The ability of flagellum-specific Proteus vulgaris bacteriophage PV22 to interact with Campylobacter jejuni flagella in culture.

BACKGROUND: There has been a recent resurgent interest in bacteriophage biology. Research was initiated to examine Campylobacter jejuni-specific bacteriophage in the Russian Federation to develop alternative control measures for this pathogen. RESULTS: A C. jejuni flagellum-specific phage PV22 from Proteus vulgaris was identified in sewage drainage. This phage interacted with C. jejuni by attachment to flagella followed by translocation of the phage to the polar region of the bacterium up to the point of DNA injection. Electron microscopic examination revealed adsorption of PV22 on C. jejuni flagella after a five minute incubation of the phage and bacteria. A different phenomenon was observed after incubating the mix under the same conditions, but for twenty minutes or longer. Phage accumulated primarily on the surface of cells at sites where flagella originated. Interestingly, PV22 did not inject DNA into C. jejuni and PV22 did not produce lytic plaques on medium containing C. jejuni cells. The constant of velocity for PV22 adsorption on cells was 7 x 10(-9) ml/min. CONCLUSION: It was demonstrated that a bacteriophage that productively infects P. vulgaris was able to bind C. jejuni and by a spot test that the growth of C. jejuni was reduced relative to control bacteria in the region of phage application. There may be two interesting applications of this effect. First, it may be possible to test phage PV22 as an antimicrobial agent to decrease C. jejuni colonization of the chicken intestine. Second, the phage could potentially be utilized for investigating biogenesis of C. jejuni flagella.

Interfacial assembly at silver nanoparticle enhances the antibacterial efficacy of nisin.

Nisin is a well-recognised antimicrobial peptide (AMP) used in food industry. However, efficacy of the peptide has been compromised due to development of resistance in different bacterial strains. Here, efficacy of the peptide upon assembly at a silver nanoparticle (AgNP) interface has been characterized. To this end, experimental and simulation studies are done to characterize the interfacial assembly of nisin and underlie antibacterial mechanism. Being an AMP, efficacy of an intact nisin is explored against Gram-positive and Gram-negative bacteria, and compared with antibacterial propensity of the interfacially assembled nisin. Antibacterial propensity, upon the assembly, increases against both kinds of bacteria. Interestingly, the growth inhibition studies of the interfacially assembled nisin indicate that the originally nisin resistant Gram-negative bacteria become sensitive to the nanomolar nisin concentrations. Furthermore, reactive oxygen species (ROS) measurements together with confocal microscopy imaging indicate that the increase in interfacial and intracellular ROS production upon the treatment is underling mechanism of enhanced antibacterial propensity of the assembled nisin. Thus, the study observed that the interfacial assembly of nisin at AgNP interface enhances the efficacy of nisin against different spectrum of bacteria, where the intact nisin is largely ineffective for the studied concentrations.

Lipopolysaccharides of flagellated and non-flagellated Proteus vulgaris strains.

Lipopolysaccharides from two strains of Proteus vulgaris were analyzed. One strain (08) was motile, giving swarming growth on solid media and the other (04)--non-flagellated, not able to swarm. Both lipopolysaccharides appeared to be heterogeneous and were separated into two fractions each. Yield, chemical composition and SDS-polyacrylamide gel electrophoresis showed differences between fractions in percentage content of 0-specific and R-core polysaccharides. Relation between ability to swarming growth of Proteus strains and heterogeneity of their lipopolysaccharides is also discussed.

[Morphologic characteristics of the large bodies in cultures of the L-forms of bacteria according to scanning electron microscope studies]. / Morfologicheskaia kharakteristika bol'shikh tel v kul'turakh L-form bakterii po dannym skaniruiushchei élektronnoi mikroskopii.

Large bodies appear at the time of protoplast and spheroplast formation and are revealed at all the L-transformation stages and at the initial stage of reversion. They can be represented both by a single giant cell and by a conglomerate of different cells connected with one another. They are not only spheroid, but can be of the most varied shape, and structurally they are connected with other L-colony elements: filamentous structures, spheroid cells, elementary bodies and the so-called acellular material. At the early L-transformation stage the large bodies probably appear as a result of coalescence of lysed cells and represent polygenome formations. Elementary bodies and spheroid cells form within the large bodies and on their surface at the late stage of L-transformation. In case of reversion bacterial cells form from them.

[Ultrastructural characteristics of Proteus vulgaris and Proteus mirabilis cells differing in the capacity for swarming]. / Ul'trastrukturnye osobennosti mikrobnykh kletok Proteus vulgaris i Proteus mirabilis, razlichaiushchikhsia po sposobnosti k roeniiu.

