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Criteria for the standardization of Salmonella mutagenicity tests: results of a collaborative study. IV. Relationship between the number of his- bacteria plated and number of his+ revertants scored in the Salmonella mutagenicity test.

Five laboratories participated in a joint ring study to investigate the role of bacterial cell number in the Salmonella mutagenicity test. A strictly standardized protocol, using sodium azide and TA 1535, was developed and employed to test the mutagenicity of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) with different dilutions of Salmonella typhimurium TA-100 and TA-1535 cultures. All laboratories detected the mutagenic activity of sodium azide with only a 2-fold variation of test results. For MNNG the interlaboratory variation was approximately 5-fold. Decreasing numbers of test bacteria employed resulted in lower numbers of MNNG-induced revertants in all laboratories. The number of preexisting revertants decreased in direct proportion to the reduced cell content, whereas the number of spontaneous revertants was not as greatly affected. A critical amount of test bacteria was required in order to obtain numbers of induced revertants which were equal to twice the number of spontaneous revertants. Two evaluation parameters which may be employed to describe the mutagenicity of a compound are compared.

Animals↗

Intracellular survival of Brucella abortus, Mycobacterium bovis BCG, Salmonella dublin, and Salmonella typhimurium in macrophages from cattle genetically resistant to Brucella abortus.

Peripheral blood monocyte-derived macrophages were obtained from a herd of cows selected, bred, and confirmed as resistant or susceptible by in vivo challenge of Brucella abortus Strain 2308. The ability to control in vitro intracellular bacterial replication of B. abortus Strain 2308, Mycobacterium bovis Bacillus Calmette-Guerin (BCG) Montreal Strain 9003, Salmonella dublin Strain 5631, and Salmonella typhimurium Strain 14028 was evaluated in a bactericidal assay. The macrophages from resistant cattle were significantly superior (P < 0.05) in controlling intracellular growth of B. abortus, M. bovis BCG, S. dublin but not of S, typhimurium than macrophages from susceptible animals. Controls of all four pathogens correlated strongly with each other in resistant or susceptible macrophages. Data from resistant cattle had a tighter grouping than that of susceptible cattle, while data from susceptible cattle overlapped considerably with data from resistant animals. Therefore, this assay was considered a phenotypic marker of the resistant trait. For each bacterial species a percent bacterial survival value was used as a cut-off point to designate animals as resistant or susceptible. These data were compared with the in vivo challenged resistant or susceptible classification by using the Chi-square analyses. A cut-off point of 70 percent bacterial survival for B. abortus designated 14 cattle as susceptible and seven as resistant and this correlated 100 percent with the number of animals designated as to the relevant category by in vivo challenge. A value of 65 percent bacterial survival for M. bovis BCG, and 100 percent bacterial survival for S. dublin correlated highly with actual numbers of animals designated as susceptible or resistant.

Animals↗

Cloning and characterization of the asd gene of Salmonella typhimurium: use in stable maintenance of recombinant plasmids in Salmonella vaccine strains.

The asd mutants of Salmonella typhimurium have an obligate requirement for diaminopimelic acid (DAP) and will undergo lysis in environments deprived of DAP. This has allowed the development of a balanced-lethal system for the expression of heterologous antigens in vaccine strains using vectors containing the wild-type asd gene from Streptococcus mutans and asd mutant Salmonella hosts [Nakayama et al., Biotechnology 6 (1988) 693-697]. We have cloned the asd gene from S. typhimurium, characterized the gene product and used this gene to construct Asd+ expression cloning vectors. In addition we have constructed an asd cassette and a transposon derived from Tn5 that allow the rapid modification of other vectors for use with delta asd vaccine strains of S. typhimurium adding versatility to the Asd+ vector/delta asd host system of plasmid maintenance.

Amino Acid Sequence↗

Persistence of Escherichia coli O157:H7, Salmonella Newport, and Salmonella Poona in the gut of a free-living nematode, Caenorhabditis elegans, and transmission to progeny and uninfected nematodes.

