Curtiss, Roy, III (Oak Ridge National Laboratory, Oak Ridge, Tenn.), Leigh J. Charamella, Claire M. Berg, and Paula E. Harris. Kinetic and genetic analyses of d-cycloserine inhibition and resistance in Escherichia coli. J. Bacteriol. 90:1238-1250.1965.-Wild-type cells of Escherichia coli growing at 37 C in mineral salts-glucose medium with vigorous aeration were lysed at maximal exponential rates by 10(-4) to 10(-2)md-cycloserine. At concentrations above 2 x 10(-2)m, d-cycloserine was bacteriostatic. Low levels of d-cycloserine (10(-5)m) and pencillin G (10 units per ml) interacted synergistically to cause a rapid exponential rate of lysis. Spontaneous mutations to d-cycloserine resistance occurred in discrete steps at frequencies of 10(-6) to 10(-7) for each step. First-, second-, and third-step d-cycloserine-resistant mutants were lysed at maximal exponential rates by d-cycloserine concentrations of 10(-3), 3 x 10(-3), and 5 x 10(-3)m, respectively. d-Alanine, l-alanine, and dl-alanyl-dl-alanine reversed d-cycloserine-induced lysis, in that order of effectiveness. On the basis of these observations, a d-cycloserine-enrichment cycling technique was developed for isolation of auxotrophic mutants. d-Cycloserine at 2 x 10(-3)m was as efficient as penicillin G (1,000 units per ml) for mutant enrichment in E. coli and should be useful for isolation of mutants in penicillin-resistant microorganisms. Bacterial conjugation experiments indicated that all three mutations conferring d-cycloserine resistance were linked to the met(1) locus. Transduction experiments showed that the mutation conferring first-step resistance was at least 0.5 min away from the mutations conferring second- and third-step resistance. The latter two mutations possibly occurred in the same gene, since they were sometimes carried in the same transducing phage. Studies on expression of d-cycloserine resistance indicated that these mutations were neither dominant nor recessive to each other nor to the d-cycloserine-sensitivity allele. Each allelic state exerted its influence on the phenotype independently of the others. These results are discussed in terms of the known inhibition of alanine racemase and d-alanyl-d-alanine synthetase by d-cycloserine.

Alanine↗

PubMedDate not supplied

Stabilization of recombinant avirulent vaccine strains in vivo.

Salmonella strains attenuated by various mutational alterations and expressing heterologous colonization and virulence antigens specified by cloned genes have begun to be widely used as vaccines for oral immunization to induce protective immunity against the pathogens supplying the genes for the colonization or virulence antigens. Problems associated with plasmid instability and/or poor expression of cloned gene products have frequently been encountered and regulatory agencies are now banning use of antibiotic resistance markers in live attenuated vaccine strains. We have therefore developed a balanced lethal host-vector system in which the chromosome of the attenuated vaccine strain contains a deletion mutation that impose a requirement for diaminopimelic acid (DAP), an essential constituent of the rigid layer of the cell wall of all Gram-negative and some Gram-positive microbes. The plasmid cloning vector contains the wild-type allele for this gene allowing the recombinant avirulent Salmonella to be independent of DAP. Since DAP is not prevalent in nature, especially in the animal host, essentially 100% of the surviving avirulent Salmonella recovered from an immunized animal host still contain the recombinant plasmid and express the foreign colonization or virulence antigen. Occasional loss of the plasmid renders the avirulent Salmonella DAP-requiring, which quickly results in DAPless death with lysis of the bacterium to release its antigenic contents, an occurrence which might further enhance the immune response to the foreign colonization or virulence antigen. We describe below strains of bacteria, recombinant vectors and the methods to make use of this system in a diversity of situations for development of live recombinant avirulent vaccines as well as for other potential applications.

Amino Acid Sequence↗

PubMedDate not supplied

Control of colonization by virulent Salmonella typhimurium by oral immunization of chickens with avirulent delta cya delta crp S. typhimurium.

Oral immunization with a delta cya delta crp Salmonella typhimurium strain has been shown to preclude colonization by wild-type, virulent S. typhimurium and induces humoral and cellular immune response in chickens. Intestinal tract colonization by the virulent challenge strain was used to determine the level of protection conferred by immunization with the delta cya delta crp mutant. The associated humoral and cellular immune responses were measured by ELISA and delayed-type hypersensitivity (DTH) tests, respectively. The levels of colonization by both Salmonella strains were determined by enumeration of viable cells in the intestinal tract. A reduction in faecal excretion of the wild-type strain was observed with a single oral immunization with the delta cya delta crp mutant, but caecal colonization was not affected. However, double oral immunization with the delta cya delta crp mutant precludes caecal colonization by the virulent strain. IgM, IgA and IgG were detected against sonicated Salmonella whole-cell antigens. Outer membrane and flagella proteins induced DTH responses, whereas lipopolysaccharide failed to do so. The effectiveness of the delta cya delta crp strain in reducing caecal colonization by the highly virulent challenge strain in chickens demonstrates that oral vaccination with the delta cya delta crp S. typhimurium should aid in eliminating Salmonella carriers in chickens. The elimination of these carriers on the poultry farm should help to control Salmonella contamination of poultry products, therapy improving public health.

