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In Vitro and In Vivo Studies of Streptomycin-Dependent Cholera Vibrios
Abstract
Streptomycin-dependent cholera vibrio strains were derived from Inaba, Ogawa, and NAG vibrios by the method of Mel. These phenotypes grew more slowly and attacked fermentable substances after a longer period of time than the streptomycin-sensitive parent strains. Rabbits injected with streptomycin-sensitive strains and their streptomycin-dependent forms showed homologous agglutinin production. Patas monkeys fed with 10(9) streptomycin-dependent strains shed them for 1 to 2 days without ill effect, whereas the same number of streptomycin-independent organisms caused disease. The possibility of the application of multiple doses of streptomycin-dependent organisms in oral immunization against cholera was considered.
... It should be noted that there is some ambiguity as to whether NCV 10125 is the same as the ATCC 25872 strain deposited by Felsenfeld (also named '280 NAG' in ATCC records). NCV 10125 was originally classified as a Heiberg group II Vibrio [28], and the description of 280 NAG is that of a Heiberg group I strain [29]. Two other V. cholerae were deposited simultaneously by Felsenfeld into ATCC [29]: ATCC 25873 (also named '281 NAG') and ATCC 25874 ('123 NAG'). ...
... NCV 10125 was originally classified as a Heiberg group II Vibrio [28], and the description of 280 NAG is that of a Heiberg group I strain [29]. Two other V. cholerae were deposited simultaneously by Felsenfeld into ATCC [29]: ATCC 25873 (also named '281 NAG') and ATCC 25874 ('123 NAG'). Like ATCC 25872, both were originally obtained from Aldová [30,31]. ...
... In addition to their historical and epidemiological significance, the laboratory strains derived from both the Sudanese and Czechoslovakian outbreaks, V52 and ATCC 25872 respectively (Table 1) [29,37], are important reference strains used in V. cholerae experimental research. For instance, ATCC 25872 was used in the original characterization of the TCP-encoding VPI element (now renamed VPI-I), work which showed that V. cholerae O37 can harbour the VPI-I genomic island encoding the CTX bacteriophage receptor and thereby become toxigenic through lysogenic conversion [4]. ...
Between 1965 and 1968, outbreaks of cholera in Sudan and former Czechoslovakia provoked considerable public health concern. These still represent important historical events that need to be linked to the growing genomic evidence describing the aetiological agent of cholera, Vibrio cholerae. Whilst O1 serogroup V. cholerae are canonically associated with epidemic and pandemic cholera, these events were caused by a clone of toxigenic V. cholerae O37 that may be more globally distributed than just to Europe and North Africa. Understanding the biology of these non-O1 strains of V. cholerae is key to understanding how diseases like cholera continue to be globally important. In this article, we consolidate epidemiological, molecular and genomic descriptions of the bacteria responsible for these outbreaks. We attempt to resolve discrepancies in order to summarize the history and provenance of as many commonly used serogroup O37 strains as possible. Finally, we highlight the potential for whole-genome sequencing of V. cholerae O37 isolates from strain collections to shed light on the open questions that we identify.
... The use of genetic techniques to derive isogenic mutants for studies of pathogenesis has been successfully applied to Vibrio cholerae by a number of researchers (4,5,12,16,18,19,28,29; D. Schneider, S. Ingram, and C. Parker, Abstr. Annu. ...
... With V. cholerae, the primary methodology used has been selection of mutants with specific defects from mutagen-induced cultures and examination of these mutants in an animal model for alterations in disease-inducing ability. Application of this genetic approach to studies of cholera pathogenesis has indicated that toxin production (18,19), motility (16), smooth lipopolysaccharide structure (15), neuraminidase production, and nutritional restrictions, (e.g., streptomycin dependence [5,12]) affect the disease-inducing capacity of V. cholerae. In addition, several other, more complex mutants have been shown to be avirulent (4,29). ...
