation was associated with surface components that bind complement regulator factor H; binding is trypsin sensitive and independent of prior complement activation. Immunoblotting of cell wall proteins of the virulent strain ATCC 6303 with anti-human factor H antibody revealed three factor H-binding proteins of 88, 150, and 196 kDa. Immunogold electron microscopy showed a close association of factor H-binding components with the outer surface of the cell wall. The role of these factor H-binding surface proteins in the virulence of pneumococci is interesting and warrants further investigation.     

Serum bactericidal effect on Pseudomonas aeruginosa isolates from cystic fibrosis patients.

The bactericidal activity against Pseudomonas aeruginosa strains isolated from cystic fibrosis patients was determined in a 10% concentration of normal serum or autologous cystic fibrosis serum. Of the 167 strains tested, 77 (46%) were sensitive (greater than 95% killed) in normal serum. Mucoid strains were more frequently sensitive than nonmucoid strains. Twenty-three sensitive strains tested in ethyleneglycoltetraacetic acid-chelated serum were resistant (less than 10% killed), suggesting only classical pathway activation. Absorption of cystic fibrosis serum with the autologous P. aeruginosa strain resulted in decreased killing by that serum. All sera, including the chelated and absorbed sera, had comparable total hemolytic complement levels. Patients in poor clinical condition (5 out of 12), in contrast to patients in good or moderate condition(1 out of 30), were more likely to have P. aeruginosa strains that were serum resistant in autologous serum but sensitive in normal serum. Sera from these five patients in poor clinical condition were capable of killing heterologous P. aeruginosa strains. These results suggest the presence of a protective or "blocking" activity in serum from some patients in poor clinical conditions. This association of a blocking activity with clinical condition may signal a transition point in the progression of cystic fibrosis lung disease and thus may be another contributory factor in the failure of the cystic fibrosis host to control infection.     

Correlation of the virulence of Klebsiella pneumoniae K1 and K2 with the presence of a plasmid encoding aerobactin.

Nine isolates of Klebsiella pneumoniae belonging to capsular serotypes K1 and K2 were assayed for virulence in mice. Virulent isolates (50% lethal dose of less than 10(3) microorganisms) and avirulent isolates (50% lethal dose of over 10(6) microorganisms) were selected. Supplementation of a defined minimal medium with transferrin markedly reduced the growth of avirulent strains but had no significant effect on the growth of virulent strains. All isolates produced enterochelin, but only production of aerobactin could be correlated with virulence. The genes encoding aerobactin and its receptor protein were located on a 180-kilobase plasmid. They were cloned into the mobilizable vector pSUP202. Homology was demonstrated with the aerobactin operon of the Escherichia coli plasmid pColV-K30. Transfer of the recombinant plasmid pKP4 into an avirulent recipient enhanced virulence by 100-fold. These experiments demonstrated that aerobactin is an essential factor of pathogenicity in K. pneumoniae.     

Assessment of protease (elastase) as a Pseudomonas aeruginosa virulence factor in experimental mouse burn infection.

The data presented indicate that in experimental Pseudomonas aeruginosa infection of mice, protease enhances the virulence of the organism. Anesthetized CBA/Lü mice were subjected to a 15-s flame burn and infected with a wild-type protease-producing strain and two of its protease-deficient mutants. The average bacterial cell mean lethal dose (LD50) of 3.8 +/- 0.3 standard deviation (log10) for mice infected with the protease-producing P. aeruginosa was at least 1 log lower than the LD50 of the protease-deficient mutants (0.02 greater than P greater than 0.01). The addition of purified protease to the infecting inoculum of protease-deficient strains reduced the LD50. Although the generation time in vitro was the same for all three bacterial strains used, there were consistently fewer viable bacteria in the blood of mice infected with protease-deficient strains than in those infected with the protease-producing strain. When a protease-deficient strain was mixed with the protease-producing wild-type strain, the number of protease-producing pseudomonas found in the blood remained constant, whereas the number of protease-deficient organisms increased, suggesting that protease contributed to the invasiveness of the organisms. The survival of mice infected with protease-producing pseudomonas was enhanced by antiprotease serum. Antiprotease serum had no effect in mice infected with protease-deficient mutants.     

