Comparative characteristics of antigenic peculiarities and several other properties of stable long-term cultures of salmonella L-forms]

Enzymatic properties, sensitivity to antibiotics and peculiarities of the antigenic structure of stable L-forms of S. paratyphi B (L-115), S. typhimurium (L-71a-16) and S. typhi (L-5761) were studied. In difference from the initial strains, the L-form under study possessed a high penicillin and ampicillin sensitivity, and also a marked sensitivity to polymyxin and detergents; this characterized them as L-forms of protoplastic type. All the L-variants differed considerably by the enzymatic properties from the initial strains, and, in the majority of cases, retained the antigenic components and the species-specificity characteristic of the initial parental cultures. Despite the loss of the cell wall the synthesis of O-antigen in the L-forms under study was undisturbed. The 6-forms contained an antigenic component not found in the initial cultures and apparently causing the neutralizing activity of the L-antiser.     

The process of the persistence of Salmonella typhi L forms in the body of experimental animals studied by immunoradiometric analysis

The use of the immunoradiometric assay made it possible to reveal a prolonged persistence of the antigen of viable cultures of S. typhi stable L-forms, as well as to study the dynamics of its spread in the body of experimental animals. After both subconjunctival and intraperitoneal infection of guinea pigs the antigen of S. typhi L-forms spread slowly in the body of experimental animals with its localization first in the lymphoid formations in the pharynx and the intestine and subsequent undulatory accumulation in the marrow, spleen and bile. The persistence of the antigen of live S. typhi L-forms lasted as long as 6 months (the term of observation); killed L-forms could be detected for not more than 17 days. Regular inoculations of samples from different organs into media for the cultivation of S. typhi bacterial and L-forms yielded no positive results, which showed the difficulty of obtaining stable L-forms and evidenced the absence of their reversion in the body of experimental animals.     

In vivo experimental modelling of the infectious process caused by stable L forms of the causative agent of typhoid

To simulate the infectious process and to study the persistence of L-forms, rabbits and guinea pigs were infected with S. typhi stable L-forms. The materials presented in this work indicate that both subconjunctival and intraperitoneal infection led to the development of the clinically indistinct, but morphologically pronounced pathological process with characteristic localization and typical changes in the gastrointestinal tract. The typical features of the process were the generalized immunomorphological reaction of the lymphoid apparatus with the appearance of light-colored reticulomacrophagal elements, the signs of the activation of humoral and cell-mediated immunity and the formation of small epitheloidocellular granulomas. The results of the investigation indicate that the stable cultures of S. typhi L-forms are highly pathogenic and capable of inducing the infectious process in experimental animals.     

Comparative Survival of Indicator Bacteria and Enteric Pathogens in Well Water

The comparative survival of various fecal indicator bacteria and enteric pathogens was studied in a stable well water supply by using membrane chambers. There was more variation in the 29 coliform cultures and they died more rapidly, as a group, than the 20 enterococcus cultures that were examined. The comparative survival of the organisms tested follows: Aeromonas sp. > the shigellae (Shigella flexneri, S. sonnei, and S. dysenteriae) > fecal streptococci > coliforms = some salmonellae (Salmonella enteritidis ser. paratyphi A and D, S. enteritidis ser. typhimurium) > Streptococcus equinus > Vibrio cholerae > Salmonella typhi > Streptococcus bovis > Salmonella enteritidis ser. paratyphi B. S. bovis had a more rapid die-off than did S. equinus, but both had significantly shorter half-lives than the other streptococci. The natural populations of indicator bacteria from human and elk fecal material declined similarly to the pure cultures tested, whereas the die-off of fecal streptococci exceeded the coliforms from bovine fecal material.     

Processes of bacterial L transformation and L form reversion in the body of argasid ticks

The experimental infection of tampan ticks (Ornithodoros moubata) with the bacterial cultures of Listeria monocytogenes and Salmonella typhimurium, as well as with their L-forms, was carried out. These experiments demonstrated that both the L-transformation of bacteria and the reversion of their L-forms into the initial bacterial culture could occur in the body of the ticks.     