... Baselski and C. D. Parker, unpublished observation). Apparently, purine auxotrophs are somehow metabolically restricted in the host and are therefore similar to streptomycin-dependent avirulent mutants (5,12). It is interesting that the avirulent mutants isolated by Bhaskaran and Sinha (4) are also Pur-. ...
Mutants of Vibrio cholerae was isolated on the basis of reduced ability to induce diarrhea in orally challenged infant mice. Nitrosoguanidine-treated clones were screened for low fluid accumulation ratios in individual mice, and presumptive mutants were confirmed in additional mouse tests. Mutants were examined for alterations in phage type, motility, toxin production, proteolytic activity, neuraminidase production, amylase production, morphology, growth requirements, carbohydrate fermentations, in vitro growth patterns, and cell surface alterations. The types of mutants found included several with previously recognized virulence-associated markers (rough, nonmotile, toxin deficient, protease deficient); several types with pleiotropic alterations (cell morphology, decreased extracellular products); and several with no previously recognized virulence-deficient phenotype (purine requiring, cell surface altered, rapid death in vitro, no defect found). Dose-response kinetics showed that most mutants could provoke diarrhea if given in 100-fold greater numbers than the dose used for screening. Recovery of viable organisms from the gut late in infection showed reduction of survival and/or multiplication capacity for the mutants, with variation in the degree of reduction for the different classes.
... doi:10.1371/journal.ppat.0030081.g004 test this prediction, we used chitin-induced natural transformation to transform the A1552 O1 serogroup strain with gDNA from an O37 serogroup strain (ATCC25872; [34]) that caused a localized outbreak of diarrhea in Czechoslovakia in 1965 [35]. We then used an O1-specific lytic phage to select transformants of the A1552 strain that lack the O1 determinant, using the phage selection method discussed above. ...
... Bacterial strains and growth conditions. Bacterial strains used in this study are V. cholerae A1552 (serogroup O1 El Tor [53]), MO10 (serogroup O139 [21,54]), VCO139-Kan (MO10 with Kan R cassette inserted between wbfB and wbfA), O139-KanDIS1358 (VCO139-Kan, deletion in IS1358), and ATCC25872 (serogroup O37 [34,35]). V. cholerae was grown either in LB medium or in defined artificial seawater medium. ...
The environmental reservoirs for Vibrio cholerae are natural aquatic habitats, where it colonizes the chitinous exoskeletons of copepod molts. Growth of V. cholerae on a chitin surface induces competence for natural transformation, a mechanism for intra-species gene exchange. The antigenically diverse O-serogroup determinants of V. cholerae are encoded by a genetically variable biosynthetic cluster of genes that is flanked on either side by chromosomal regions that are conserved between different serogroups. To determine whether this genomic motif and chitin-induced natural transformation might enable the exchange of serogroup-specific gene clusters between different O serogroups of V. cholerae, a strain of V. cholerae O1 El Tor was co-cultured with a strain of V. cholerae O139 Bengal within a biofilm on the same chitin surface immersed in seawater, and O1-to-O139 transformants were obtained. Serogroup conversion of the O1 recipient by the O139 donor was demonstrated by comparative genomic hybridization, biochemical and serological characterization of the O-antigenic determinant, and resistance of O1-to-O139 transformants to bacteriolysis by a virulent O1-specific phage. Serogroup conversion was shown to have occurred as a single-step exchange of large fragments of DNA. Crossovers were localized to regions of homology common to other V. cholerae serogroups that flank serogroup-specific encoding sequences. This result and the successful serogroup conversion of an O1 strain by O37 genomic DNA indicate that chitin-induced natural transformation might be a common mechanism for serogroup conversion in aquatic habitats and for the emergence of V. cholerae variants that are better adapted for survival in environmental niches or more pathogenic for humans.
... Strain O37-Kan is a derivative of ATCC25873 [5,6]. The kanamycin resistant gene was inserted between ORF6 and ORF7 in the O37 specific region [7]. ...