A study was undertaken to determine the persistence of Escherichia coli O157:H7 and salmonellae in the gut of a free-living nematode, Caenorhabditis elegans, as affected by temperature and relative humidity and to determine if infected worms transmit Salmonella enterica serotype Newport to progeny and uninfected worms. Worms were fed cells of a non-pathogenic strain of E. coli (OP50), E. coli O157:H7, S. enterica serotype Newport, and S. enterica serotype Poona, followed by incubating at 4, 20, or 37 degrees C for up to 5 days. Initial populations of ingested pathogens significantly increased by up to 2.93 log(10) cfu/worm within 1 day at 20 degrees C on K agar and remained constant for an additional 4 days. When worms were placed on Bacto agar, populations of ingested pathogens remained constant at 4 degrees C, decreased significantly at 20 degrees C, and increased significantly at 37 degrees C within 3 days. Worms fed E. coli OP50 or S. Newport were incubated at 4 or 20 degrees C at relative humidities of 33%, 75%, or 98% to determine survival characteristics of ingested bacteria. Fewer cells of the pathogens survived incubation at 33% relative humidity compared to higher relative humidities. Populations of ingested E. coli OP50 and S. Newport decreased by up to 1.65 and 3.44 log(10) cfu/worm, respectively, in worms incubated at 20 degrees C and 33% relative humidity. Placement together on K agar of adult worms, labeled with green fluorescent protein (gfp) in the pharynx area, that had ingested gfp-labeled S. Newport and uninfected wild type worms resulted in transfer of the pathogen to gut of wild type worms. S. Newport was isolated from C. elegans two generations removed from exposure to the pathogen. Results of these studies show that C. elegans may serve as a temporary reservoir of foodborne pathogens, and could perhaps be a vector for contaminating preharvest fruits and vegetables, thus potentially increasing the risk of enteric infections associated with consumption of raw produce.

Animals↗

Evaluation of Salmonella enterica serovar Typhi (Ty2 aroC-ssaV-) M01ZH09, with a defined mutation in the Salmonella pathogenicity island 2, as a live, oral typhoid vaccine in human volunteers.

Salmonella enterica serovar Typhi strains with mutations in the Salmonella pathogenicity island-2 (SPI-2) may represent an effective strategy for human vaccine development, and a vectoring system for heterologous antigens. S. Typhi (Ty2 aroC-ssaV-) M01ZH09 is an attenuated, live, oral typhoid vaccine harboring defined deletion mutations in ssaV, which encodes an integral component in the SPI-2 type III secretion system (TTSS), as well as a mutation in an aromatic biosynthetic pathway needed for bacterial growth in vivo (aroC). SPI-2 mutant vaccines have yet to be evaluated in a large, randomized human trial. A simplified or single-oral dose oral typhoid vaccine using the SPI-2 strategy would offer significant advantages over the currently licensed typhoid vaccines. We performed a double-blinded, placebo-controlled, dose-escalating clinical trial in 60 healthy adult volunteers to determine the tolerability and immunogenicity of a single dose of M01ZH09. Three groups of 20 healthy adult volunteers were enrolled; 16 in each group received a single oral dose of the freeze-dried vaccine at 5 x 10(7), 5 x 10(8) or 5 x 10(9)CFU in a bicarbonate buffer. Four volunteers in each cohort received placebo in the same buffer. Adverse events were infrequent and not statistically different between vaccine and placebo recipients, although two subjects in the mid-range dose and three subjects in the highest dose had temperature measurements >37.5 degrees C. No blood or urine cultures were positive for M01ZH09, and fecal shedding was brief. The immune response was dose-related; the highest vaccine dose (5 x 10(9)CFU) was the most immunogenic. All tested subjects receiving the highest dose had a significant ASC response (mean 118 spots/10(6) cells). A >or=4-fold increase in antibody titer for S. Typhi LPS or flagellin was detected in 75% of volunteers in the highest-dose cohort by day 28. The SPI-2 mutant vaccine, M01ZH09, is a promising typhoid vaccine candidate and deserves further study as a vectoring system for heterologous vaccine antigens.

Administration, Oral↗

The structure of the antigenic lipopolysaccharide O-chains produced by Salmonella urbana and Salmonella godesberg.