Virulence and vaccine potential of phoP mutants of Salmonella typhimurium

We have constructed Salmonella typhimurium phoP mutants and found them to be avirulent and able to induce a protective immune response. BALB/c mice survived challenge with phoP derivatives of the highly virulent S. typhimurium strains SR-11 and SL1344 when inoculated intraperitoneally and per oral with doses equivalent to 10(4) 50% lethal doses (LD50) of the parent virulent strains. The avirulent mutants were able to establish an infection of the Peyer's patches of orally infected animals for up to 10 days after inoculation but were very inefficient at reaching the spleens. Despite the low level of infectivity of these mutants, immunized animals developed a delayed-type hypersensitivity (DTH) response to Salmonella antigens and resisted challenge with up to 10(4) LD50 of the virulent parent strain 30 days after immunization.     

Selection of virulent mutants of Streptococcus pneumoniae. Utilization of a murine model of septicemia]

Genetic construction of virulence deficient mutant is a strategy to analyse virulence genes of Streptococcus pneumoniae and was used to virulence factors as capsule, pneumolysin, autolysin and PspA. We perform a model allowing the in vivo positive selection of virulent S. pneumoniae mutants. Mice which are the most susceptible animals to pneumococcal infection, offer the best model for screening virulent S. pneumoniae. Indeed, after intraperitoneal injection of bacterial mix which was composed to a lot of avirulent bacteria (6 log10 CFU per mouse) (V1015 strain, DL50 = 7.05) and few virulent pneumococci (1 to 2 log10 CFU per mouse) (P4241 strain, DL50 < 1), mice cleared all avirulent bacteria but not virulent pneumococci. Thus, mice dead in 3 to 4 days with septicaemia and positive hemoculture contained only virulent strain. This model was validated by in vivo selection of a virulent mutant (V1042, DL50 = 4.1) which was obtained after transformation of avirulent strain V1015 with the genomic fragment of virulent strain P4241. Our model of screening was the only one allowing detection of virulent S. pneumoniae mutants. This new genetic strategy which consisted in gene addition and used mouse as selection agent, could be used to discover new virulence genes required to in vivo bacterial development.     

Rapid and sensitive method for evaluating Pseudomonas aeruginosa virulence factors during corneal infections in mice.

A murine corneal scratch model has been used extensively to study various aspects of the pathogenesis of Pseudomonas aeruginosa, a common etiologic agent of corneal infections. This model uses mild inhalation anesthetics which keep the animals immobile for a relatively short time and promote the interaction between the infecting organisms and the corneal wound. Under these circumstances, only a small number of P. aeruginosa isolates delivered at inocula of > 10(7) CFU are infectious. We determined that this model is useful for studying other P. aeruginosa strains given at lower doses if injectable anesthetics are administered prior to infection to keep the animals immobile for 15 to 30 min. Under these conditions, eight clinical isolates of P. aeruginosa tested at doses of 10(8) CFU per eye induced corneal perforation and/or phthisis in C3H/HeN mice. The 50% infective doses of several strains were between 3 x 10(2) and 1 x 10(5) CFU per mouse eye. When this modified anesthetic procedure was used to evaluate the roles of different P. aeruginosa virulence factors in eye infections, pathology was not observed when eyes were inoculated with 10(8) CFU of strains deficient in production of a complete lipopolysaccharide or the RpoN sigma factor. A strain with a point mutation in the fur gene, involved in production of iron-regulated factors, showed decreased virulence, while a mutant deficient in both hemolytic and nonhemolytic phospholipase C was fully virulent. By modifying the anesthesia procedure, the corneal scratch model allows rapid evaluations of the roles of P. aeruginosa virulence factors in corneal infections.     

Relationships among capsular structure, phagocytosis, and mouse virulence in Klebsiella pneumoniae.