Background
A fundamental clinical and scientific concern is how lytic bacteriophage, as well as antibiotics, impact diagnostic positivity.
Methods
Cholera was chosen as a model disease to investigate this important question. Patients with diarrheal disease were enrolled at two remote hospitals in Bangladesh. Diagnostic performance was assessed as a function of lytic bacteriophage detection, as well as exposure to the first-line antibiotic azithromycin detected by mass spectrometry.
Results
Among diarrheal samples positive by nanoliter quantitative PCR for Vibrio cholerae (n=78/849), the odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by 89% (OR 0.108; 95%CI 0.002-0.872) and 87% (OR 0.130; 95%CI 0.022-0.649) when lytic bacteriophage were detected, respectively. The odds that a rapid diagnostic test (RDT) or qPCR was positive was reduced by more than 99% (OR 0.00; 95% CI: 0.00-0.28) and 89% (OR 0.11; 95% CI: 0.03-0.44) when azithromycin was detected, respectively.
Conclusions
Estimations of cholera burden may improve by accommodating for the negative effect of antimicrobial exposure on diagnostic positivity. Furthermore, the findings herein challenge our current approach to interpreting and developing bacterial diagnostics given variable rates of lytic bacteriophage and antibiotic exposure.
Vibrio cholerae, the causative agent of cholera, is a model organism for studying virulence regulation, biofilm formation, horizontal gene transfer, and the cell-to-cell communication known as quorum sensing (QS). As in any research field, discrepancies between data from diverse laboratories are sometimes observed for V. cholerae. Such discrepancies are often caused by the use of diverse patient or environmental isolates. In this study, we investigated the inability of a few laboratories to reproduce high levels of natural transformation, a mode of horizontal gene transfer that is specifically induced on chitinous surfaces. This irreproducibility was mostly related to one specific isolate of V. cholerae: the O1 El Tor C6706 strain. C6706 was previously described as QS proficient, an important prerequisite for the induction of natural competence for transformation. To elucidate the underlying problem, we collected seven isolates of the same C6706 strain from different research laboratories in North America and Europe and compared their phenotypes. Importantly, we observed a split response with respect to QSrelated gene expression, including chitin-induced natural competence and type VI secretion (T6S). While approximately half of the strains behaved as reported for several other O1 El Tor pandemic isolates that are commonly studied in the laboratory, the other half were significantly impaired in QS-related expression patterns. This impairment was caused by a mutation in a QS-related gene (luxO). We conclude that the circulation of such QS-impaired wild-type strains is responsible for masking several important phenotypes of V. cholerae, including natural competence for transformation and T6S.
Natural competence for transformation is a common mode of horizontal gene transfer and contributes to bacterial evolution. Transformation occurs through the uptake of external DNA and its integration into the genome. Here we show that the type VI secretion system (T6SS), which serves as a predatory killing device, is part of the competence regulon in the naturally transformable pathogen Vibrio cholerae. The T6SS-encoding gene cluster is under the positive control of the competence regulators TfoX and QstR and is induced by growth on chitinous surfaces. Live-cell imaging revealed that deliberate killing of nonimmune cells via competence-mediated induction of T6SS releases DNA and makes it accessible for horizontal gene transfer in V. cholerae.
Copyright © 2015, American Association for the Advancement of Science.
Au cours du deuxième semestre 1971, treize souches de vibrions cholériques, biotype El Tor, sérotype Inaba, ont été isolées par le Service de bactériologie-virologie du Centre hospitalo-universitaire de Dakar, au Sénégal (Pr agr. Mme M. Castets), dans des selles de malades.
Nous avons étudié la sensibilité aux antibiotiques de ces treize souches; nous en rapportons les résultats dans cette étude.
