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M M Levine

Publications and source records attributed to M M Levine.

At least 109 records · Page 6Linked to original sources
PubMed1995

Prevalence and association of the longus pilus structural gene (lngA) with colonization factor antigens, enterotoxin types, and serotypes of enterotoxigenic Escherichia coli.

Human enterotoxigenic Escherichia coli (ETEC) produces a plasmid-encoded type IV pilus termed longus (for long pilus). Regardless of the geographic origins of ETEC strains, the longus structural gene lngA was found to have the highest level of association with ETEC producing colonization factor antigen (CFA) CFA/II, followed by ETEC producing CFA/I and CFA/IV. ETEC bearing the less prevalent CFA/III and putative colonization factors and ETEC negative for CFA and putative colonization factor also contained lngA-related sequences. lngA was found in a considerable number of ETEC serotypes and was more often associated with ETEC producing heat-stable enterotoxins than with ETEC producing both heat-labile and heat-stable enterotoxins or heat-labile enterotoxin alone. lngA was found more often in strains isolated from children with diarrhea than in strains from healthy children, suggesting an association with intestinal disease. We conclude that longus is a widely distributed antigenic determinant in ETEC that is highly associated with known plasmid-encoded virulence factors, namely, CFAs and enterotoxins. A longus-specific probe may be a helpful epidemiological tool to assist in the identification of ETEC.

Bacterial Proteins↗
PubMed1995

Identification and cloning of a novel plasmid-encoded enterotoxin of enteroinvasive Escherichia coli and Shigella strains.

We have employed a molecular genetic approach to characterize the nature of enteroinvasive Escherichia coli (EIEC) enterotoxic activity, as previously observed in Ussing chambers (A. Fasano, B.A. Kay, R.G. Russell, D.R. Maneval, Jr., and M.M. Levine, Infect. Immun. 58:3717-3723, 1990). The screening of TnphoA mutants of EIEC yielded a single insertion mutant which had significantly reduced levels of enterotoxic activity in the Ussing chamber assay. DNA flanking the insertion was used as a probe to screen for EIEC cosmid clones which conferred secretogenic activity. Such screening resulted in the identification of two overlapping cosmid clones which elicited significant changes in mucosal short-circuit current (Isc). Subcloning and nucleotide sequence analysis of a DNA fragment from one of the cosmid clones led to the identification of a single open reading frame which conferred this enterotoxic activity. By DNA hybridization, this gene (designated sen for shigella enterotoxin) was found in 75% of EIEC strains and 83% of Shigella strains and was localized to the inv plasmid of Shigella flexneri 2457T. By PCR, a sen gene with 99.7% nucleotide identity was cloned and sequenced from 2457T. A deletion in the EIEC sen gene was constructed by allelic exchange, resulting in significantly lower rises in Isc than were elicited by the wild-type parent; however, significant enterotoxic activity remained in the sen deletion mutant. To purify the Sen protein, the gene was cloned into the multiple cloning site of the expression vector pKK223-3. Purification of the sen gene product yielded a protein with a molecular mass of 63 kDa which elicited rises in Isc in the Ussing chamber. We believe that the sen gene product may constitute all or part of a novel enterotoxin in EIEC and Shigella spp.

Amino Acid Sequence↗
PubMed1995

Immune responses to novel Escherichia coli and Salmonella typhimurium vectors that express colonization factor antigen I (CFA/I) of enterotoxigenic E. coli in the absence of the CFA/I positive regulator cfaR.

An asd-stabilized plasmid carrying enterotoxigenic Escherichia coli cfaABCE genes was constructed and called pJGX15C-asd+. Expression of colonization factor antigen I (CFA/I) by this plasmid occurs independently of the cfaABCE positive regulator cfaR in attenuated Salmonella delta aro delta asd strain H683 and nonpathogenic laboratory E. coli asd strain chi 6212. Oral immunization of mice with nonpathogenic E. coli chi 6212 (pJGX15C-asd+) does not elicit significant serum or mucosal responses against CFA/I. In contrast, oral immunization with a single dose of attenuated S. typhimurium H683(pJGX15C-asd+) elicits a 10(5)-fold increase in CFA/I-specific serum immunoglobulin G and significant elevation of CFA/I-specific immunoglobulin A-secreting B cells in the lamina propria, mesenteric lymph nodes, and spleen. Thus, only the Salmonella-CFA/I construct effectively delivered CFA/I to the inductive sites of the gut-associated and systemic lymphoid tissues.

