Analysis of coccal cell formation by Campylobacter jejuni using continuous culture techniques, and the importance of oxidative stress

Campylobacter jejuni is an important human gastro-intestinal pathogen. In hostile environments it may adapt its physiology to prolong survival, potentially including the adoption of a viable, non-culturable form and a change to coccal cell morphology. By independently controlling the individual parameters of continuous cultures of Camp. jejuni (e.g. pH, nutrient limitation, growth rate, etc.), coccal cell formation was shown to be elicited only by high oxygen tension in conjunction with reduced carbon concentration. Electron microscopy revealed degradative changes in these cells. This occurred as a transient response over 48 h coincident with a large reduction in maximum growth rate and viable count. Kinetic analysis of the biomass reduction of the cultures demonstrated that significant underlying growth was maintained, with the subsequent selection of a more oxygen-resistant population of cells and reversion to spiral morphology. Coccal cells appear to be predominantly a degenerate form of Camp. jejuni resulting from oxidative damage. While some of these coccal cells may recover, the more interesting population of cells is probably that which retains, or regains, spiral morphology during adaptation to oxidative stress.     

Recovery of viable but non-culturable Campylobacter jejuni.

Suspensions of Campylobacter jejuni became non-culturable after storage in sterilized pond water at 4 degrees C for periods between 18 and 28 d, depending on the strain. Suspensions of four strains of C. jejuni that had been in water for 6 weeks, and shown to be non-culturable, were fed to suckling mice. Colonization of mice was established with two of the strains and failed with the other two strains. Examination of these suspensions under the electron microscope showed some cocci having the appearance of being viable, but most cocci and all remaining spiral forms showed extensive degeneration. The results indicate that non-culturable coccal forms of C. jejuni are capable of infecting mice but that this property may differ between strains.     

A scanning electron microscope study of mycobacterial developmental stages in commercial BCG vaccines

Scanning electron microscopy (SEM) studies were performed on freshly prepared and freeze-dried Tice-substrainMycobacterium bovis BCG vaccine as well as Tice BCG grown on Middlebrook 7H10 agar. Intact colonies of the Tice and Glaxo BCG substrains growing on agar were also examined. The presence of developmental stages of the mycobacterial life cycle previously reported in the literature was confirmed in actively growing BCG and in commercial vaccine preparations. The pleomorphic forms consisted of various size coccal and bacillary cells. Propagation appeared to occur by fission of both forms to produce aggregate bodies and by a coccal-bacillary cycle. Filterable (30–200 nm) granular cocci and coccal microcolonies were also observed in commercially prepared BCG vaccines. The implications of pleomorphism on the biologic activities of various BCG vaccines are discussed.     

Spatial Distribution of Circadian Clock Phase in Aging Cultures of Neurospora crassa

Neurospora crassa has been utilized extensively in the study of circadian clocks. Previously, the clock in this organism has been monitored by observing the morphological and biochemical changes occurring at the growing front of cultures grown on solid medium. A method has been developed for assaying the clock in regions of the culture behind the growing front, where no apparent morphological changes occur during the circadian cycle. Using this assay with Petri dish cultures that were 2 to 7 days old, the presence of a functional circadian clock not only at the growing front but in all other regions of the culture as well was demonstrated. Furthermore, the entire culture is not in the same phase, but shows a gradient of phases which is a function of the length of time the clock in a given part of the culture has been free-running. This gradient may be the result of a somewhat longer period of the oscillator behind the growing front compared to that at the growing front. The phase differences within a single culture of interconnected mycelium demonstrate the absence of total internal synchronization between adjacent regions of the hyphae under these conditions.     

Influence of growth conditions on diverse polysaccharide production by Campylobacter jejuni

Campylobacter jejuni is the leading bacterial cause of gastroenteritis worldwide. The present study was undertaken to determine the forms of polysaccharide-related compounds (PRCs) produced by C. jejuni and the culture conditions influencing their production. Expression of polysaccharides by C. jejuni was influenced by culture medium composition and growth phase. In addition to the production of lipooligosaccharide and capsular polysaccharide, a previously undescribed polysaccharide, not related to capsular polysaccharide, was shown to occur in C. jejuni in batch liquid and chemostat cultures. Thus, a variety of PRCs are produced by C. jejuni, and this should be considered when growing the bacterium in vitro for pathogenesis studies.     

Morphology and adhesion of Campylobacter jejuni to chicken skin under varying conditions

The adhesion of Campylobacter jejuni to chicken skin, along with the associated morphological changes under aerobic conditions at 4, 25, and 37 degrees C and microaerobic (O2 5%, CO2 10%, N2 85%) conditions, were investigated using confocal laser scanning microscopy (CLSM), flow cytometry, and plate counting. The morphological change of C. jejuni from a spiral shape to a coccoid form or VBNC form (viable but nonculturable form) progressed rapidly under aerobic conditions at 25, 37, and 4 degrees C. As regards adhesion, the C. jejuni cells were mostly located in the crevices and feather follicles of the chicken skin, where the cells in the feather follicles floated freely in the entrapped water, even after the skin was rinsed quite thoroughly. CLSM also revealed the penetration of some spiral-shaped C. jejuni cells into the chicken skin. Even after changing their shape at various temperatures, coccoid-form C. jejuni cells were still found in the crevices and feather follicles of the chicken skin.     