Administration, Oral↗

PubMedDate not supplied

Recombinant avirulent Salmonella vaccine strains with stable maintenance and high level expression of cloned genes in vivo.

Salmonella typhimurium strains with deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes are avirulent for mice and induce a high level of protective immunity to oral challenge with up to 10,000 times what would be a lethal dose of wild-type virulent S. typhimurium cells. This immunity begins as early as seven days after immunization and lasts for at least four months. S. typhimurium delta cya delta crp mutants stably maintain plasmids and give high-level expression of cloned gene products; in this they appear superior to other avirulent S. typhimurium strains. S. typhimurium delta cya delta crp strains with a delta asd mutation (abolishing production of aspartate beta-semialdehyde dehydrogenase), have an obligate requirement for diaminopimelic acid (DAP). This strain can be used in conjunction with plasmid vectors lacking antibiotic resistance markers but having the wild-type asd+ gene from Streptococcus mutans to complement the delta asd chromosomal mutation. The Asd+ plasmid vector can be used to express a diversity of colonization and virulence antigens from other pathogens. In the delta cya delta crp delta asd S. typhimurium vaccine strain, the plasmid is completely stable in the absence of any exogenous selective pressure either in vitro or in vivo.

Adenylyl Cyclases↗

PubMedDate not supplied

Pathogenic diversity of Escherichia coli and the emergence of 'exotic' islands in the gene stream.

Escherichia coli is a highly adaptive bacterial species that is both a member of the commensal intestinal flora and a versatile pathogen associated with numerous types of intestinal and systemic infections in humans and other animals. The spectrum of diseases caused by E. coli is due to the acquisition of specific virulence genes harbored on plasmids, bacteriophages, or within distinct DNA segments termed pathogenicity islands (PAIs) that are absent from the genomes of commensal E. coli strains. PAIs are likely to have been transferred horizontally and may have integrated into the E. coli chromosome through bacteriophage or plasmid integration or transposition. The contribution of intergenic inheritance to the adaptation and evolution of E. coli, types of PAIs associated with different groups of pathogenic E. coli and approaches to identify unique sequence islands (USIs), some of which might confer pathogenicity, in E. coli and other bacteria are presented.

Animals↗

PubMedDate not supplied

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.

Air Sacs↗

PubMedDate not supplied

Reaction of the avian respiratory system to intratracheally administered avirulent Salmonella typhimurium.

Chickens were inoculated intratracheally (IT) with the SR-11 Salmonella typhimurium deletion mutant x4062 strain. Data collected for 8 days postinoculation (PI) were: signs of respiratory and gastrointestinal disease; histological lesions; the influx, phagocytic proportion, and phagocytic capacity of avian respiratory phagocytes (ARPs); and the proportion of granulocytes vs. macrophages in the lung tissues and lavage fluids of the lungs and air sacs. S. typhimurium-inoculated chickens had no clinical signs of gastrointestinal or respiratory disease but had various degrees of inflammatory changes in the lungs. At 5 hr PI, S. typhimurium-inoculated chickens had approximately 53-fold more ARPs than mock-inoculated controls. Between 26 hr and 8 days PI, the number of ARPs from S. typhimurium-inoculated birds was not significantly higher than the number from the mock-inoculated controls. Flow cytometric analysis of ARPs demonstrated that the proportion of phagocytic ARPs and the phagocytic capacity of ARPs from S. typhimurium-inoculated chickens were significantly higher between 5 and 26 hr PI than those of the ARPs from mock-inoculated chickens. Kinetic changes over 8 days in the granulocyte/macrophage ratios in the lavage fluids, as compared with kinetic changes in the lung tissues, suggested that the granulocytes generally represent a much higher proportion of the ARPs, and egress earlier and in much larger numbers from the tissues to the lumen of lungs and air sacs than do macrophages.

Animals↗

PubMedDate not supplied

Virulence of Salmonella typhimurium mutants for White Leghorn chicks.

A strain of Salmonella typhimurium that is highly virulent for 1-day-old white leghorn chicks was genetically modified by deletion (delta) of the adenylate cyclase (cya) and cyclic AMP receptor protein (crp) genes or by removal (curing) of the 91-kilobase virulence plasmid. These mutants were then compared with the wild-type S. typhimurium strain for virulence in 1-day-old chicks and for their ability to colonize chicks of various ages. The plasmid-cured mutant showed a slight reduction in virulence, whereas the delta cya delta crp mutant was completely avirulent. The wild-type strain and both mutant strains were capable of colonizing various organs within the chicks. At all time points, the delta cya delta crp strain colonized chicks at lower levels than the wild-type strain. Titers of the plasmid-cured strain increased more slowly in visceral organs than did those of the wild type.