The lipopolysaccharides of Salmonella urbana and Salmonella godesberg, which belong in group N (O:30) of the Kauffmann-White system, were shown by SDS-PAGE electrophoresis, glycose analysis, periodate oxidation, methylation, and 1H- and 13C-n.m.r. analyses to have identical O-chains composed of repeating, branched pentasaccharide units having the structure: [----4)-beta-D-Glcp-(1----3)-alpha-D-GalNAcp-(1----2)-alpha-D-P erNAcp-(1----3)-alpha-L-Fucp-(1----]n 4 increases 1 beta-D-Glcp. The serological cross-reactivity of S. urbana and S. godesberg with Brucella abortus and Yersinia enterocolitica (O:9) can now be related to the presence of a 1,2-glycosylated N-acyl derivative of 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residues in their respective lipopolysaccharide O-chains.

Carbohydrate Conformation↗

A role for the leucine-responsive regulatory protein and integration host factor in the regulation of the Salmonella plasmid virulence (spv ) locus in Salmonella typhimurium.

The Salmonella plasmid virulence (spv ) genes of Salmonella typhimurium are activated at the level of transcription as the bacteria enter stationary phase in vitro or in response to signals received during intracellular growth. Activation requires the LysR-like transcription factor SpvR and the alternative sigma factor RpoS. In this report, we show by biochemical and genetic analyses that two chromosomally encoded DNA-binding proteins contribute to the control of spv expression. These are the integration host factor (IHF), which binds to DNA sequences upstream of the spvR regulatory gene, and the leucine-responsive regulatory protein (Lrp), which binds to sequences upstream of the spvABCD operon. Under all conditions tested, inactivation of IHF expression reduces the level of spvR transcription by twofold. It also alters the response of the spv regulon to loss of DNA gyrase activity, consistent with a role for IHF in organizing DNA structure in the vicinity of the spvR promoter. Lrp represses spvA gene expression by up to fivefold and Lrp-mediated repression is antagonized by leucine. The Lrp binding site upstream of the spvA gene overlaps one of the binding sites for the positive regulator SpvR, suggesting a mechanism by which Lrp repression is exerted. This is a first demonstration of a role for Lrp in controlling genes that are also subject to intracellular regulation. These data show that the spv virulence genes belong simultaneously to several regulons in the cell, raising the possibility that spv expression can be fine-tuned in response to multiple environmental inputs.

Bacterial Proteins↗

Penetration of human intestinal epithelial cells by Salmonella: molecular cloning and expression of Salmonella typhi invasion determinants in Escherichia coli.

Salmonella typhi, the causative agent of typhoid fever, must invade the human gastrointestinal tract and multiply within the host to cause disease. We have cloned from S. typhi Ty2 a chromosomal region that confers upon Escherichia coli HB101 the ability to invade cultured human intestinal epithelial cells. Three invasion-positive recombinant cosmids were isolated and restriction endonuclease analyses of the inserts showed a 33-kilobase region of identity. Transmission electron microscopy of epithelial cells invaded by S. typhi Ty2 or E. coli HB101 carrying an invasion cosmid showed intracellular bacteria contained within endocytic vacuoles. One of the invasion cosmids was mutagenized with transposon Tn5 to identify the cloned sequences that are required for the invasive phenotype. Seven of 92 independent Tn5 insertions within the common 33-kilobase region eliminated invasive ability and revealed at least four separate loci that are required for invasion. Penetration of epithelial cells by Ty2 and HB101 carrying the cloned invasion determinants was inhibited by cytochalasin B and D, indicating that epithelial cell endocytosis of S. typhi is a microfilament-dependent event. The invasion cosmids were found to carry the recA and srlC genes indicating that the cloned invasion determinants are located at about 58 minutes on the S. typhi chromosome. With a segment of the cloned S. typhi invasion region used as a probe, homologous sequences were isolated from Salmonella typhimurium. Two independent S. typhimurium recombinant cosmids containing the entire 33-kilobase common region identified in S. typhi were isolated, but these cosmids did not confer upon HB101 the ability to invade epithelial cells.

Cell Line↗

Idiotypy of rabbit antibodies. I. Comparison of idiotypy of antibodies against Salmonella typhi with that of antibodies against other bacteria in the same rabbits, or of antibodies against Salmonella typhi in various rabbits.