Spontaneous and chemically induced mutants with reduced ability to produce cholera enterotoxin (choleragen) as an extracellular protein were isolated from Vibrio cholerae strains 569B Inaba, a classical cholera vibrio, and 3083-2 Ogawa, an El Tor vibrio. By qualitative and quantitative immunological assay in vitro such mutants could be separated into different classes characterized either by production of no detectable choleragen (tox minus), or of small quantities of extracellular choleragen, or of large quantities of cell-associated choleragen but little extracellular choleragen. Analysis of proteins in concentrated culture supernates by electrophoresis in polyacrylamide gels showed that cultures from tox minus strains lacked proteins with electrophoretic mobilities corresponding with choleragen or the spontaneously formed toxoid (choleragenoid). Infant rabbits infected with the tox minus strains remained asymptomatic or developed milder symptoms than rabbits infected with the tox+ parental strains. When symptoms of cholera developed after inoculation with tox minus mutants, detectable numbers of tox+ revertants could be isolated from the intestines of the infected animals. Two tox minus strains, designated M13 and M27, caused no sumptoms and showed no evidence of reversion to tox+ during single passage in infant rabbits, and mutant M13 also remained avirulent and stably tox minus during six cycles of serial passage in infant rabbits. Strains M13 and M27 were also noncholeragenic in acult rabbit ileal loops. Quantitative cultures of the intestines from infected infant rabbits demonstrated that the avirulent mutant M13 can multiply in vivo and can persist in the intestinal tract for at least 48 h.
The polyphenylalanine chain, made by poly U-directed† synthesis in the cell-free system from Escherichia coli, is studied. This nascent polypeptide chain is bound to the 50 s subunit of the 70 s ribosome. Furthermore, the growing chain is covalently linked to an S-RNA molecule. The bond between the polypetide chain and the S-RNA is similar to that in amino acyl S-RNA but is an order of magnitude more stable against alkaline hydrolysis or hydroxylamine treatment. When puromycin releases the chain from the ribosome, it breaks the bond to the S-RNA. This terminal S-RNA is used as an end group method to measure the molecular weight of the nascent chain. Chains of the order of 40 amino acids are made, and new chains are initiated throughout the reaction.The behavior of the nascent proteins found after cell-free synthesis in E. coli differs from that of the polyphenylalanine only in that the bound nascent proteins stabilize the 70 s ribosome to a higher degree and bind less tightly to the 50 s subunit at low magnesium ion concentrations.
The poly U-directed† synthesis of polyphenylalanine in the cell-free system from Escherichia coli is used as a model system in which to investigate the interaction of messenger RNA with ribosomes. It is shown that both the 50 s and the 30 s ribosome are necessary for polypeptide synthesis. The ribosomes accept the messenger RNA under conditions in which the dominant form is the 70 s particles, but the particles involved will dimerize more readily than the rest of the 70 s particles.All of the synthetic activity of the poly U-ribosome mixture appears as a rapidly sedimenting complex, 140 to 200 s. This active complex depends upon RNA for its integrity and contains an amount of poly U consistent with one molecule for several ribosomes. Furthermore, all of the synthetic capacity of the usual crude extract from E. coli is in the form of rapidly sedimenting complexes in the 100 to 200 s range.
A bacteriophage-typing scheme for Vibrio cholerae has been developed on the basis of the pattern of susceptibility of V. cholerae strains to four groups of freshly isolated cholera bacteriophages. Some 4066 strains of V. cholerae isolated in Calcutta during the period 1955-61 have been classified into seven types and subtypes. Less than 1% of the strains were untypable. No correlation was found to exist between the phage-types and serological types of V. cholerae. In lysogenic strains, however, a correlation was observed between the phage-types of V. cholerae and their lysogenic phages. Groups of infection deriving from a single source were found to be caused by single phage-types. The author also discusses the practical value of the phage-typing scheme for the epidemiological investigation of cholera.