Animals↗
PubMed1995

Attenuated live cholera vaccine strain CVD 103-HgR elicits significantly higher serum vibriocidal antibody titers in persons of blood group O.

Persons of blood group O are at increased risk of developing cholera gravis. In a randomized, placebo-controlled, double-blind safety-immunogenicity trial of live oral cholera vaccine CVD 103-HgR in 5- to 9-year-old Chilean children, vibriocidal antibody seroconversion (74% overall) did not differ by blood group. However, the reciprocal geometric mean titer (GMT) in blood group O vaccines (GMT = 486) was higher than that in non-O vaccines (GMT = 179) (P < 0.02).

ABO Blood-Group System↗
PubMed1995

Safety and immunogenicity of a live oral bivalent typhoid fever (Salmonella typhi Ty21a)-cholera (Vibrio cholerae CVD 103-HgR) vaccine in healthy adults.

The safety and immunogenicity of the live oral attenuated vaccine strains vibrio cholerae CVD 103-HgR and Salmonella typhi Ty21a were evaluated alone or in a combined bivalent formulation in four groups composed of 185 healthy European adults. All presentations were well tolerated. The serum anti-S. typhi lipopolysaccharide immunoglobulin G and immunoglobulin A antibody responses were comparable for all groups (66 to 72% seroconversion). The serum vibriocidal antibody seroconversion rate ranged from 78 to 92.5% (P > 0.05) among the groups. However, the peak and geometric mean vibriocidal antibody titers were significantly higher (P < 0.005) in the groups which received the bivalent formulation along with two doses of Ty21a than in the group which received CVD 103-HgR followed by two doses of killed Escherichia coli K-12 placebo. The ingestion of a placebo shortly after CVD 103-HgR may have suppressed the magnitude of the immune response. These findings demonstrate the feasibility of producing multivalent live oral attenuated vaccines.

Administration, Oral↗
PubMed1995

Sorbitol-negative phenotype among enterohemorrhagic Escherichia coli strains of different serotypes and from different sources.

Enterohemorrhagic Escherichia coli (EHEC) strains detected with DNA probes (for virulence plasmid and Shiga-like toxins) from subjects with hemolytic-uremic syndrome (n = 19) or diarrhea (n = 41) or asymptomatic carriers (n = 29) were examined for sorbitol fermentability, as were enterotoxigenic (n = 40), enteropathogenic (n = 40), and enteroinvasive (n = 40) E. coli and urinary tract infection (n = 40) strains and normal flora E. coli strains (n = 40). Sorbitol negativity was common only in EHEC, particularly among strains from severe clinical infections. All 19 EHEC strains from patients with hemolytic-uremic syndrome, irrespective of O:H serotype or Shiga-like toxin genotype, were sorbitol negative.

Carrier State↗
PubMed1995

Shigella enterotoxin 1: an enterotoxin of Shigella flexneri 2a active in rabbit small intestine in vivo and in vitro.

Culture filtrates of Shigella flexneri 2a strain M4243 grown in iron-depleted medium, caused significant fluid accumulation in rabbit ileal loops. Also, when tested in Ussing chambers, a greater rise in potential difference and short circuit current was seen with such filtrates compared with the medium control. Analogous filtrates from two M4243 derivatives lacking the 140-MD invasiveness plasmid (either M4243avir or BS103) retained 60-65% of the wild-type enterotoxic activity. Ultrafiltration and gel exclusion size fractionation of M4243 filtrate revealed that the activity was approximately 60 kD. SDS-PAGE performed on this fraction showed 18 bands, 5 of which reacted with human convalescent sera. Genes encoding this enterotoxin, named ShET1 for Shigella enterotoxin 1, were cloned from the S. flexneri 2a chromosome, and two separate open reading frames of 534 and 186 bp were sequenced. These observations suggest that S. flexneri 2a elaborates two distinct enterotoxins: ShET1, encoded by genes located on the chromosome, and ShET2, encoded by a gene on the 140-MD invasiveness plasmid. ShET1, which is composed of two distinct subunits and is elaborated in vivo, where it elicits an immune response, may be important in the pathogenesis of diarrheal illness due to S. flexneri 2a.