Sera of rabbits immunized against Salmonella typhi have been studied for the idiotypy of certain of their components, i.e., the property of these components to possess an antigenic specificity which is different in individual rabbits, and which varies with the antigens against which these rabbits have been immunized. The reagent used (precipitating anti-idiotypic sera) have been prepared by injecting rabbits with bacteria agglutinated by anti-S. typhi sera (immunizing sera) as was done in the first observations by the authors of the phenomenon in the rabbit. These first observations have been confirmed and extended. In contrast to allotypy, the anti-idiotypic sera precipitate the corresponding immunizing sera, but not the sera taken in the immunizing rabbits prior to their immunization against S. typhi, nor the immunizing sera absorbed with the somatic antigen of S. typhi, demonstrating that idiotypes are antibodies. The idiotypic specificities of the antibodies of one rabbit against S. typhi are not detected in the antibodies of the same rabbit against another noncross-reacting Salmonella (S. tranoroa) and vice versa; nor are they detected in the anti-pneumococcal antibodies of the same rabbit. Each anti-idiotypic serum fails to precipitate anti-S. typhi sera of rabbits other than the immunizing one except for certain extremely faint reactions, the significance of which has not been established. The idiotypic specificities of anti-S. typhi antibodies of three rabbits were not found in anti-S. typhi antibodies of their parents. This lack of a sign of hereditary transmission of idiotypic specificities contrasts with allotypy. The apparent role of random chance in the determinism of the idiotypic patterns or of the idiotypic determinants has been discussed. Unless it were admitted that antibodies with similar functions do not exist in different individuals, idiotypy apparently adds an order of magnitude to the antibody variability which had been previously envisaged. In one given individual, the heterogeneity of the idiotypic specificities seems to be less extended than that of the antibody functions. The possible relationships between these two levels of molecular variability and between the corresponding levels of cellular variability have been discussed.

Animals↗

Comparison of the effects of dietary selenium, zinc, and selenium and zinc supplementation on growth and immune response between chick groups that were inoculated with Salmonella and aflatoxin or Salmonella.

The effects of four diets (basal diet, Se, Zn, and Se- and Zn-enriched diets) fed to chicks that were administered one of three treatments [Salmonella and aflatoxin inoculation (T1), Salmonella inoculation (T2), or uninoculated (T3)] were investigated for growth and immune responses. We found a significant improvement in growth performance represented by relative body gain (RBG) and feed efficiency (FE), for the Zn- and Se + Zn-enriched diets fed to the T1 and T2 groups. The antibody immune response was significantly improved for the Se enrichment diet in the T1 and T2 groups. The weight of the bursa and thymus, which relate to the level of the immune response, showed significant decreases, whereas the spleen had a significantly increased relative weight (RW) in the T1 group. The variable dietary trace elements supplement increased the thymic RW in the T2 group.

Aflatoxins↗

Tumor vaccination by Salmonella typhimurium after transformation with a eukaryotic expression vector in mice: impact of a Salmonella typhimurium gene interfering with MHC class I presentation.

Transformed attenuated Salmonella typhimurium (ST) have been suggested as an efficient means of tumor vaccination. However, ST themselves might be immunosuppressive, and the question has arisen as to whether this impedes vaccination efficacy even if ST are transformed with a eukaryotic expression vector such that "tumor antigen" will be transcribed by the host. The question was evaluated using a mutant SL7207, where the yej operon, which interferes with MHC I-mediated presentation, had been inactivated (SL7207DeltayejE). Mice were vaccinated with SL7207 or SL7207DeltayejE transformed with a eukaryotic expression vector carrying the lacZ or the gp100 gene and later received lacZ-transfected RENCA or YC8 or gp100-expressing B16F1 tumor cells. In vaccinated mice, tumor growth started with a delay and some animals remained tumor-free; however, the tumor growth rate remained unaltered. No significant difference was seen between SL7207DeltayejE versus SL7207 vaccinated mice. The latter finding contrasted with ex vivo analyses where vaccination with SL7207DeltayejE, compared with SL7207, induced a significantly stronger response, including nonadaptive defense mechanisms. The failure to detect a superior vaccination efficacy of SL7207DeltayejE in vivo could be attributed to a stronger effect of the yej operon on MHC-mediated antigen presentation when driven by a prokaryotic promoter. Also, additional Salmonella genes apparently interfere with maintenance of a sustained immune response. Thus, the immunosuppressive yej operon affects innate and adaptive immunity. However, when ST are carriers for eukaryotic-expressed tumor antigens, yej does not severely hamper induction of an immune response.

