Interaction of Salmonella typhi strains with cultured human monocyte-derived macrophages.

Human monocyte-derived macrophages (MDM) provided this laboratory with a tool to develop a primary-cell assay for evaluating the relative virulence of newly constructed Salmonella typhi carrier strains. In this study, the interaction with and survival within MDM were compared for delta aroA143-attenuated strains, wild-type virulent strains, and the current oral-vaccine strain, Ty21a.

Cloning of a Brucella melitensis group 3 antigen gene encoding Omp28, a protein recognized by the humoral immune response during human brucellosis.

Brucella group 3 antigens (Ags) are outer membrane proteins (OMPs) with a molecular mass ranging from 25 to 30 kDa. The OMPs are of interest partially because of their potential use as vaccine and diagnostic reagents. We used human convalescent antibody (Ab) to clone a gene that encoded a 28-kDa protein from a lambdagt11 library of Brucella melitensis 16M genomic DNA. DNA sequence analysis revealed a single open reading frame that would encode a protein of 26,552 Da. The 28-kDa protein had a primary amino acid sequence that was 43% similar to a previously described Brucella abortus group 3 Ag, Omp25 (P. de Wergifosse, P. Lintermans, J. N. Limet, and A. Cloeckaert, J. Bacteriol. 177:1911-1914, 1995). The similarity to a known group 3 OMP, immunoreactivity with Ab prepared against B. abortus group Ags, immunolabeling of whole cells, and Southern hybridization led to our conclusion that the B. melitensis 28-kDa protein was a group 3 protein distinct from B. abortus Omp25. We designated the B. melitensis protein Omp28. Human convalescent sera from patients infected with B. abortus and Brucella suis as well as rabbit antisera prepared against killed B. abortus whole cells recognized B. melitensis Omp28 on Western blots (immunoblots). Furthermore, mice and goats infected with smooth strains of B. melitensis produced Abs against Omp28. Our results may begin to explain the variability in molecular weight seen in Brucella group Ags and point toward their possible use in vaccination against infection as well as diagnosis of the disease.

Epidemiology and control of acute respiratory diseases with emphasis on group A beta-hemolytic streptococcus: a decade of U.S. Army experience.

OBJECTIVE: To summarize the experiences of the U.S. Army regarding prevention and control, and frequencies, rates, trends, and determinants of febrile acute respiratory diseases (ARDs), particularly Group A beta-hemolytic streptococcus (GABHS). METHODOLOGY: Since 1966, the U.S. Army has conducted routine surveillance of ARDs among basic trainees. Since 1985, all trainees with fever and respiratory tract symptoms have been cultured for GABHS: Field investigations were conducted when outbreaks of acute respiratory or GABHS-associated illnesses were detected. Mass plus tandem benzathine penicillin prophylaxis were used to interdict and control training center GABHS outbreaks. RESULTS: During the period 1985 to 1994, there were 65,184 hospitalizations for acute febrile respiratory illnesses among Army trainees. The crude hospitalization rate was 0.45 per 100 trainees per week. The rate consistently declined over the period. Incremental declines were temporally associated with increased use of adenovirus immunizations and broader use of benzathine penicillin prophylaxis. During the period, 10,789 of 59,818 (18%) pharyngeal cultures were positive for GABHS: GABHS outbreaks were associated with diverse clinical manifestations including streptococcal toxic shock, acute rheumatic fever, and pneumonia. The emergence of mucoid colony morphology in clinical isolates was a consistent indicator of circulating virulent strains with epidemic potential. Outbreak-associated M types were M1, M3, M5, and M18. In response to six GABHS outbreaks, mass plus tandem benzathine penicillin chemoprophylaxis produced rapid and sustained GABHS control. ARD and GABHS recovery rates were lowest when benzathine penicillin prophylaxis was widely used. CONCLUSIONS: ARD rates among Army trainees have consistently declined to unprecedented levels. GABHS has reemerged as an important threat to military trainees. Benzathine penicillin chemoprophylaxis is safe and effective for interdicting and preventing GABHS outbreaks in closed, healthy young adult populations.

Flagellar serotypes of Salmonella typhi in Indonesia: relationships among motility, invasiveness, and clinical illness.