Amino Acid Sequence↗
PubMed1995

Vibrio cholerae CVD103-HgR live oral attenuated vaccine: construction, safety, immunogenicity, excretion and non-target effects.

In many controlled studies, CVD103-HgR has been shown to be safe and immunogenic and to offer a significant degree of protection against experimental cholera after a single dose. Its minimal excretion and limited ability to compete and survive in various ecosystems indicate that this strain presents little risk to the environment. Furthermore, the potential of CVD103-HgR to regain virulence by acquisition of the CT A or LT A gene is extremely remote even under optimal conditions. Therefore, CVD103-HgR possesses those traits desired in a live oral attenuated vaccine produced by recombinant DNA technology.

Adult↗
PubMed1995

[In vitro susceptibility of enterohemorrhagic Escherichia coli to 11 antimicrobials. Relationship between antibiotic resistance and toxigenic genotype].

Enterohemorrhagic E coli (EHEC) has been recognized as the main etiologic agent of hemorrhagic colitis and hemolytic uremic syndrome (HUS). The usefulness of antibiotic treatment in patients with EHEC infections is a matter of current debate. Knowledge on EHEC antimicrobial susceptibility patterns in different geographic areas is important for both treatment considerations and for strain characterization. We studied by diffusion disk agar technique the antibiotic susceptibility of 83 EHEC strains obtained from stools of patients with HUS or diarrhea. Eleven antimicrobials were tested (ampicillin, cotrimoxazole, tetracycline, chloramphenicol, furazolidone, gentamycin, amikacin, ciprofloxacin, erythromycin, vancomycin, and metronidazol). Resistant strains by disk diffusion were tested for MIC (mg/ml) by agar dilution. SLT-I and SLT-II were detected with specific biotinylated gene probes. All 83 strains were susceptible to furazolidone, ciprofloxacin, gentamycin and amikacin. Resistance was detected to tetracycline 4%, chloramphenicol 5%, cotrimoxazole 24% and ampicillin 25%. As expected for EHEC strains all were resistant to erythromycin, vancomycin, and metronidazol. Resistant strains were significantly more common in non toxigenic and SLT-I producing strains (p = 0.01). Resistant strains were similarly distributed among patients who had diarrhea only and those who developed HUS (p = 0.3). In Chile, resistant EHEC strains seem to be more common and of different genotypes than those reported in more developed countries. Regional differences of EHEC antibiotic susceptibility patterns indicate a need for continuous monitoring, specially if antibiotics prove to be useful in disease prevention.

Child, Preschool↗
PubMed1995

A review of the current status of enteric vaccines.

Much progress has been made in developing vaccines against the most important enteric infections. Two new vaccines against typhoid fever (oral Ty21a and parenteral Vi polysaccharide) have been licensed in many countries. Newer, more sophisticated typhoid vaccines undergoing clinical testing include recombinant attenuated Salmonella typhi strains and Vi polysaccharide-carrier-protein conjugate vaccines. Two inactivated oral cholera vaccines, consisting of inactivated Vibrio cholerae O1 bacteria alone or in combination with B subunit of cholera toxin, each conferred 50-53% protection over three years in a field trial in Bangladesh where subjects were immunized with a three-dose regimen. An engineered live oral cholera vaccine, strain CVD 103-HgR, has been shown in extensive clinical trials to be well tolerated by children and adults in less developed countries and highly immunogenic following administration of just a single oral dose; a large-scale field trial of the efficacy of this vaccine is underway. In experimental challenge studies in volunteers, a single dose of CVD 103-HgR confers significant protection against challenge with wild-type V. cholerae O1 of either classical or El Tor biotype and either Inaba or Ogawa serotype. Several candidate vaccines against Shigella and enterotoxigenic Escherichia coli are in clinical trials. A multivalent rotavirus vaccine (rhesus reassortant vaccine) is undergoing extensive field testing in developed and less developed countries.

