Loss of sulfonamide resistance in Neisseria meningitidis

Four strains of Neisseria meningitidis were studied during serial passage. From two strains which originally were sulfonamide resistant, variants developed that had altered susceptibility to sulfonamides. One of the variants became relatively highly sulfonamide-sensitive, the other exhibited merely reduced sulfonamide resistance. There was a difference in the resistance pattern for two sulfonamides (sulfaisodimidine and sulfamethoxazole), and the effect of inoculum size and growth conditions in three different media could be demonstrated. Although the patterns of susceptibility to other antibacterial agents were different for the strains studied, no further susceptibility alterations occurred in parallel to the sulfonamide sensitivity changes. The variants also lost their ability to liberate free endotoxin.     

Variability in LPS composition, antigenicity and reactogenicity of phase variants of Bordetella pertussis

Comparison of lipopolysaccharides (LPS) from phase variants of different strains of Bordetella phase variants of different strains of Bordetella pertussis has shown a difference in their composition, antigenicity and reactogenicity. Phase I variants of B. pertussis, with the exception of strain 134, contain a preponderance of LPS I whereas the major component of LPS of phase IV variants is LPS II. Sera raised to LPSs of phase I strains, other than 134, cross-react with each other but not with phase IV LPSs; and similarly all sera raised to phase IV LPSs cross-react with each other and with LPS from 134 phase I. The LPSs of all phase I variants, including that of 134, are approximately ten-fold or more reactive in the limulus amoebocyte lysate assay (LAL) than phase IV LPSs. In the human mononuclear cell pyrogen assay phase IV LPSs also stimulated a lower response than phase I LPSs. The B. pertussis phase I LPSs are 10-times more reactive than Escherichia coli standard endotoxin in the LAL assay but 100-times less reactive than E. coli LPS in the monocyte test for pyrogen. The SDS-PAGE profiles of B. pertussis LPSs are quite different from those of B. parapertussis and B. bronchiseptica strains. B. pertussis LPSs produced a typical lipo-oligosaccharide (LOS) pattern. B. bronchiseptica LPS produced a similar pattern but was antigenically distinct from B. pertussis LPSs I and II. B. parapertussis in contrast produced a ladder pattern typical of smooth type LPS.     

A comparison of the febrile responses of the Brattleboro and Sprague-Dawley strains of rats to endotoxin and endogenous pyrogens

The febrile responses of homozygous (di/di) Brattleboro rats, to both intravenous endogenous pyrogen and to a lipopolysaccharide endotoxin, were compared with those of normal Sprague-Dawley rats. There were no detectable differences between the fever curves of the two strains in response to endogenous pyrogen. Brattleboro rats, which are deficient in the neuropeptide arginine vasopressin (AVP), displayed fevers that were both qualitatively and quantitatively indistinguishable from those of normal Sprague-Dawley rats that do not suffer from congenital diabetes insipidus. It is concluded that the absence of AVP-containing cells in Brattleboro rats is not an important factor in determining the nature of their febrile responses to endogenous pyrogen. More remarkable, however, were the divergent febrile responses of the two strains to intravenously injected endotoxin. Normal rats displayed hypothermic responses, whereas the Brattleboro rats became febrile. By 2 h after the injection of endotoxin, body temperatures in both strains had returned to normal. Three hours after the rats had been exposed to endotoxin, both strains were found to be totally refractory to endogenous pyrogen. However, when both strains of rats were tested to endogenous pyrogen 3 days later, their febrile responses were more than double the magnitude of their initial control responses. These alterations in the febrile responsiveness of rats occurring at different times after the injection of endotoxin appear to be related to the effects that endotoxin has on the cells of the reticuloendothelial system, over the same time course.     