Bacteriophage Typing of Salmonellae. II. New Bacteriophage Typing Scheme

A phage-typing technique for salmonellae is described. One battery of phages was used to type three serotypes of Salmonella, namely, S. typhimurium, S.typhimurium var. copenhagen, and S. heidelberg. In all, 443 S. heidelberg cultures were typed into 22 phage types, 185 S. typhimurium cultures into 35 phage type, and 92 S. typhimurium var. copenhagen cultures into 26 phage types. The stability of the phage types was established by retyping 168 cultures belonging to all three serotypes. The epidemiological significance of the phage types demonstrated was evaluated by comparing phage types obtained from the University of Minnesota and those from the National Animal Disease Laboratory. Further investigation of the S. heidelberg phage types has shown that the cultures represented repeated isolates from the same birds or from a group of birds in the same flock.     

Enterotoxic properties of salmonellae and their neurotoxins

Most of live S. typhimurium cultures are capable of intraintestinal proliferation and possess enterotoxic activity. The capacity of S. typhimurium strains for producing enterotoxins is not connected with their origin. The parenteral immunization of rabbits with corpuscular vaccines prepared from S. typhimurium induced changes in the sensitivity of different sections of the small intestine of the animals to the enterotoxic action of live homologous cultures. Neurotoxin isolated from S. typhi was found to possess enterotoxic activity.     

Ultrastructure of Salmonella typhimurium forms with unbalanced growth induced by lithium chloride

The action of 1.0 and 1.5 M LiCl on S. typhimurium induces the appearance of unbalanced growth forms capable of growing and multiplication, when subcultured in a medium with this preparation. In this culture the prevalence of cells differing in their structure from the initial Salmonella cells and from stable L-form cultures is observed. Cells characteristic of the initial culture and cells resembling the L-forms occur in lesser numbers. LiCl seems to affect peptidoglycan and the cytoplasmic membrane, which brings about disturbances in the permeability of the surface structures of the cell.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

Abstract The susceptibility of Salmonella typhimurium LT2 and of S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae . The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Susceptibility of Salmonella typhimurium and Salmonella typhi to oxygen metabolites

The susceptibility of Salmonella typhimurium LT2 and S. typhi 1079 to oxygen metabolites were compared. S. typhimurium LT2 and S. typhi 1079 were killed to an equal extent (about 40%) by the xanthine-xanthine oxidase (200 mU/ml) system. Among the various scavengers of oxygen metabolites, catalase alone inhibited the killing of S. typhimurium LT2 and S. typhi 1079 by the xanthine-xanthine oxidase system, indicating that hydrogen peroxide contributed to the killing of Salmonellae. The respiratory burst of murine macrophages was efficiently triggered by the ingestion of S. typhimurium LT2, S. typhimurium SL1102, and S. typhi 1079 and all to the same extent. However, in the range of the concentration of hydrogen peroxide produced by murine macrophages, neither S. typhimurium LT2 nor S. typhi 1079 were killed. Only S. typhimurium SL1102, a rough mutant of S. typhimurium LT2, was markedly susceptible under these conditions. The findings suggest that both S. typhimurium LT2 and S. typhi 1079 are resistant to oxygen-dependent killing mechanisms.     

Variation in Plating Efficiency of Salmonellae on Eight Lots of Brilliant Green Agar

The plating efficiency of Salmonella anatum, S. cubana, S. dublin, S. tennessee, and S. typhimurium was determined for eight lots of Brilliant Green Agar made by two manufacturers. Washed cells were used as the inoculum and cultures were incubated at 41.5 C. All lots of Brilliant Green Agar were supplemented with 12 mg of sulfadiazine per 100 ml of medium. Of the eight lots of Brilliant Green Agar tested, average recovery of the test salmonellae in three did not differ from recoveries with Trypticase Soy Agar, which was used as a control to indicate the number of viable salmonellae in the test suspension capable of growth on a nonselective medium. Two lots of Brilliant Green Agar gave salmonellae recoveries with geometric means about 25% lower than, and significantly different from, those of the control agar. The remaining three lots of Brilliant Green Agar were generally unproductive.     

The possibilities of using variants of immunoenzyme analysis for detecting L-form Salmonella typhi bacteria in biological fluids

The possibility of using, on principle, the enzyme immunoassay (EIA) in different modifications for the detection of S. typhi L-forms in biological fluids (blood, urine) was established. The inhibiting variant of EIA showed the highest sensitivity: 1 ng/ml. The direct sandwich variant permitted the quantitative determination of the antigen of S. typhi L-form in the widest range of 20-500 ng/ml. The indirect enzyme immunometric variant permitted the detection of S. typhi L-forms with a sensitivity of 10(5) colony-forming units per ml only in urine.     