Specific differences in the structure of colonies and the location of microbial cells in colonies, characteristic for aggregating and nonaggregating genetically related pairs of P. vulgaris and P. mirabilis strains, have been demonstrated by means of transmission and scanning electron microscopy. In calculating the number of flagellae per 100 outlines of microbial bodies revealed in negatively stained preparations, the fact that both aggregating and nonaggregating bacteria possess practically the same number of flagellae, on the average 4-8 flagellae per microbial cell outline, has been established. This fact indicates that the presence of flagellae in microbial cells is unrelated to their capacity for swarming.

[Morphologic characteristics of spheroid elements in a culture of bacterial L-forms according to scanning electron microscopy findings]. / Morfologicheskaia kharakteristika sharovidnykh élementov v kul'ture L-form bakterii po dannym skaniruiushchei élektronnoi mikroskopii.

The authors studied stable L-cultures of Proteus valgaris, Bac. subtillis, Staphylococcus aureus, Streptococcus pyogenes of group A, and also unstable cultures of the L-forms of Proteus vulgaris and Proteus vulgaris culture at the stage of spheroplasts. Spheroid cells proved to appear at the stage of spheroplasts, prevailed at the log phase in stable and unstable L-cultures, but were less frequent at the stationary phase. Cross section of L-colonies showed that they were located at the surface. The size of spheroid elements was from 1 to 5 micron; their surface was smooth or slightly wrinkled with numerous protrusions and individual sockets. The spheroid cells were distributed in the colonies freely, in clusters, or were connected to one another by anastomosis. Several methods of reproduction of spheroid cells are described, including equal and unequal binary fission, budding, and formation of elementary bodies within the cell. Morphological connection of spheroid cells with large bodies, filamentous structures and structureless matrix of the L-colony apparently pointed to their origin from the corresponding elements of the L-cultures.

[A study of Proteus L forms during reversion by means of electron microscopy and ultrathin sections]. / Izuchenie L-form protea v protsesse reversii metodom skaniruiushchei élektronnoi mikroskopii i ul'tratonkikh srezov

The method of scanning electron microscopy was applied to the study of the Proteus L-forms which reversed directly on the millipore filters. It appeared that reversion was accompanied by elongation of the cell, piriform cells forming as the result. Filamentous cells segmented into short fragments and large bodies with a spongy surface structure with numerous ruptures were also revealed in the reversing culture. The method of ultrathin sections showed the other membrane in many cells to be fragile and readily twisting forming vesicles collecting into chains. As supposed, this is connected with degenerative changes involving the outer membrane of the paternal L-forms at the early reversion periods. An interrupted structure of the outer membrane was also demonstrated in many cells. It is supposed that in this case a synthesis of the outer membrane de novo at the later stages of reversion is dealt with. Phenomenon of "extrusion" of the multilayer structures into the periplasmic space, characteristic of spheroplasts, is described. A marked similarity of the morphological picture of reversion and at the initial stages of L-transformation, i.e. of the processes representing a looking glass reflection of one another, is described.

[Electron microscopic study of pathogenic bacteria on environmental objects]. / Elektronno-mikroskopicheskoe issledovanie patogennykh bakterii na ob"ektakh vneshnei sredy.

The morphological picture of different bacteria (Salmonella typhimurium, Proteus vulgaris, Klebsiella pneumoniae, Pseudomonas aeruginosa, Yersinia enterocolitica O3, Y.pseudotuberculosis 1, Y.frederiksenii, Y.intermedia, Y.kristensenii) on environmental objects was studied with the use of scanning electron microscopy (SEM). Bacteria adhered to the surface of pieces of fodder, egg shell, cabbage leaves and form microcolonies, whose morphology was similar to colonies, grown on nutrient media. The cells produced extracellular substances, seen in SEM as integuments. These integuments were gourd to protect the population from the action of unfavorable factors.

[Modes of the formation of elementary bodies and their isolation from the cell in L-form bacteria]. / Sposoby obrazovaniia élementarnykh tel i ikh vydeleniia iz kletki u L-form bakterii.

Elementary bodies are formed on the cell surface and inside the cell body in all cell types characteristic of L-form cultures, i. e. spherical cells, large bodies and filament structures. The following ways of elementary body formation are described: by budding on the cell surface, appearance immediately in the cytoplasm, in the vacuole, as a result of cytoplasmic fragmentation accompanied by the lysis of the cell, as well as in cases of the separation of cytoplasmic areas surrounded by the membrane or the myelin-like structure. The release of elementary bodies from the cell occurs as a result of the lysis or death of the mother cell, the thinning of the vacuole wall, and possibly due to small transient defects in the membrane, not accompanied by the death of the mother cell. The scheme of the formation and release of elementary bodies from the cell is presented.