Animals↗

Differentiation of Salmonella gallinarum and Salmonella pullorum by their whole-cell fatty acid methyl ester profiles.

The whole-cell fatty acid methyl ester (FAME) profiles of Salmonella (S.) gallinarum and Salmonella (S.) pullorum strains were compared in the computer-linked gas-liquid chromatography. The profiles of whole cellular FAMEs allow the separation and identification S. gallinarum and S. pullorum by the Microbial Identification System and so can be used for their differentiation.

Animals↗

Variant Salmonella genomic island 1 antibiotic resistance gene cluster containing a novel 3'-N-aminoglycoside acetyltransferase gene cassette, aac(3)-Id, in Salmonella enterica serovar newport.

Salmonella genomic island 1 (SGI1) harbors an antibiotic resistance gene cluster and was previously identified in the multidrug-resistant Salmonella enterica serovars Typhimurium DT104, Agona, Paratyphi B, and Albany. This antibiotic resistance gene cluster is a complex class 1 integron and most often confers resistance to ampicillin (Ap), chloramphenicol (Cm)/florfenicol (Ff), streptomycin (Sm)/spectinomycin (Sp), sulfonamides (Su), and tetracycline (Tc) (ApCmFfSmSpSuTc profile). Recently, variant SGI1 antibiotic resistance gene clusters conferring different antibiotic resistance profiles have been identified in several S. enterica serovars and were classified as SGI1-A to -G. We identified a new variant SGI1 antibiotic resistance gene cluster in two multidrug-resistant S. enterica serovar Newport strains isolated from humans in France. In these strains, the Sm/Sp resistance gene cassette aadA2 inserted at the first attI1 site was replaced by two other aminoglycoside resistance gene cassettes. The first one contains a new resistance gene encoding an AAC(3)-I aminoglycoside 3-N-acetyltransferase that confers resistance to gentamicin (Gm) and sisomicin (Sc). This gene has been named aac(3)-Id. The second one harbors the Sm/Sp resistance gene aadA7. This gene cassette replacement in the SGI1 complex integron of serovar Newport strains constitutes a new variant SGI1 antibiotic resistance gene cluster named SGI1-H. The occurrence of SGI1 in different S. enterica serovars, now including serovar Newport, strengthens the hypothesis of horizontal transfer of SGI1.

Acetyltransferases↗

Salmonella gene rma (ramA) and multiple-drug-resistant Salmonella enterica serovar typhimurium.

MarA and its homologue, RamA, have been implicated in multidrug resistance (MDR). RamA overexpression in Salmonella enterica serovar Typhimurium and Escherichia coli conferred MDR independently of marA. Inactivation of ramA did not affect the antibiotic susceptibilities of wild-type S. enterica serovar Typhimurium or 15 unrelated clinical MDR isolates. Thus, ramA overexpression is not a common MDR mechanism in Salmonella.

Bacterial Proteins↗

Heat resistance of Salmonella: the uniqueness of Salmonella senftenberg 775W.

Of approximately 300 cultures of Salmonella, representing 75 different serotypes, none was found to be as heat-resistant as S. senftenberg 775W. However, S. blockley 2004 was 5 times more heat-resistant and S. senftenberg 775W was 30 times more heat-resistant than S. typhimurium Tm-1, the reference strain in this study. All other strains of Salmonella tested, including 19 strains of S. senftenberg and 7 strains of S. blockley, had decimal reduction times at 57 C of about 1 min, equivalent to that of the reference organism, Tm-1. As observed in other bacterial species, strain 775W is more heat-sensitive in the log phase than in the stationary phase of growth. Cells from cultures grown at 44 C were more heat-resistant than those grown at either 35 or 15 C; the medium of growth, whether minimal or complex, made no appreciable difference in heat resistance. Cells from cultures limited by a carbon source were killed at a much slower rate than those limited by a nitrogen source and exhibited a 1-hr lag at 55 C before a significant rate of kill was attained. For any given set of growth conditions, strain 775W was always more heat-resistant than another strain of S. senftenberg, 197B, which has normal heat resistance.

Culture Media↗