Ganglioside expression in macrophages from endotoxin responder and nonresponder mice

Peritoneal macrophage ganglioside patterns and ganglioside sialic acid content were compared for two congenic strains of mice having differing responses to bacterial lipopolysaccharide. Resident macrophage ganglioside patterns from C3H/HeJ mice (endotoxin hyporesponsive) and C3H/HeN mice (endotoxin responsive) were similar. Macrophages elicited with phenol-extracted or butanol-extracted endotoxin showed distinctly more complex ganglioside patterns in C3H/HeN mice. C3H/HeJ macrophages showed distinct, but less complex changes when elicited with butanol-extracted endotoxin. As expected, there were minimal alterations induced by phenol-extracted endotoxin in the C3H/HeJ patterns. When injected with whole killed E. coli, both strains of mice exhibited complex ganglioside patterns; however, there were relative differences in the quantities of multiple gangliosides. Differences in ganglioside patterns were mirrored in the relative ratios of N-acetyl- to N-glycolylneuraminic acid. When macrophages were activated by administration of either endotoxin preparation, macrophage gangliosides from C3H/HeN mice always contained a higher proportion of N-acetylneuraminic acid compared with C3H/HeJ macrophage gangliosides. Oxidative metabolism of the macrophage populations was assessed by PMA-induced H2O2 release. This indicated that endotoxin activation produced an increase in PMA-induced H2O2 release as well as a shift of sialic acid class from the N-glycolyl type to the N-acetyl type. However, no direct correlation could be made between ganglioside composition, sialic acid content, and macrophage function. These data indicate that both ganglioside composition and sialic acid composition of macrophages are profoundly altered with endotoxin activation. The data further indicate that under conditions which C3H/HeJ mice respond to Gram-negative bacteria, their macrophage ganglioside patterns still differ from normal mice.     

Neuraminidase activity in representatives of the genus Francisella]

In two Francisella species (F. tularensis and F. novicida) neuraminidase activity, heretofore unknown, was detected. The enzyme exhibited specificity with respect to the substrates used in the investigation, neutralizing natural mucins, but not other compounds (glycoproteins and glycoproteins). All F. tularensis strains were found to have enzymatic activity irrespective of their subspecies, but neuraminidase activity was higher in the strains belonging to the American subspecies. Experimentally obtained F. tularensis noncapsular variants possessed higher neuraminidase activity than the capsular parent strains. The conclusion on the possible role of this enzyme in F. tularensis colonization of the host body was made.     

Type 1 and Type 2 Herpes Simplex Viruses: Serological and Biological Differences

Forty isolates of herpes simplex virus were compared by means of cross-neutralization curves. The 11 oral isolates were serotype 1, and all 29 genital/anal isolates were serotype 2. The cytopathic effects of the two serotypes were consistently different. Passage of strains of type 1 and type 2 in mice and in rabbits yielded two variants, although the majority of the strains remained unchanged serologically and in their cytopathic effects. The two variants were derived from type 1 strains and differed from the parent strains in their cytopathic effects, each of them producing syncytia and enlarged plaques. They had, however, retained the serotypic properties and the deoxyribonucleic acid (DNA) densities of their parent strains. The Roizman syncytial/macroplaque strain of herpes simplex virus was also included in the study; the density of its DNA (1.727 g/ml) was typical of type 1 strains, and serologically it seemed to be basically a type 1 strain, although it was neutralized by type 2 antiserum slightly better than were other type 1 strains. Growth curves were performed of the two serotypes in rabbit kidney, human fibroblast, and mouse embryo tissue cultures. The type 2 strains attained lower titers of infectivity in these three cell systems; the levels of infectivity of type 2 virus in the culture fluid decreased much more rapidly after the maximum had been attained than did the levels of infectivity of the type 1 strains, due to the greater instability of the type 2 virus. Parallel titrations of different strains in tissue cultures and intracerebrally in mice indicated that the latter assay system was usually more sensitive for type 2 strains than it was for type 1 strains. The paralytic sequelae and inflammatory changes of lumbar ganglia and spinal cord in young rabbits inoculated extraneurally with strains of the two serotypes also indicate that the type 2 virus is more virulent in laboratory animals than is type 1 virus.     

Changes in internal and external pH accompanying growth of Candida albicans: studies of non-dimorphic variants

Non-dimorphic variants of Candida albicans, which were unable to form germ tubes or mature hyphae in media containing amino acids, glucose and salts or N-acetylglucosamine or serum, were prepared from two hyphal positive laboratory strains using a physical separation method. The hyphal-minus phenotype was stable and may be due to mutations or phenotypic variation. The variant strains maintained their internal pH within narrower bounds as compared to their parental wild-types. When exposed to conditions that normally supported the induction of germ tubes the cytoplasmic pH of the wild type strains increased from 6.8 to over pH 8.0 within 5 min while in the variants the rise in internal pH was only about 0.3 pH units. The wild type strains acidified the growth medium more rapidly than the variants. The results suggest that the control of internal pH is directly or indirectly associated with the regulation of dimorphism. The variants had unaltered cell volumes and specific growth rates. The hyphal-minus phenotype was however fully reversible since revertants occurred spontaneously on serum containing agar.     