Morphological changes of synchronized Campylobacter jejuni populations during growth in single phase liquid culture

Campylobacter jejuni strains demonstrate a variety of growth phase-linked distinct morphological forms when grown in liquid culture. The typical spiral form of the organism, evident during logarithmic phase, undergoes elongation during stationary phase before becoming coccoid via the formation of membrane blebs and budded forms in decline phase. Cellular elongation and coccoid formation occurred despite the inhibition of protein synthesis and without a detectable change in the protein components of the inner and outer cell membranes.     

Morphological forms and viability of Campylobacter species studied by electron microscopy.

Electron microscopic studies of Campylobacter revealed that different morphological forms predominate at different parts of a colony. At the periphery, cells were almost all spirals, while in the center of the colony cells were mainly coccus shaped. Unusual ring-shaped cells, "donuts", were observed in the raised, peripheral region of the colony. Donut or ring forms have not previously been reported for Campylobacter organisms. Our data indicate that young or actively growing cells are mainly spiral shaped. Older cells undergo a degenerative change to coccoid forms. The donut shape appears to be an intermediate stage between spirals and cocci. Comparisons of plate counts of actively growing and inactive cells confirmed that coccoid cells are probably nonviable.     

Double-staining method for differentiation of morphological changes and membrane integrity of Campylobacter coli cells

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

Structural studies of peptidoglycans in Campylobacter species.

Peptidoglycans (PG) from Campylobacter coli, Campylobacter jejuni, and Campylobacter fetus were composed of muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid in a molar ratio of 1.1:1:1.7:1.1:09. Thirty percent of the amino groups of diaminopimelic acid were involved in cross-linkages between peptides. During cultivation, C. coli and C. jejuni changed from a spiral to a coccoid form. In C. coli, we could isolate PG only from the spiral forms in yields of 0.8-1.2% by dry weight. C. fetus did not change to a coccoid form, and always contained PG. Thus, it is possible that the morphological transformation from the spirals to the coccoid forms of C. coli and C. jejuni is accompanied by, and probably due to, the degradation of PG.     

A comparative study of the rod and coccoid forms of Campylobacter jejuni ATCC 29428

Coccoid forms in cultures of a strain of the enteric pathogen Campylobacter jejuni were investigated. A culture containing 100% coccoid forms was non-viable. Coccoid forms had a lesser content of cytoplasmic components and nucleic acids than rods of C. jejuni. During the conversion to coccoid forms nucleotides leaked from the cells. The results of treatments with ionic and non-ionic detergents, and lysozyme and ethylenediaminetetraacetic acid indicated a changed cell wall in coccoid forms compared with rods. Using rate-zonal centrifugation coccoid forms were found to be less dense than rods. The results of this study indicate that the coccoid form of C. jejuni ATCC 29428 is a degenerate cell form which is undergoing cellular degradation.     

Phase variable genes of Campylobacter jejuni exhibit high mutation rates and specific mutational patterns but mutability is not the major determinant of population structure during host colonization

Phase variation of surface structures occurs in diverse bacterial species due to stochastic, high frequency, reversible mutations. Multiple genes of Campylobacter jejuni are subject to phase variable gene expression due to mutations in polyC/G tracts. A modal length of nine repeats was detected for polyC/G tracts within C. jejuni genomes. Switching rates for these tracts were measured using chromosomally-located reporter constructs and high rates were observed for cj1139 (G8) and cj0031 (G9). Alteration of the cj1139 tract from G8 to G11 increased mutability 10-fold and changed the mutational pattern from predominantly insertions to mainly deletions. Using a multiplex PCR, major changes were detected in 'on/off' status for some phase variable genes during passage of C. jejuni in chickens. Utilization of observed switching rates in a stochastic, theoretical model of phase variation demonstrated links between mutability and genetic diversity but could not replicate observed population diversity. We propose that modal repeat numbers have evolved in C. jejuni genomes due to molecular drivers associated with the mutational patterns of these polyC/G repeats, rather than by selection for particular switching rates, and that factors other than mutational drift are responsible for generating genetic diversity during host colonization by this bacterial pathogen.     

Double-Staining Method for Differentiation of Morphological Changes and Membrane Integrity of Campylobacter coli Cells

We developed a double-staining procedure involving NanoOrange dye (Molecular Probes, Eugene, Oreg.) and membrane integrity stains (LIVE/DEAD BacLight kit; Molecular Probes) to show the morphological and membrane integrity changes of Campylobacter coli cells during growth. The conversion from a spiral to a coccoid morphology via intermediary forms and the membrane integrity changes of the C. coli cells can be detected with the double-staining procedure. Our data indicate that young or actively growing cells are mainly spiral shaped (green-stained cells), but older cells undergo a degenerative change to coccoid forms (red-stained cells). Club-shaped transition cell forms were observed with NanoOrange stain. Chlorinated drinking water affected the viability but not the morphology of C. coli cells.     