Detection of microbial variants of Salmonella typhi in the bone marrow of typhoid patients and carriers

The immunofluorescence test with the use of antisera to S. typhi and its L-forms permits the detection of the infective agent in bone marrow smears. This diagnostic method is particularly important in cases of carrier state in the latent phase and in differential diagnosis. As revealed in this study, the microbial variants of S. typhi, reacting with antiserum to its L-forms, are present in the bone marrow of carriers. They are localized, as a rule, on the membranes of lymphoid and erythroid cells, which probably leads to the formation of rosettes.     

Genetic Basis of Vi Antigen Expression in Salmonella paratyphi C

Analysis of hybrids formed in a cross between a Salmonella paratyphi C Hfr and an S. typhimurium recipient indicated that the structural genetic determinants of the S. paratyphi C Vi antigen are located closely adjacent to the mel determinant, between this marker and purA. A similar location was indicated for the structural genetic determinants of the S. typhi Vi antigen (the viaB locus) by the results of a mating in which a hybrid S. typhimurium Hfr bearing the S. typhi viaB determinants was used to transfer these genes to an S. typhimurium recipient. Mating experiments with a Vi-antigen-expressing S. typhi Hfr and an S. typhimurium hybrid recipient expressing the Vi antigen of S. paratyphi C yielded no recombinants in which loss of Vi antigen expression occurred, indicating that the chromosomal locus occupied by the genetic determinants of the S. paratyphi C Vi antigen is the same one at which, in S. typhi, the viaB genes reside. Introduction of a mutant S. typhi viaA gene into an S. typhimurium hybrid expressing the Vi antigen, as the consequence of prior receipt of the S. paratyphi C viaB determinants, resulted in that hybrid's loss of Vi antigen expression, demonstrating that the viaA determinant plays a role in Vi antigen expression in S. paratyphi C, as well as in S. typhi. Although the percentages of coinheritance of the viaB and mel determinants in the mating experiments suggested that their linkage is sufficiently close to allow cotransduction by P22, attempts to accomplish this with lysates prepared on S. typhimurium hybrids expressing either S. typhi or S. paratyphi C viaB determinants were not successful.     

Detection of lymphocytes binding erythrocytes conjugated with Salmonella antigens

Immune reagents for the detection of specific antigen-binding lymphocytes (ABL) with respect to different Salmonells antigens were developed. Rabbits were immunized with killed S. typhi and other salmonellae containing cross-reacting antigens, and the dynamics of the formation of ASL of each specificity was studied. Differences in the time of the appearance of ASL with receptors to thymus-independent (09, 12 or Vi) and thymus-dependent (Hd) antigens were studied. The relative content of ASL, determined with the use of immune reagents prepared from S. typhi antigens, was higher, on the whole, in rabbits immunized with S. typhi than in rabbits immunized with salmonellae containing one of cross-reacting antigens (S. enteritidis--09, 12; S. paratyphi C--Vi; S. virginia--Hd).     

Antibiotic sensitivity of S. typhi cultures that have reverted from L forms isolated from the blood of patients]

L-forms of bacteria were isolated in 18 out of 300 fever patients with diagnoses of typhoid-paratyphoid fever, grippe, virus respiration disease and others in the Diagnostic Department of an Infection Hospital during bacteriological tests of the blood. Among the cultures tested 13 were instable and reversed to the bacterial form. The type identification showed that only 9 revertants possessed properties characteristic of the typhoid fever microbes and belonged to S. typhi. Sensitivity of the typhoid fever revertants to levomycetin, sintomycin, streptomycin, pencillin and tetracycline was studied. The studies showed that the typhoid fever revertants from the L-forms isolated from the patients were sufficiently sensitive to levomycetin, sintomycin, penicillin and tetracycline. The minimum bactericidal concentrations of the above antibiotics ranged within 12.5--100 gamma/ml.     

Immunoelectrophoretic analysis of the antigenic composition of Salmonella typhi in the process of L transformation and reversion

In the study of the antigenic composition of S. typhi L-forms and their revertants were determined. The stable L-forms were characterized by profound disturbances in the synthesis of Vi- and H-antigens. After reversion to bacterial forms all the revertants under study showed the complete restoration of their bacterial antigenic structure.     