[Various data concerning the ultrastructure of Proteus spheroplasts]. / Nekotorye dannye po ul'trastrukture sferoplastov u protea

Spheroplasts were obtained under the action of penicillin on the same medium which was later used to obtain the L-forms. Spheroplast formation started 15 to 20 minutes after the addition of penicillin and reached the maximum in 2 hours. The first dividing forms were revealed at that time, and this division continued for at least 24 hours. The majority of the cells represented spheroplasts surrounded by outer and cytoplasmic membrane, and only some--true protoplasts--had cytoplasmic membrane alone. Division was anomalous in comparison with the bacterial forms with the cell wall: it was noted that either both daughter cells were surrounded by a common outer membrane, or one daughter cell had two membranes serving as a spheroplast and the other--one membrane, serving as a true protoplast. Individual vesicles and myelin-like structures were found to be extruded into the periplasmic space or directly into the surrounding environment. In the latter case pearl-like structures described by Ryter in the formation of protoplasts in bacilli were observed. However, in the Proteus such structures were largely formed by the material of the plastic layer of the cell wall, and to a lesser extent--by the lipoproteid membrane.

[The ultrastructure of L-forms. III. The L-forms of S. typhi and Pr. vulgaris]. / Ul'trastruktura L-form. Soobshchenie III. L-formy S. typhi i Pr. vulgaris

A marked polymorphism was revealed in the colonies of 5--7 day L-forms of S. typhi and Pr. vulgaris: among different types of the cells there prevailed spherical or oval forms with a distinct nucleoid. Three-layer cytoplasmic membranes--the site of formazan TNBT deposition in the reaction for succinic dehydrogenase--were detected on the greater part of the cell surface. Sometimes there were revealed on the surface of the membrane residues of the cell wall in the form of structureless material of low electron density or pieces of external membrane. Myelin-like structures, mesosomes of ring or lamellar structure and two-contour vesicles located directly in the cytoplasm were found in the cytoplasm. L-form cells displayed binary fission or reproduced by budding; in the latter case there formed elementary bodies surrounded by a three-layer membrane, not infrequently with a visible nucleoid and structures resembling mesosomes of the lamellar type.

[Characteristics of various morphological and biological properties of bacteria of the genus Proteus]. / Kharakteristika nekotorykh morfologicheskikh i biologicheskikh svoistv bakterii roda Proteus.

The stable O-form of Proteus has been found to differ from the initial H-form in the absence of flagellae or in a few weakly developed flagellae, in decreased capacity for agglutination with polyvalent and typing sera and in virulence for laboratory animals. As revealed in this study, the conversion of the H-form of Proteus into the stable O-form leads to the loss of resistance to streptomycin, chloramphenicol, tetracycline, monomycin, ampicillin and neomycin.

[Study of the sequential stages of Proteus vulgaris L-transformation by scanning electron microscopy]. / Izuchenie posledovatel'nykh étapov L-transformatsii u Proteus vulgaris metodom skaniruiushchei élektronnoi mikroskopii.

Consecutive stages of L-transformation caused by penicillin in Proteus vulgaris were studied, from spheroplasts and unstable, easily reversing L-forms to stable L-forms which did not reverse. To maintain natural topography of the culture on the growth medium, millipore filters were placed on the surface of the medium and then were fixed with the culture. All the main elements of L-cultures were found at the stage of spheroplast formation, before the first passage onto the medium for cultivating L-forms, i.e. spherical cells having a diameter from 3.5 to 1 mcm, elementary bodies 1.0-0.6 mcm large, filamentous structures with a diameter to 4 mcm, large bodies having dimensions of 20 mcm and more, and "cell-free" masses. The composition of the structural elements and their reproduction are supposed to be almost the same in the bacterial culture and L-forms at the stage of spheroplast formation.

[New data on the karyology of formation of L forms of Proteus vulgaris]. / Novye dannye o kariologii obrazovaniia L-form Proteus vulgaris

The growth and development of the Proteus vulgaris culture as well as the process of formation of its L-forms were studied following the Pitzurra method on slides covered with an agar layer. The latter were kept upon inoculation in a vertical position in a moist chamber at 37 degrees C. On fixed and stained after Giemza preparations, it was possible to follow the consecutive stages of the cytoplasmic "hernia" formation due to the local disruption of the bacillary cell wall. It was easy to disern the penetration of the nucleoid into this evagination. The growth of the formed cytoplast was followed by multiple reduplication of the nucleoid which ultimately grew out into a scalloped ribbon whose length served to estimate the ploidy of the developing speroplast. Gigantic individuals with massive nuclei were observed. The ploidy of such nuclei could reach 2000--4000 haploid units. The data of light microscopy were confirmed by electronograms of the ultrathin sections of the L-forms of Proteus vulgaris. Frame-by-frame microcinematography of the multiplying Proteus cells grown in the Fonbrun oil chamber and photographed with the aid of the Peshkoff loop-hole objectives has made it possible to register the division of the nuclear apparatus of bacillary forms. On penicillin-containing media, it was easy to observe the formation of the cytoplasmic "hernia", the penetration into it of the nuceloid, and its successive multiplication in the growing cytoplast to form a massive nucleal mass.