Genetic variability of proteins from mitochondria and mitochondrial fractions of mouse organs

Proteins of whole mitochondria from mouse liver and brain and proteins of liver mitochondrial fractions (plasma and rough membrane fraction) were separated by two-dimensional electrophoresis. Protein patterns of two inbred strains of mouse, C57BL/6J and DBA/2J, and of F₁ mice of these two strains were studied. The protein patterns obtained from the different mitochondrial materials were analyzed with regard to their protein composition and the genetic variability of proteins (qualitative and quantitative protein variants). Included in this analysis are data previously obtained from the cytosols and plasma membranes of the same organs and mouse strains. The results showed the following. (1) Mitochondria and organelle-free cell components (cytosol and plasma membranes) have only a few percent of their proteins in common, while two organs, liver and brain, reveal up to approximately 50% organ-nonspecific proteins. The frequency of proteins common to solubilized and structure-bound proteins ranges below 20%. (2) Genetic variability in protein amount occurs much more frequently than genetic variability in protein structure. Liver proteins reveal more genetic variants than brain proteins. Proteins solubilized in the cell show more genetic variation than structure-bound proteins. Furthermore, the results show that with regard to the composition and the genetic variability of proteins, liver and brain differ more in their mitochondria than in their cytosol and plasma membranes.This work was supported by grants from the Deutsche Forschungsgemeinschaft awarded to Sonderforschungsbereich 29.     

Factors influencing the chlorine susceptibility of Mycobacterium avium,Mycobacterium intracellulare,and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30 degrees C was increased when cells were exposed to chlorine at 40 degrees C compared to susceptibility after exposure at 30 degrees C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

Composition and genetic variability of heparin-sepharose CL-6B protein fractions obtained from the solubilized proteins of mouse organs

The solubilized proteins of liver and brain from mice of two inbred strains (C57BL/6J and DBA/2J) and their hybrids were subfractionated by heparin Sepharose (H-S) CL-6B affinity chromatography. The H-S binding and nonbinding proteins were separated by two-dimensional electrophoresis. The protein patterns obtained were analyzed with regard to their protein composition and their genetic variability (qualitative and quantitative variants). Eighty to ninety percent of the H-S binding proteins were unique to this class of proteins. This class was rich in organ-specific proteins. Compared to the nonbinding proteins the portion of basic proteins was only slightly increased, suggesting that most of the H-S binding proteins interact specifically with heparin. The frequency of qualitative protein variants revealed that H-S binding proteins are more conservative than H-S nonbinding proteins. The quantitative genetic variability was higher in liver than in brain. Quantitative protein variants occurred more frequently than qualitative variants.     

Haplopappus gracilis cell strains resistant to pyrimidine analogues

Summary Strains of Haplopappus gracilis (Nutt.) Gray cells resistant to 6-azauracil have been isolated from cultures of diploid cells. These strains are also resistant to 8-azaguanine, as is their parent. The variants are 100- to 125-fold more resistant to 6-azauracil than their parent, and they exhibit different spectra of cross resistance to other pyrimidine analogues. The phenotype of each variant is stable in the absence of selection. The majority of cells in cultures of the variants are diploid; all others examined were tetraploid. Initial rates of uptake of uracil are not reduced in the variants. Fluorouracil, to which two variants are resistant, is taken up by one of them as well as by the parent. Responses of the other two to fluorouracil are not correlated with decreased ability to accumulate this analogue.     

Hydrogen Sulfide-Producing Variants of Escherichia coli

Seventeen strains of H(2)S-producing variants of Escherichia coli were isolated from specimens submitted for microbiological study (ten from stool, five from urine, and two from postmortem material). Production of H(2)S was unstable in several strains; however, other than their production of H(2)S, all strains closely resembled typical E. coli in their biochemical reactions. In vitro susceptibilities of the H(2)S-producing variants to antimicrobics closely resembled those of typical E. coli in this laboratory.     