: A streptomycin-dependent mutant of Salmonella typhosa injected live in rabbits stimulated the production of 0 antibodies. Sera from these rabbits protected mice against challenge with virulent S. typhosa. Mutation to the dependent state did not result in loss of antigenicity. Streptomycin dependency of the mutant was demonstrated in vivo in the mouse. Antibiotic treated mice succumbed to challenge with dependent bacilli, apparently because of presence of lethal quantities of endotoxin produced by multiplication of the mutant in the presence of streptomycin.
There is no reason to doubt that textbooks of immunology are correct when they teach that the immune response to antigenic stimulation is a reaction in which all the lymphatic tissues of the body have their share. However, work initiated shortly after the turn of the century and gaining increasing momentum in recent years has brought to light that nearly all individual tissues and organs, when confronted with an antigen, display the capacity to set up their own local immune response which is largely independent of the systemic reaction.
Post-irradiation protein and RNA synthesis were studied in Escherichia coli cells exposed to ultraviolet light doses ranging from 250 to 5000 erg/mm2. The following conclusions were drawn. (1) In u.v.-irradiated cells abnormal, shortened polypeptide chains are synthesized in addition to normal ones, presumably on shortened messenger RNA molecules which are reportedly produced when transcription is prematurely terminated at the site of u.v. lesions in DNA. (2) In u.v.-irradiated cells the number of polypeptide chains synthesized per unit time is also reduced, which is attributed to a reduced rate of messenger RNA synthesis. (3) The release of nascent proteins from polysomes is not greatly delayed in u.v.-irradiated cells, indicating that the termination of protein synthesis at the 3′-end of the messenger RNA molecule does not necessarily require a special termination sequence. (4) After u.v. irradiation, polysomes contain defective ribosomal subunits.
Results of tests made in 1964 confirm the previous findings that live oral vaccine, prepared from streptomycin-dependent strains of shigellae, confers a strong, type-specific protection against acute bacillary dysentery. This vaccine did not reduce the carrier rate of shigellae. Observations on soldiers treated with a vaccine of Shigella flexneri serotypes 2a and 3 combined revealed no antagonizing effects from the type 3 component upon the protective effect of the 2a component contained in the same vaccine.
In view of reports of inconsistent results with the haemolytic test for identification of the El Tor biotype of Vibrio cholerae, a study was made of the experimental variables involved in order to achieve more precise standardization of the procedure. It was found that different types of media and different incubation times of a culture in a particular medium exerted a profound influence on the results. The authors describe the materials and conditions for performance of a reliable haemolytic test, and consider that, when properly performed, the haemolytic test should be a valuable epidemiological tool.The persistence of haemolysin in cultures of strains from the El Tor Quarantine Station during incubation is in contrast to its early disappearance in cultures of more recent isolates. Evidence is presented that the haemolytic activity of a strain may become altered during subculture, since rugose variants of recent strains resemble the old El Tor strains in haemolysin persistence.
Since 1922 when Wu proposed the use of the Folin phenol reagent for
the measurement of proteins (l), a number of modified analytical pro-
cedures ut.ilizing this reagent have been reported for the determination
of proteins in serum (2-G), in antigen-antibody precipitates (7-9), and
in insulin (10).
Although the reagent would seem to be recommended by its great sen-
sitivity and the simplicity of procedure possible with its use, it has not
found great favor for general biochemical purposes.
In the belief that this reagent, nevertheless, has considerable merit for
certain application, but that its peculiarities and limitations need to be
understood for its fullest exploitation, it has been studied with regard t.o
effects of variations in pH, time of reaction, and concentration of react-
ants, permissible levels of reagents commonly used in handling proteins,
and interfering subst.ances. Procedures are described for measuring pro-
tein in solution or after precipitation wit,h acids or other agents, and for
the determination of as little as 0.2 y of protein.
Riboprotein particles from E. coli
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Outline for the isolation and identification of V. cholerae
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Dept. of Health Education and Welfare, Atlanta.
Media, tests, reagents
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