Proteomics analysis of the causative agent of typhoid fever

Typhoid fever is a potentially fatal disease caused by the bacterial pathogen Salmonella enterica serotype Typhi ( S. typhi). S. typhi infection is a complex process that involves numerous bacterially encoded virulence determinants, and these are thought to confer both stringent human host specificity and a high mortality rate. In the present study, we used a liquid chromatography-mass spectrometry (LC-MS)-based proteomics strategy to investigate the proteome of logarithmic, stationary phase, and low pH/low magnesium (MgM) S. typhi cultures. This represents the first large-scale comprehensive characterization of the S. typhi proteome. Our analysis identified a total of 2066 S. typhi proteins. In an effort to identify putative S. typhi-specific virulence factors, we then compared our S. typhi results to those obtained in a previously published study of the S. typhimurium proteome under similar conditions ( Adkins, J. N. et al. Mol. Cell. Proteomics 2006, 5, 1450-1461 ). Comparative proteomics analysis of S. typhi strain Ty2 and S. typhimurium strain LT2 revealed a subset of highly expressed proteins unique to S. typhi that were exclusively detected under conditions that are thought to mimic the infective state in macrophage cells. These proteins included CdtB, HlyE, and gene products of t0142, t1108, t1109, t1476, and t1602. The differential expression of T1108, T1476, and HlyE was confirmed by Western blot analysis. When our observations are taken together with the current literature, they suggest that this subset of proteins may play a role in S. typhi pathogenesis and human host specificity.     

Roles of the complement receptor type 1 (CR1) and type 3 (CR3) on phagocytosis and subsequent phagosome-lysosome fusion in Salmonella-infected murine macrophages

Abstract The receptors involved in the recognition of Salmonella typhimurium and S. typhi by murine macrophages were identified, and their relevance to phagosome-lysosome fusion was also investigated. Phagocytosis of S. typhimurium by murine macrophages was dependent on the opsonization with normal fresh serum, although the opsonin had no triggering activity in phagosome-lysosome fusion. In contrast, the opsonization of S. typhi with normal fresh serum efficiently triggered both phagocytosis and following phagosome-lysosome fusion. Anti-murine CR1 antibody suppressed phagocytosis of S. typhimurium by 36%, whereas anti-CR3 antibody, mannan, and advanced glycosylation endproducts (AGE)-BSA all failed to prevent phagocytosis of S. typhimurium , suggesting that CR1 may only contribute to the recognition of S. typhimurium and may possibly play a minor role. Other receptors involved may also influence the outcome phagocytosis in terms of phagosome-lysosome fusion. In the case of S. typhi , only anti-CR3 antibody significantly inhibited not only phagocytosis of S. typhi but also following phagosome-lysosome fusion. Treatment with K76COONa, an inhibitor of C3bINA (I factor), resulted in a marked inhibition of phagosomelysosome fusion in S. typhi -infected macrophages, although no significant inhibition was observed on phagocytosis of S. typhi . These results suggest that S. typhimurium and S. typhi may be recognized at least in part by CR1 and CR3, respectively, and that the recognition by CR3 but not CR1 is functionally associated with subsequent phagosomelysosome fusion in murine macrophages.     

Inactivation of mismatch repair overcomes the barrier to transduction between Salmonella typhimurium and Salmonella typhi.

P22 transduction of chromosomal genes from Salmonella typhimurium into Salmonella typhi occurs at a low frequency. Transduction of plasmids from S. typhimurium into S. typhi occurs at a frequency similar to that between S. typhimurium strains, indicating that the barrier to transduction of chromosomal genes is not due to an inability of P22 to inject DNA into S. typhi or a restriction endonuclease that rapidly degrades foreign DNA. Furthermore, transduction of mutS and mutL derivatives of S. typhi with chromosomal genes from S. typhimurium occurs efficiently. These results indicate that the transduction barrier is due to activity of the recipient mismatch repair system, which senses sequence divergence and disrupts heteroduplexes in favor of recipient sequences. Inactivation of the mismatch repair system allows P22 transduction to be used as an effective tool for constructing S. typhi-S. typhimurium hybrids.