Immunoelectron microscopy of Proteus vulgaris by the plasma polymerization metal-extraction replica method: differential staining of flagellar (H) and somatic (O) antigens by colloidal golds.

Flagellar (H) and somatic (O) antigens of Proteus vulgaris were differentially stained with antibodies coupled to different sizes of colloidal gold particles, and the distribution of these antigens was visualized by the plasma polymerization metal-extraction replica (PMR) method. The H antigen, labeled with 5 nm colloidal gold, was almost exclusively located on the flagella, whereas the O antigen, labeled with 10 nm colloidal gold, was almost exclusively located on the bacterial body. The marker gold particles were clearly observed as electron-dense particles on the relatively low contrast background of three-dimensional replica image of the flagellated bacteria. Thus, the PMR method may prove to be a useful tool for studying the localization of multiple substances on the cell surface, at a high resolution and in three dimensions. The diameter of the flagella measured by the replica method was about 15 nm, close to the value obtained by negative staining (16 nm). When treated with anti-flagellar (H) factor serum and protein A-gold, the diameter of flagella was significantly increased to about 35 nm. This increase in diameter was presumably caused by binding of immunoglobulins to H antigens of flagella.

Growth and cell structure of Proteus vulgaris when cultivated in weightlessness in the Cytos apparatus.

Growth data and electron-microscopic analyses are presented for Proteus vulgaris cultures which were grown during space flight in polyethylene packets in a semisolid medium with Tryptose for 96 h. In the suboptimal culture conditions the growth and morphological characteristics of the flight and ground control variants were nearly identical, but we were able to detect a number of differences between the cellular ultrastructure of these variants. These differences testify to changes in the bacterial cell metabolism during space flight.

[Spheroplasts of Proteus studied by scanning electron microscopy, freeze etching and ultrathin sections]. / Izuchenie sferoplastov protea metodom skaniruiushchei élektronnoi mikroskopii, zamorazhivaniia--travleniia i ul'tratonkikh srezov

Spheroplasts are formed 15 min after the addition of penicillin. Swellings appear at the end or in the middle of the cell. Buds were found on the surface of the spheroplasts. As was found by scanning electron microscopy, the surface of the spheroplasts is folded and, during later stages, wrinkled. "Expulsion" of mesosomes into the periplasmic space was found, as well as separation of vesicles, formed by the outer and cytoplasmic membranes, from the cell surface. The two fracture faces of the outer membrane are uncomplementary; the convex fracture face is mainly smooth but contains occasional dents of the rod-like shape while the concave face does not contain them. The outer (true) surface of the cell wall is smooth. The similarity and differences between the protoplasts, spheroplasts and L forms are discussed . The protoplasts and spheroplasts may divide, grow and multiply under definite conditions of cultivation, like the L forms do, but they do not possess the well developed intracytoplasmic membrane system typical of the L forms.

Surface characteristics of gram-negative and gram-positive bacteria in an atomic force microscope image.

Bacterial images can be obtained rather easily with an atomic-force microscope (AFM) in the magnification range of 5,000 to 30,000 times without any pretreatment of the specimens for such observations as chemical fixation, dehydration or staining. The bacterial shapes or the presence of flagella can be clearly recognized in these magnification ranges. In addition, we were also able to distinguish between gram-negative and gram-positive bacteria based on the specific wavy surface appearance of the former. AFM could thus be a useful tool for the identification of bacteria in the resolution range between electron and light microscopy.

Demonstration of cell division by septation in a variety of gram-negative rods.

Through use of an initial fixative employing a combination of crotonaldehyde and glutaraldehyde, septa were preserved in thin sections of dividing cells of strains of Pseudomonas aeruginosa, Salmonella typhimurium, Shigella sonnei, and Escherichia coli when grown at 30 C in a dilute basal medium. The same procedures, however, revealed only a constrictive division process in Proteus vulgaris and Erwinia sp. This adds to the evidence that septation, although difficult to demonstrate, is the process of cell division in the enteric gram-negative rods and the pseudomonads and that constriction is a fixation artifact in these organisms.