Allele Variants of Enterotoxigenic Escherichia coli Heat-Labile Toxin Are Globally Transmitted and Associated with Colonization Factors

Enterotoxigenic Escherichia coli (ETEC) is a significant cause of morbidity and mortality in the developing world. ETEC-mediated diarrhea is orchestrated by heat-labile toxin (LT) and heat-stable toxins (STp and STh), acting in concert with a repertoire of more than 25 colonization factors (CFs). LT, the major virulence factor, induces fluid secretion after delivery of a monomeric ADP-ribosylase (LTA) and its pentameric carrier B subunit (LTB). A study of ETEC isolates from humans in Brazil reported the existence of natural LT variants. In the present study, analysis of predicted amino acid sequences showed that the LT amino acid polymorphisms are associated with a geographically and temporally diverse set of 192 clinical ETEC strains and identified 12 novel LT variants. Twenty distinct LT amino acid variants were observed in the globally distributed strains, and phylogenetic analysis showed these to be associated with different CF profiles. Notably, the most prevalent LT1 allele variants were correlated with major ETEC lineages expressing CS1 + CS3 or CS2 + CS3, and the most prevalent LT2 allele variants were correlated with major ETEC lineages expressing CS5 + CS6 or CFA/I. LTB allele variants generally exhibited more-stringent amino acid sequence conservation (2 substitutions identified) than LTA allele variants (22 substitutions identified). The functional impact of LT1 and LT2 polymorphisms on virulence was investigated by measuring total-toxin production, secretion, and stability using GM1–enzyme-linked immunosorbent assays (GM1-ELISA) and in silico protein modeling. Our data show that LT2 strains produce 5-fold more toxin than LT1 strains (P < 0.001), which may suggest greater virulence potential for this genetic variant. Our data suggest that functionally distinct LT-CF variants with increased fitness have persisted during the evolution of ETEC and have spread globally.     

Spontaneous variability in a population of Candida albicans strains]

The spontaneous variability of the populations of C. albicans strains of different genesis in the morphological properties of their colonies and in the potential of the activity of their extracellular proteolytic and phospholipid enzymes has been studied. The isolated types of colonies, differing in their morphology, have the phenotypic character of variability. Different populations of strains exhibited variability in the activity of enzymes, depending on morphological variants isolated from these populations. Selected morphological variants with high potential of their proteolytic enzymes retained stability in this property for 5 generations and can be used in medical practice for the isolation of C. albicans antigens.     

DNA relatedness among Erysipelothrix rhusiopathiae strains representing all twenty-three serovars and Erysipelothrix tonsillarum.

The levels of relatedness among strains of Erysipelothrix rhusiopathiae (serovars 1 through 23 and type N) were estimated by performing DNA-DNA hybridization experiments with the type strains of E. rhusiopathiae and Erysipelothrix tonsillarum, which are the two Erysipelothrix species that have been described. Two distinct DNA relatedness groups were identified. The group 1 strains, representing serovars 1, 2, 4 through 6, 8, 9, 11, 12, 15 through 17, 19, and 21 and type N, exhibited more than 73% hybridization with the type strain of E. rhusiopathiae but less than 24% hybridization with the type strain of E. tonsillarum. Group 2 included serovar 3, 7, 10, 14, 20, 22, and 23 strains, and these strains exhibited more than 66% hybridization with the type strain of E. tonsillarum but less than 27% hybridization with the type strain of E. rhusiopathiae. Strains representing serovars 13 and 18 exhibited low levels of hybridization (16 to 47%) with both of the type strains, indicating that these serovars may be members of a new genomic species. The members of the E. rhusiopathiae and E. tonsillarum groups resembled each other in many phenotypic characteristics, but differed in their ability to produce acid from saccharose and in their pathogenicity for swine. Our results confirm that the genus Erysipelothrix contains two main genomic species, E. rhusiopathiae and E. tonsillarum, which can be differentiated into serovars.     