Klebsiella pneumoniae strains of the K2 capsular serotype are usually highly virulent in mice, which is in contrast to the low virulence of most other serotypes. Here we used a genetic approach to examine the relative contribution of capsule type to the virulence of K. pneumoniae in mice. We used wild-type strains expressing capsular polysaccharide (CPS) serotypes K2 (strain KPA1) and K21a (strains KPB1 and KPC1), which were then used to construct capsule-switched derivatives. The close proximity of the cps gene cluster to selectable his markers made it possible to mobilize the cps genes by conjugation from one serotype (donor) to another (recipient) and to obtain recombinants in which interserotype switching had occurred by reciprocal recombination. Each capsule-switched derivative examined of the KPA and KPC strain backgrounds produced a CPS that was immunologically and structurally identical to that of the donor. Strain background was confirmed by demonstrating restriction fragment length polymorphism patterns identical to those of the respective recipients. The parent strains were then compared with capsule-switched recombinants for phenotypic properties associated with virulence. Clearance from the bloodstreams of mice was rapid in serotype K21a strains of either wild-type or recombinant origin, whereas K2 strains remained viable in the blood during the period examined. These differences appeared to be dependent upon the CPS type but independent of strain background. Binding to macrophages was higher in K21a strains than in those with the K2 capsule and was also independent of the strain background. Both blood clearance and macrophage-binding activities were completely inhibited by yeast mannan, suggesting that they were mediated via the macrophage mannose receptor. The K2 parent strain was highly virulent to mice (50% lethal dose [LD50], 3 x 10(3)), while the K21a parent strains demonstrated low virulence (LD50, > 2 x 10(8)). Interestingly, the virulence of recombinant KPC10(cpsK2), originally of the KPC1(cpsK21a) background, was intermediate (LD50, 4 x 10(5)). In contrast, both cpsK21a recombinants of the originally virulent KPA1 (cpsK2) background became nearly avirulent (LD50, > 2 x 10(8)). Six additional serotypes (K12, K24, K32, K55, K62, and K67) were examined, and all showed a positive correlation between the ability of the Klebsiella serotype to interact with a human mannose receptor, as expressed by Cos I cell recombinants, and the LD50 of the serotype. These results suggest that expression of a capsule which is recognized by the mannose receptor markedly affects the interaction with macrophages and blood clearance.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of pyochelin on the virulence of Pseudomonas aeruginosa.

A virulent isolate of Pseudomonas aeruginosa PAO1, which had been obtained from eight sequential intraperitoneal infections in mice compromised with iron and methotrexate, expressed greater lethality than the avirulent parent strain when both strains were injected into mice treated with iron. The present study demonstrates that pyochelin, a siderophore produced by P. aeruginosa, also increases the lethality of the virulent bacteria but not of the avirulent bacteria. Analysis of the growth and clearance of both virulent and avirulent strains in mice revealed that pyochelin increased the growth and lethality of virulent bacteria but only increased the survival of the avirulent bacteria. A streptomycin-dependent mutant of strain PAO1 (strd1) was used to demonstrate that pyochelin did not affect the clearance activity of mice. This strongly suggests that the effects of pyochelin in stimulating the persistence of avirulent bacteria and in increasing the lethality of virulent bacteria are due solely to the promotion of bacterial growth. Since the virulent bacteria were equivalent to the avirulent bacteria in utilizing pyochelin during in vitro growth in the presence of transferrin, it appears that the stimulation of growth by pyochelin allows the expression of additional virulence properties by the virulent bacteria.     

Virulence for mice of Klebsiella strains belonging to the O1 group: relationship to their capsular (K) types.

Eighty-two Klebsiella O1 strains belonging to various K types were tested for virulence for mice by intraperitoneal inoculation. They comprised 49 strains newly isolated from various clinical specimens, 31 reference strains, and 2 laboratory strains. Of 9 Klebsiella O1:K2 strains, 7 were highly virulent inasmuch as their 50% lethal doses per mouse were less than 10 CFU, whereas the other 2 strains were avirulent even when encapsulated just like the virulent strains as revealed by the quellung test. Klebsiella K2 reference strain B5055 and strain Chedid, which were maintained in vitro for a long time, were highly virulent. Of 8 Klebsiella O1:K1 strains, 2 showed moderate virulence, and the other 6 strains had relatively low or no virulence. We found no definite correlation between the virulence of Klebsiella K1 and K2 strains for mice and the kind of clinical specimens in which they originated. All of the Klebsiella O1 strains tested belonging to K types other than K1 and K2 showed very low or no virulence for mice.     

Cytopathogenic effects in enterocytelike Caco-2 cells differentiate virulent from avirulent Listeria strains.

We have developed a simple test that differentiates between virulent and avirulent Listeria species as defined by the mouse 50% lethal doses (LD50S). The assay is based on trypan blue-revealed cytopathogenic effects that are produced during the infection of the human enterocytelike cell line Caco-2. These effects were elicited only by Listeria strains that had an intraperitoneal mouse LD50 less than 10(8) and were not produced by nonhemolytic, avirulent strains of Listeria monocytogenes generated spontaneously or by Tn916 mutagenesis or by avirulent Listeria species. A negative test was also obtained with hemolysin-producing, avirulent L. monocyotogenes NCTC5105 and Listeria ivanovii KC1786. The test was negative with avirulent L. monocytogenes strains which are strong inducers of opacity in egg yolk agar. However, a strain which has a low LD50, such as 10(4), may show less severe cytopathogenic effects than a strain having a higher LD50, such as 10(6). The test has been effectively used to screen for virulent listerial isolates, spontaneous mutants, and transposon-induced mutants.     