While the H1-d flagellar serotype of Salmonella typhi has been found worldwide, the H1-j serotype occurs only in Indonesia. A cross-sectional survey in Indonesia compared epidemiologic, clinical, and pathogenetic characteristics of these two serotypes. S. typhi isolates were collected from patients with acute typhoid fever in four Indonesian cities. Flagellar serotype was determined by polymerase chain reaction amplification of the fliC locus of the flg gene. Of 321 isolates, 51 (15.9%) were H1-j. Patients with H1-j infection were older than those with H1-d (P < .001). Among 30 patients with known clinical outcomes, H1-j infection was associated with milder clinical illness than H1-d (P = .06). In vitro, H1-j isolates were both less motile on semi-solid agar plates (P = .004) and less invasive of HEp-2 cells (P = .002) than H1-d isolates. The association of decreased severity of illness with decreased motility and invasiveness suggests that flagellar properties are a component of S. typhi's virulence.

Molecular evolutionary genetics of the cattle-adapted serovar Salmonella dublin.

An electrophoretic analysis of allelic variation at 24 enzyme loci among 170 isolates of the serovar Salmonella dublin (serotype 1,9,12[Vi]:g,p:-) identified three electrophoretic types (Du 1, Du 3, and Du 4), marking three closely related clones, one of which (Du 1) is globally distributed and was represented by 95% of the randomly selected isolates. All but 1 of 114 nonmotile isolates of serotype 1,9,12:-:- recovered from cattle and swine in the United States were genotypically Du 1. The virulence capsular polysaccharide (Vi antigen) is confined to clone Du 3, which apparently is limited in distribution to France and Great Britain. For all 29 isolates of Du 3, positive signals were detected when genomic DNA was hybridized with a probe specific for the ViaB region, which contains the structurally determinant genes for the Vi antigen; and 23 of these isolates had been serologically typed as Vi positive. In contrast, all 30 isolates of Du 1 tested with the ViaB probe were negative. These findings strongly suggest that the ViaB genes were recently acquired by S. dublin via horizontal transfer and additive recombination. The clones of S. dublin are closely similar to the globally predominant clone (En 1) of Salmonella enteritidis (serotype 1,9,12:g,m:-) in both multilocus enzyme genotype and nucleotide sequence of the fliC gene encoding phase 1 flagellin. Comparative sequencing of fliC has revealed the molecular genetic basis for expression of the p and m flagellar epitopes by which these serovars are distinguished in the Kauffmann-White serological scheme of classification.

Evolutionary genetic relationships of clones of Salmonella serovars that cause human typhoid and other enteric fevers.

Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not. Although the clones of S. decatur and S. choleraesuis are serologically and biochemically similar, they are genotypically very distinct. Two clones of S. typhi were distinguished, one globally distributed and another apparently confined to Africa; both clones are distantly related to those of all other serovars studied.

Rapid diagnosis of typhoid fever through identification of Salmonella typhi within 18 hours of specimen acquisition by culture of the mononuclear cell-platelet fraction of blood.

Detection of Salmonella typhi in blood by culture of the mononuclear cell-platelet layer was compared with other methods currently used for the diagnosis of typhoid fever. Colonies of S. typhi were present in all mononuclear cell-platelet layer-positive cultures within 18 h of plating and were identified within an additional 10 min by a coagglutination technique. In contrast, identification of all positive cultures by conventional blood culture required 3 days.

Construction and characterization of a Vi-positive variant of the Salmonella typhi live oral vaccine strain Ty21a.

The viaB locus coding for the Vi antigen of Salmonella typhi Ty2 was cloned on a 40.6-kilobase fragment into the cosmid vector pHC79. The live, oral, attenuated Vi-negative S. typhi Ty21a vaccine strain was transformed with the recombinant cosmid encoding the viaB locus. Homologous recombination of the viaB locus into the chromosome of S. typhi Ty21a was induced by UV irradiation, and Vi-positive recombinants were selected in the presence of D-cycloserine. One such isolate, termed WR4103, contained no plasmids or the attendant antibiotic resistance markers and expressed the Vi antigen stably. Vi antigen extracted from WR4103 was immunologically indistinguishable from Vi antigen purified from S. typhi Ty2. The only detectable difference between Ty21a and WR4103 was in the production of Vi antigen. The mean lethal doses of Ty21a and WR4103 for mice were nearly identical. Immunization of mice with WR4103 engendered a Vi antibody response and afforded complete protection against fatal infection with virulent S. typhi Ty2. Thus, S. typhi WR4103 may serve as an improved oral vaccine for protection against typhoid fever.