Autolysins and shape change in rodA mutants of Bacillus subtilis.

The biochemical phenotype of rodA mutants was not affected by the simultaneous presence in double mutants of the lyt gene which makes them 90 to 95% deficient in autolysin action. The only morphological effect of this deficiency on the expression of the rod gene was that both the rod and the coccal forms of the mutant failed to separate and grew as long chains of cells. Inhibition of protein synthesis stopped the increase in peptidoglycan that occurred when the growth temperature for the mutants was raised to 45 degrees C. These observations support the idea that a derepression of peptidoglycan synthesis occurs at this temperature. The increased amount of cellular peptidoglycan at the higher growth temperature is not likely to be the result of the concomitant switching off of autolytic enzyme action.     

Electron microscopic study of the cytopathogenic action of meningococci in a continuous human amnion cell culture

The electron-microscopic study of the interaction of meningococci with continuous human amnion cell culture F1 has revealed that this process comprises 3 stages. The study has shown that, following the adhesion of meningococci to the surface of cells F1, these cells are invaded by individual coccal forms of meningococci. In response to infection vacuoles appear in the cytoplasm of the cells. Meningococci are either phagocytosed inside these vacuoles, or their release into the intercellular space and the death of the infected by meningococci are observed. When the cells are infected by cytopathogenic strains, the infectious process results in the appearance of degenerative changes in the cells.     

Scanning-Beam Electron Microscopy of Mycoplasma pneumoniae

The morphology and the existence of a growth cycle of Mycoplasma pneumoniae have not been clearly established. There is disagreement as to whether this organism exists as a spherical or filamentous form, and whether it progresses from filamentous to spherical forms as the organism ages. A scanning-beam electron microscope (SEM) was utilized to provide detailed observations of the cycle of morphological changes during growth phases of M. pneumoniae. Cultures of cells grown and fixed in liquid suspension displayed morphological changes from spherical to filamentous and then to larger round forms. After 8 hr to 2 days of growth (phase I), spherical forms and aggregates were revealed. Two- to 6-day-old growth (phase II) was composed of both straight and branching filaments with bulbous elements situated at intervals along their lengths, and microcolonies composed predominantly of intertwined filaments. Six- to 10-day-old growth (phase III) was characterized by flattened, spherical organisms larger than those observed in phase I, occasional membranes or ghosts, and a paucity of aggregates or microcolonies. Thus, stereo-scan electron microscopic studies suggest that M. pneumoniae undergoes an orderly and sequential metamorphosis during its life cycle.     

Electron microscopy and autoradiography study of bronchoalveolar lavage cells in nonspecific pneumonia

Cells obtained by bronchoalveolar lavage from patients with chronic nonspecific pulmonary inflammatory diseases were studied using light and electron microscopy and radioautography. Five morphological forms of alveolar macrophages, distinct in their structure and 3H-uridine content were described. A higher level of RNA synthesis has been revealed in alveolar macrophage forms 2 and 3 than in forms 1, 4 and 5; with it being lower in polymorphonuclear leukocytes and lymphocytes. It was shown that changes in the number of lavage cells and the structure-to-function characteristics in each cellular population depended on the phase of the inflammatory process. It was postulated that structural and metabolic heterogeneity of alveolar macrophages reflected the successive stages of cellular development from cell-precursors (through activation of protein synthesis) to cells with complete lysosomal cycle and the following phagocytosis.     

Differences in penicillin-binding proteins of Streptococcus pyogenes and two derived, stabilized L forms.

The penicillin-binding proteins (PBPs) of Streptococcus pyogenes and two of its derived, stabilized (i.e., nonreverting) L forms, an osmotically fragile L form and a physiologic isotonic L form, were compared. The numbers of PBPs in the membranes of these organisms were 6, 4, and 2 for the coccus and the osmotically fragile and physiologic isotonic L forms, respectively. Likewise, the relative amounts of total PBPs were 1.00: 1.48:0.32 for this coccus and the osmotically fragile and physiologic isotonic L forms, respectively. The two largest PBPs (PBPs 1 and 2) of the coccus were absent in both L forms, while the smallest PBPs (PBPs 5 and 6) were found in all three membranes. Deacylation (half-life) of three of the four PBPs in the osmotically fragile L form membrane required a significantly longer time than did deacylation of these presumed identical enzymes in the parental coccal membrane. Conversely, there was no such difference between the only two PBPs of the physiologic isotonic L form and the same coccal membrane proteins. Intact cells of all three organisms secreted PBPs and what appeared to be penicilloic acid and a minimal amount of free penicillin. A greater amount of these PBPs was secreted by both L forms than by the coccus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns and ratios of secreted PBPs were identical to those from labeled membrane preparations. These differences are correlated with some of our previous findings and are discussed in terms of inhibition of cell wall synthesis and resulting membrane changes in these two derived, stabilized coccal L forms.