Adjuvants, Immunologic↗
PubMed1994

Structural studies of the O-antigenic polysaccharides of Escherichia coli O3 and the enteroaggregative Escherichia coli strain 17-2.

The polysaccharide part of the lipopolysaccharide isolated from an enteroaggregative Escherichia coli isolated from a young child with diarrhoea in Santiago, Chile (strain 17-2), has been investigated. Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H-NMR and 13C-NMR spectroscopies revealed that the polysaccharide is composed of pentasaccharide repeating units. The structure of the repeating unit of E. coli strain 17-2 O-polysaccharide is: [formula: see text] The structure of the O-polysaccharide from E. coli O3 was shown to be identical to that of E. coli strain 17-2 by sugar and methylation analyses and by 1H-NMR and 13C-NMR spectroscopies.

Carbohydrate Sequence↗
PubMed1994

Changes in human ecology and behavior in relation to the emergence of diarrheal diseases, including cholera.

Human populations throughout the world can be found in diverse conditions. A proportion of the population of developing countries lives in deprived conditions characterized by ramshackle housing, lack of piped water and sanitation, and widespread fecal contamination of the environment. Enteric infections, particularly due to bacterial pathogenes, are readily transmitted under these circumstances. In contrast, the majority of inhabitants of industrialized countries live in a sanitary environment that generally discourages the transmission of enteric pathogenes, particularly bacteria. In both these ecologic niches, changes in human ecology and behavior are leading to the emergence of certain enteric infections. Relevant factors in developing areas include urbanization (leading to periurban slums), diminished breastfeeding, and political upheaval that results in population migrations. In industrialized areas, large-scale food production (e.g., enormous poultry farms), distribution, and retailing (e.g., fast-food chains) create opportunities where widespread and extensive outbreaks of food-borne enteric infection can ensue if a breakdown in food hygiene occurs.

Behavior↗
PubMed1994

Adaptive acid tolerance response by Salmonella typhi and candidate live oral typhoid vaccine strains.

The data presented here demonstrate that Salmonella typhi is capable of expressing an acid tolerance response (ATR) and that effective induction of this response (in nutrient-rich medium) occurs at pH 5.0 in anaerobic conditions. The candidate live oral S. typhi vaccine strains made by precise genetic methods and which carry auxotrophic mutations were CVD 906 (carries defined attenuating deletion mutations: delta aroC, delta aroD), CVD 908 (carries defined attenuating deletion mutations: delta aroC, delta aroD), 541Ty (carries attenuating deletion mutations: aroA, purA), and galE, Vi-negative (via) strain EX462. All generate an effective ATR. In contrast, nitrosoguanidine-derived live oral typhoid vaccine strain Ty21a only weakly expresses acid tolerance. This further demonstrates that the non-specific mutagenesis process used to produce Ty21a affects genetic loci outside the intended target genes for mutagenesis, galE and via, and further emphasizes the importance of using precise genetic techniques when developing live oral S. typhi vaccines.

Acids↗
PubMed1994

Kinetics of the vibriocidal antibody response to live oral cholera vaccines.

The best correlate of protection against cholera is the level of serum vibriocidal antibodies, which are primarily directed against the O antigen of Vibrio cholerae O1 and lyse V. cholerae in the presence of complement. We established the timing of peak vibriocidal antibody response using sera from safety/immunogenicity studies of live oral cholera vaccines CVD 103-HgR, CVD 103-HgR2 and CVD 110 among immunologically naive North Americans and Colombians. The serum reciprocal vibriocidal antibody titre was consistently higher 10 days postimmunization than on either day 7 or day 14. This study suggests that recent phase 2 studies of CVD 103-HgR may have underestimated the peak vibriocidal titre by collecting serum on days 7-8 rather than on day 10; future studies of live oral cholera vaccines should take these results into account to obtain the best measurement of peak immunological responses. Because of the rapid drop in vibriocidal antibody titres about 2 weeks after immunization, care must be exercised in comparing immunogenicity of different vaccine candidates, formulations, dosage levels and immunization schedules.