Antimicrobial activity of lactic acid bacteria isolated from goat's milk and artisanal cheeses: characteristics of a bacteriocin produced by Lactobacillus curvatus IFPL105

A total of 203 lactic acid bacteria isolated from raw goat's milk and artisanal cheese were tested for antibacterial activity. Only two strains of Lactococcus lactis , one strain of Enterococcus faecalis and one strain of Lactobacillus curvatus were shown to produce a bacteriocin-like substance. Lactobacillus curvatus IFPL105 produced a heat-stable bacteriocin, which was hydrolysed by α-chymotrypsin, proteinase K and pancreatin and exhibited a broad spectrum of inhibitory activity. The bactericidal activity of the bacteriocin was more potent when sensitive strains were in the logarithmic growth phase, inducing cell lysis, as observed by decreases in optical density and release of intracellular marker enzymes. Curing experiments resulted in variants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of the parental strain and the bacteriocin-negative variants indicated that a plasmid of about 46 kbp may be involved in bacteriocin production and immunity to this antibacterial compound.     

Factors Influencing the Chlorine Susceptibility of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum

The susceptibility of representative strains of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium scrofulaceum (the MAIS group) to chlorine was studied to identify factors related to culture conditions and growth phase that influenced susceptibility. M. avium and M. intracellulare strains were more resistant to chlorine than were strains of M. scrofulaceum. Transparent and unpigmented colony variants were more resistant to chlorine than were their isogenic opaque and pigmented variants (respectively). Depending on growth stage and growth rate, MAIS strains differed in their chlorine susceptibilities. Cells from strains of all three species growing in early log phase at the highest growth rates were more susceptible than cells in log and stationary phase. Rapidly growing cells were more susceptible to chlorine than slowly growing cells. The chlorine susceptibility of M. avium cells grown at 30°C was increased when cells were exposed to chlorine at 40°C compared to susceptibility after exposure at 30°C. Cells of M. avium grown in 6% oxygen were significantly more chlorine susceptible than cells grown in air. Chlorine-resistant MAIS strains were more hydrophobic and resistant to Tween 80, para-nitrobenzoate, hydroxylamine, and nitrite than were the chlorine-sensitive strains.     

The stability of toxigenicity in Clostridium botulinum types C and D.

Several type C and D strains of Clostridium botulinum, which had been converted to the toxgenic state by phages, were serially transferred through cooked meat medium with and without specific anti-phage serum. Most of the converted strains lost their toxigenicity even during transfer without antiserum, and the non-toxigenic variants that appeared were resistant to lysis and conversion by the original phage. However, in some combinations of phage and host bacteria toxigenicity was stable after ten transfers, though it showed a transient decrease, and the non-toxigenic variants that arose remained sensitive to lysis and conversion. When converted strains were transferred in medium containing anti-phage serum, toxigenicity was lost more rapidly than in the absence of serum and the non-toxigenic variants that appeared remained sensitive to lysis and conversion by the parent phage. Filtrates of the supernatants of culture fluids of strains transferred without anti-phase serum converted non-toxigenic strains to toxigenicity at varying rates.     

Synthesis and antibacterial activity of oxazolidinones containing pyridine substituted with heteroaromatic ring

A series of oxazolidinone derivatives, which morpholino group of linezolid was replaced with heteroaromatic ring substituted pyridine moiety, were newly synthesized, and their substituted effects on in vitro and in vivo antibacterial activities were evaluated against four problematic gram-positive strains including drug resistant strains and two gram-negative strains. Most compounds exhibited the enhanced in vitro activities with 4-16-fold and three compounds exerted more than 2-fold increased in vivo efficacies than linezolid.     

Method for reducing endotoxin in Moraxella catarrhalis UspA2 protein preparations

The UspA2 protein from the bacterium Moraxella catarrhalis is a potential vaccine candidate for preventing human diseases caused by this organism. Before a vaccine can be administered parentally, the level of endotoxin must be reduced as much as possible. However, in this case the endotoxin was very tightly complexed with the UspA2 protein and could not be dissociated with Triton X-100. It was found that it dissociated from the protein with the zwitterionic detergents Zwittergent 3-12 and Zwittergent 3-14. The endotoxin could then be separated from the protein by either ion-exchange or gel filtration chromatography. Using the limulus amoebocyte lysate assay for quantitation, the endotoxin was reduced approximately 20 000-fold. The removal of residual endotoxin from UspA2 preparations had no detrimental effect on the immunological properties of the protein. Mouse antisera raised against UspA2 prior to, and following endotoxin reduction exhibited comparable antibody and bactericidal titers against the tested strains. Further, mice immunized with both preparations, followed by pulmonary challenge with either a homologous or a heterologous isolate, exhibited comparable levels of clearance.