Animals↗

PubMedDate not supplied

Effect of infective dose on humoral immune responses and colonization in chickens experimentally infected with Salmonella typhimurium.

The influence of infective dose on chicken immunogenicity was examined in 1-week-old chickens. Chickens were infected orally with various doses of chi 3761 or chi 3985. Fecal shedding, colonization of the cecum, and induction of Salmonella-specific serum immunoglobulin isotypes were analyzed over a 5-week period. The delta cya delta crp Salmonella typhimurium vaccine strain chi 3985 was used to assess the effect of vaccination dose on protection after oral vaccination of chickens at 1 day and 2 weeks of age. Wild-type S. typhimurium strain chi 3761 was used to challenge vaccinated and unvaccinated chickens at 6 weeks of age, and the recovery of Salmonella from the cecum was used as a measure of protection. Infection of 1-week-old chickens with chi 3985 was more effective in reducing fecal excretion and cecal colonization than was infection with chi 3761. Double vaccination with 10(8) or 10(7) CFU of chi 3985 at 1 day and 2 weeks of age protected vaccinated chickens against cecal colonization by the challenge strain chi 3761. Immunogenicity of Salmonella is dose and genotype-dependent.

Administration, Oral↗

PubMedDate not supplied

The effect of bacterial surface structures on the pathogenesis of Salmonella typhimurium infection in chickens.

Mutants of Salmonella typhimurium strain SR11 containing Tn10 insertions in genes encoding for the motility and fimbriation phenotypes were evaluated for adhesion, invasion, and virulence in avian models. Mutations abolishing mannose-sensitive or mannose-resistant hemagglutination did not influence adherence or invasion in epithelial cells in vitro. A double hemagglutinin-deficient mutant, lacking both mannose-sensitive and mannose-resistant hemagglutinins, was diminished in ability to adhere to chick kidney epithelial cells in vitro, but invasion in vitro was not significantly affected. Compared with the wild-type parent, mutations that decreased motility reduced invasion levels in vitro and increased the peroral LD50 in one-day-old chicks. A mutant deficient in both motility and mannose-sensitive hemagglutination was greatly reduced in its ability to invade epithelial cells in vitro and persist in the liver and spleen of orally challenged chicks. Results of this study indicate that loss of motility in S. typhimurium attenuates peroral virulence in chicks.

Animals↗

PubMedDate not supplied

Effect of inv mutations on Salmonella virulence and colonization in 1-day-old White Leghorn chicks.

Invasion of Salmonella into the cells of the intestinal epithelium is an important step in the infection process. This initial invasion is followed by colonization of other organs throughout the body. In an attempt to better understand this process, we moved defined mutations in several genes of the inv locus into Salmonella typhimurium UK-1 and two strains of Salmonella enteritidis. These mutant strains were evaluated for their oral and intraperitoneal virulence as determined by 50% lethal dose in 1-day-old white leghorn chicks. These inv mutants were also studied for their ability to colonize orally infected chicks. The invA, invB, and invC mutations all caused a reduction in oral virulence and colonization by UK-1 and the S. enteritidis strains. Mutation of the invH gene had little or no effect on oral virulence or colonization. None of the inv genes tested had any effect on virulence of these Salmonella strains when administered intraperitoneally.

Adhesins, Bacterial↗

PubMedDate not supplied

Efficacy of a live avirulent Salmonella typhimurium vaccine in preventing colonization and invasion of laying hens by Salmonella typhimurium and Salmonella enteritidis.

An avirulent live delta cya delta crp Salmonella typhimurium strain chi 3985 that precludes colonization and invasion of chickens by homologous and heterologous Salmonella serotypes was evaluated for its long-term protection efficacy. Chickens vaccinated orally at 2 and 4 wk of age were assessed for protection against oral challenge with wild-type S. typhimurium and Salmonella enteritidis strains at 3, 6, 9, and 12 mo of age. A comparison of Salmonella isolation from vaccinated and nonvaccinated layers after challenge with S. typhimurium or S. enteritidis showed that delta cya delta crp S. typhimurium chi 3985 induced excellent protection against intestinal, visceral, reproductive tract, and egg colonization, invasion, and/or contamination by Salmonella. The duration of protection lasted for 11 mo after vaccination, at which time the experiment was terminated. S. enteritidis and S. typhimurium were isolated from the yolk, albumen, and shells of eggs laid by nonvaccinated chickens challenged with Salmonella. S. typhimurium caused pathological lesions in nonvaccinated chickens, whereas vaccinated and nonvaccinated chickens challenged with S. enteritidis showed no pathological lesion in the visceral and reproductive organs. Vaccination with chi 3985 prevented transmission of S. typhimurium or S. enteritidis into eggs laid by vaccinated layers with no effect on egg production. To our knowledge, this is the first publication confirming that vaccination with live avirulent Salmonella can induce long-term protection against Salmonella infection in layers.

Administration, Oral↗