Administration, Oral↗
PubMed1994

Salmonella typhi vaccine strain CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum: strain construction and safety and immunogenicity in humans.

rcsp, encoding amino acids 21-398 of Plasmodium falciparum circumsporozoite protein (CSP), under control of tacP was integrated into the chromosomal delta aroC locus of attenuated delta aroC, delta aroD Salmonella typhi CVD 908. By immunoblot and ELISA, rCSP expression was greater from a multicopy plasmid than from the single chromosomal gene. CVD 908 omega (delta aroC1019::tacP-rcsp) was well tolerated by 10 volunteers who were fed two doses of 5 x 10(7) organisms 8 days apart. Seven subjects excreted the vaccine strain for 1-3 days. All subjects developed serologic responses to O and H antigens of the live vector, whereas 3 vaccinees responded to the foreign antigen: 1 developed an 80-fold rise in serum anti-sporozoite antibody, another had a 4-fold rise in antibody to a recombinant portion of CSP (residues 309-345), while a third vaccinee developed CSP-specific CD8+ cytotoxic T lymphocyte activity. This is the first report of attenuated S. typhi eliciting a human serologic or a cytotoxic T lymphocyte response to a foreign protein. Improved foreign gene expression should enhance immunogenicity.

Administration, Oral↗
PubMed1994

Cytokine production patterns and lymphoproliferative responses in volunteers orally immunized with attenuated vaccine strains of Salmonella typhi.

New recombinant strains of attenuated Salmonella typhi used as live oral vaccines elicit potent immune responses. This study examined the patterns of cytokine production and proliferation to specific S. typhi antigens in subjects orally immunized with attenuated S. typhi vaccines CVD 906, CVD 908, and CVD 908 expressing the circumsporozoite protein of Plasmodium falciparum. After immunization, sensitized lymphocytes were found in subjects' blood that exhibited significantly increased proliferative responses and interferon-gamma production to purified S. typhi flagella when compared with preimmunization levels. Significant negative correlations were observed between interleukin-4 production and both interferon-gamma production and proliferation to S. typhi flagella. These results demonstrate that oral immunization with attenuated S. typhi strains alone or with those carrying a foreign gene elicits strong systemic cell-mediated immunity to purified S. typhi antigens, including the production of cytokines compatible with T1-type responses.

Animals↗
PubMed1994

Construction and characterization of isogenic O-antigen variants of Salmonella typhi.

A 7.5 kb KpnI-generated fragment, from within the rfb cluster of Salmonella typhimurium LT2 that encodes abequose synthase (the rfbJ gene) which is necessary for O4 antigen synthesis, and flanking sequences, was inserted into a suicide vector. Using allelic exchange techniques, these rfb sequences of S. typhimurium were integrated into the rfb clusters of wild-type Salmonella typhi Vi-positive strain ISP 1820 (i.e. serotype O9,12; Vi+; H-d), S. typhi Vinegative strain H400 (i.e. serotype O9,12; Vi-; H-d), and a double aro mutant of S. typhi ISP 1820, strain CVD 906, resulting in the isolation of strains H325, H404 and CVD 906-O4, respectively. Immunoblot analysis of lipopolysaccharide (LPS) purified from H325, H404 and CVD 906-O4 demonstrated that these strains express the O4 antigen (an abequose residue) in place of the O9 antigen (a tyvelose residue) in the LPS molecule. Hence, the serotype of H325 is O4,12; Vi+; H-d and the serotype of H404 is O4,12; Vi-; H-d. DNA hybridization analysis of chromosomal DNA from H325, H404 and CVD 906-O4 confirmed that a precise recombination event within sequences flanking rfbSE of S. typhi (which encodes the enzymes necessary for cytidine diphosphate-tyvelose synthesis) resulted in replacement of rfbSE with rfbJ (which encodes abequose synthase and is necessary for O4 synthesis) of S. typhimurium in strains H325, H404 and CVD 906-O4. The resistance of each strain to the bactericidal effects of guinea-pig serum (GPC) was assessed. Whereas ISP 1820, H325 and H404 exhibit similar resistance patterns in GPC, strain H400 is sensitive to the bactericidal effects of GPC.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