Use of a DNA probe to detect Salmonella typhi in the blood of patients with typhoid fever.

A DNA probe was used to detect Salmonella typhi from blood samples from 14 of 33 patients with culture-confirmed typhoid fever, using the equivalent of 2.5 ml of blood. In contrast, S. typhi was detected in 17 of the same 33 patients by culture of 8 ml of blood. The probe hybridized to blood samples of 4 of 47 patients from whom S. typhi was not isolated.

Specific insertion and deletion of insertion sequence 1-like DNA element causes the reversible expression of the virulence capsular antigen Vi of Citrobacter freundii in Escherichia coli.

Citrobacter freundii strain WR7004 reversibly expresses the virulence capsular antigen Vi, whose production is controlled by two distinct chromosomal loci, viaA and viaB. The rate of oscillation between the Vi-producing (Vi+) state and the Vi-nonproducing (Vi-) state in strain WR7004 ranges from 2 X 10(-4) to 7 X 10(-3) transitions per bacterium per generation. A similarly high conversion rate from Vi+ to Vi- occurs in Escherichia coli HB101 harboring pWR127, a plasmid that contains the 18-kilobase-pair (kb) viaB region cloned from WR7004. However, the Vi- state in HB101 harboring pWR127 was so stable that transition to the Vi+ state was not detected (less than 10(-10) per bacterium per generation). When pWR127 DNA derived from Vi- strains of HB101 harboring pWR127 was transformed in HB101 recipient, a small number of Vi+ transformants were seen among the transformants, which were predominantly Vi-. The viaB region consists of two EcoRI digestion fragments, A (8.6 kb) and B (9.4 kb). A discrete 700- to 800-base-pair DNA element was found to be inserted in the EcoRI B fragment of the viaB region of pWR127 when derived from Vi- strains. However, no such DNA element was found in the pWR127 DNA isolated from Vi+ strains. This discrete DNA element inserts into a recombinational hot spot 1.1 kb from the end of the EcoRI B fragment and behaves as an insertion sequence 1 (IS1)-like element. The insertion of this IS1-like element in the viaB region thus disrupts expression of the Vi antigen. Restoration of Vi expression results when this element is excised.

Development of a DNA probe to detect Salmonella typhi.

This study was undertaken to identify a DNA sequence that could be used to facilitate the diagnostic identification of Salmonella typhi, the causative agent of typhoid fever. All virulent S. typhi strains encode a relatively unique capsular antigen termed the virulence (Vi) antigen. Two distinct genetic loci, viaA and viaB, are involved in the synthesis of this antigen. The structural genes, located at viaB, were considered as a possible specific DNA probe. The viaB locus, contained in a recombinant cosmid, was subcloned to various plasmid vectors for this purpose. Selected viaB-region DNA fragments were then analyzed for specificity in DNA colony hybridization reactions with more than 170 strains representing a variety of enteric bacteria. An 8.6-kilobase EcoRI fragment was highly specific for the viaB gene region and was considered a good hybridization probe. This DNA probe should prove useful in rapid diagnostic assays set up to detect S. typhi in mixed bacterial samples (e.g., stools) within a few hours of specimen collection.

Characterization of R factor beta-lactamases by the acidimetric method.

The properties and regulation of beta-lactamases produced by certain R factors derived from ampicillin-resistant strains of Escherichia coli and Klebsiella were characterized. beta-Lactamase activity was determined by the acidimetric method with phenol red as indicator. The sensitivity of this assay was increased by employing phosphate buffer at a final concentration of 0.4 mm; as little as 0.05 unit (1 unit equals the amount of beta-lactamase that hydrolyzes 1 mumole of benzyl penicillin per hr at pH 7.6 and 25 C) could be detected. This assay is rapid and convenient and appears to be superior to other methods currently employed to assay R factor beta-lactamases. Two classes of beta-lactamases were distinguished on the basis of substrate profile, heat inactivation, and K(m) values. The regulation of most of these R factor beta-lactamases, like certain other R factor enzymes, is subject to cyclic adenosine monophosphate-mediated catabolite repression.