Treatment of experimental infections of mice with bacteriophages.

Bacteriophages for Acinetobacter baumanii, Pseudomonas aeruginosa and Staphylococcus aureus were tested in experimental infections of mice to investigate their potential for the treatment of infections of man. As few as 10(2) particles of an acinetobacter phage protected mice against 5 LD50 (1 x 10(8)) of a virulent strain of A. baumanii, and phage was demonstrated to have multiplied in the mice. A pseudomonas phage protected mice against 5 LD50 of a virulent strain of P. aeruginosa, with a PD50 of 1.2 x 10(7) particles. A staphylococcal phage failed to protect mice infected with a strain of S. aureus. These studies support the view that bacteriophages could be useful in the treatment of human infections caused by antibiotic-resistant strains of bacteria.     

Test of the virulence and live-vaccine efficacy of auxotrophic and galE derivatives of Salmonella choleraesuis.

Aromatic compound-dependent (aro) derivatives of three mouse-virulent strains of Salmonella choleraesuis (Salmonella cholerae-suis) were constructed and shown to be nonvirulent for mice (intraperitoneal [i.p.] 50% lethal dose [LD50], greater than 5 X 10(6) CFU). A pur derivative, and a thy derivative, each of a different virulent parent, remained moderately virulent (i.p. LD50S for BALB/c mice, ca. 10(5) and 5 X 10(4) CFU, respectively). Tested as live vaccines i.p., the aro strains were ineffective in salmonella-susceptible BALB/c and C57BL/6 mice but were somewhat effective in salmonella-resistant CBA/J mice and in outbred CD-1 mice. The pur and thy strains were effective as live vaccines in BALB/c mice when given in sublethal doses. Two previously isolated nonvirulent galE derivatives of S. choleraesuis (i.p. LD50 in BALB/c mice, greater than 10(6) CFU) were also ineffective as live vaccines in BALB/c and C57BL/6 mice. The main antigenic difference between S. choleraesuis (O-6,7) and S. typhimurium (O-4,12) is in O-antigen character, thought to largely determine the specificity of protection in salmonellosis. Paired, nearly isogenic O-6,7 and O-4,12 derivatives were constructed from an aro S. typhimurium strain of proven efficacy as a live vaccine. Used as live vaccines, the O-4,12 member protected BALB/c mice against challenge with virulent S. typhimurium, whereas the O-6,7 member did not protect against virulent S. choleraesuis. However, BALB/c mice vaccinated with the O-6,7 member and mice vaccinated with an aro S. choleraesuis strain were protected against challenge with a moderately virulent (LD50, 5 X 10(4) CFU) O-6,7 derivative of an S. typhimurium strain.     

Conversion of Pseudomonas aeruginosa to the phenotype characteristic of strains from patients with cystic fibrosis.

Isolates of Pseudomonas aeruginosa from cystic fibrosis patients are unusual; they are often susceptible to the bactericidal effect of human serum, have a rough lipopolysaccharide, and produce an exopolysaccharide that is responsible for the characteristic mucoid phenotype. In contrast, strains from the environment and from patients with other diseases usually have smooth lipopolysaccharide, do not produce very much mucoid exopolysaccharide, and are phenotypically nonmucoid. The predominance of mucoid strains of P. aeruginosa in infections of patients with cystic fibrosis has not been explained. In the lower airways, where P. aeruginosa persists in cystic fibrosis, nutrients for bacterial growth may be limited. We investigated whether growth of P. aeruginosa under conditions of suboptimal nutrition causes conversion to the characteristic cystic fibrosis phenotype. Ninety-two strains of P. aeruginosa were maintained for up to 90 days in a minimal medium with acetamide as the sole carbon source. In 56 (52%) of 107 cultures, isolates with rough lipopolysaccharide emerged, and in 20 (19%) of 104 nonmucoid cultures, mucoid isolates were recovered. Strains with rough lipopolysaccharide also were sensitive to the bactericidal effect of normal human serum. Under conditions of suboptimal nutrition in vitro, isolates of P. aeruginosa emerged that produced rough lipopolysaccharide and were mucoid, typical of many isolates from cystic fibrosis patients. This peculiar phenotype may arise as a consequence of nutritional limitation within the cystic fibrosis respiratory tract rather than from features unique to these strains of bacteria.