Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Adhesion of bacteria to epithelial cell surfaces within the reticulo-rumen of cattle.

Blocks of tissue were removed from various locations in the bovine digestive tract and fixed and processed for transmission and scanning electron microscopy by techniques that retained adherent bacteria. The distribution of bacteria on the surface of epithelial cells was examined by scanning electron microscopy. This showed intermittent colonization of the epithelia with the formation of occasional microcolonies of morphologically similar bacterial cells. Transmission electron microscopy of ruthenium red-stained material showed the presence of both the glycocalyx of the bovine epithelial cells and fibrous carbohydrate coats surrounding adherent bacteria. The carbohydrate coats appeared to mediate the attachment of bacteria to the epithelium, to food particles, and to each other so that microcolonies were formed. Careful examination of the bacterial colonization of keratinized cells in the process of being sloughed from the surface of the stratified squamous epithelium of the rumen showed that these dead cells were digested by adherent bacteria of a limited number of morphological types. The spatial relationship of this mixed, adherent, microbial population to living and dead epithelial cells and to food particles indicates that digestive processes of some importance may be accomplished by this stationary component of the microbial flora of the digestive tract.     

Transfer of members of the genus Falcivibrio to the genus Mobiluncus, and emended description of the genus Mobiluncus

It has long been thought that the genera Mobiluncus and Falcivibrio contain the same organisms. Using a polyphasic approach, it was found that Mobiluncus curtisii and Mobiluncus mulieris were the same as Falcivibrio vaginalis and Falcivibrio grandis, respectively. As the genus name Mobiluncus takes precedence, it is proposed that F. vaginalis and F. grandis be transferred to the genus Mobiluncus. In agreement with previous studies, results from phenotypic tests did not support the separation of M. curtisii strains into its two subspecies, M. curtisii subsp. curtisii and M. curtisii subsp. holmesii. Phenotypic complexity within M. curtisii dictates that the species should be treated as a complex until more in-depth analyses of the species have been performed. Phylogenetic analyses, based on 16S rRNA gene sequences, demonstrated that the genus Mobiluncus was associated with Varibaculum cambriense and the two subspecies of Actinomyces neuii, and that A. neuii is only distantly related to Actinomyces sensu stricto.     

The range of antagonistic effects of Lactobacillus bacterial strains on etiologic agents of bacterial vaginosis

Bacterial vaginosis is caused by uncontrolled sequential overgrowth of some anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia, Bacteroides spp., Peptostreptococcus spp., Mobiluncus sp. usually occurring in stable numbers in the bacterial flora of healthy women. On the other hand, different species of bacteria belonging to the genus Lactobacillus, most frequently L. plantarum, L. rhamnosus and L. acidophilus, form a group of aerobic bacteria dominating in the same environment. The diversity and density of their populations depend on the age and health conditions. Thanks to their antagonistic and adherence properties bacteria of the genus Lactobacillus can maintain a positive balance role in this ecosystem. The aim of this study was to assess the antagonistic properties of Lactobacillus strains isolated from the vagina of healthy women against most common agents of bacterial vaginosis. It was found that nearly all of the tested Lactobacillus strains exerted distinct antagonistic activity against anaerobic bacteria: Gardnerella vaginalis, Prevotella bivia and Peptostreptococcus anaerobius and quite a number also against Gram-negative rods, while only some of them were able to inhibit Gram-positive aerobic cocci as Enterococcus faecalis or Staphylococcus aureus.     

A numerical taxonomic study of anaerobic gram-negative bacilli classified as Bacteroides ureolyticus isolated from patients with non-gonococcal urethritis

A numerical taxonomic study of 64 strains of anaerobic Gram-negative bacilli isolated from men with non-gonococcal urethritis, two unclassified laboratory strains of 'corroding bacilli', and 12 other strains of anaerobic Gram-negative bacilli, including nine received as anaerobic curved rods and three as 'Bacteroides corrodens' (B. ureolyticus), isolated from women with bacterial vaginosis, was undertaken. Seventeen reference anaerobic strains belonging to the genera Bacteroides, Fusobacterium, Mobiluncus, Mitsuokella and Wolinella were included. Morphological, biochemical and physiological characteristics were examined in 103 tests. The resemblance between the 95 strains was calculated using the SSM, SJ and DP coefficients for cluster analyses based on the UPGMA method. All three approaches gave similar groupings, and the estimated average probability of test error was 2.46%. The strains fell into 10 phenons. The unclassified strains from men and three from women with lower genital-tract infections, and the laboratory strains of 'corroding bacilli' clustered in one phenon with the reference strains of B. ureolyticus, indicating that they correspond to B. ureolyticus. The other unclassified strains of anaerobic curved rods clustered as a distinct phenon. They correspond to species of the newly described genus Mobiluncus. The taxonomic data and the compilation of diagnostic tables serve as a useful guide for the laboratory identification of clinical isolates regarded as B. ureolyticus.     

Light and electron microscopic study of the bacterial adhesion to termite flagellates applying lectin cytochemistry

Summary Many of the flagellates inhabiting the hindgut of lower termites are associated with ectobiotic, rod-like bacteria or spirochetes. Different types of attachment sites are present. Electron dense material underlies, e.g., the plasma membrane ofJoenia annectens at the contact site, whereas other attachment sites do not show any visible specializations. The host cell's glycocalyx may, however, be reduced at the attachment sites as it is the case inDevescovina glabra. The thick glycocalyx ofStephanonympha nelumbium is not changed at the sites where bacterial rods attach, but spirochetes penetrate to a certain extent. Bacteria which colonize the extracellular surface structures ofMicrorhopalodina multinucleata express their own glycocalyx to mediate a contact. In this study we focussed on the examination of one common mode of interaction between bacteria and their host cells, i.e., adhesion via lectins and sugars. The sugar composition was analysed by light and electron microscopic labelling experiments using the lectins Con A, WGA and SBA. In general, only the posterior body surface ofJoenia which is colonized with bacteria is labelled. The demonstrated sugars are found in fibrous glycocalyx portions surrounding the attachment sites of the bacteria. Such glycocalyx fibres in combination with the electron dense material supporting the attachment sites seem to be the prerequisites for bacterial attachment. InD. glabra, however, a role for sugars in mediating the attachment could not be demonstrated. Removal of the ectobiotes using antibiotics revealed that the specialized contact sites ofJoenia are present in the absence of bacteria and thus possibly serve to attract bacteria. Nothing, however, remains of the former attachment sites in bacteria-freeDevescovina cells. Attachment sites in this case could be induced by bacterial contact. There is not one general mechanism for bacterial attachment to termite flagellates; rather, adhesion seems to follow different strategies.Abbreviations Con A concanavalin A - DAB 3,3-diaminobenzidine tetrahydrochloride - DAPI 4,6-diamidino-2-phenylindole - DIC differential interference contrast - FA formaldehyde - FITC fluorescein isothiocyanate - GA glutaraldehyde - PB Soerensen's phosphate buffer - PC phase contrast - pen/strep penicillin and streptomycin - SBA soybean agglutinin - SEM scanning electron microscope - TBS Tris buffer saline - TEM transmission electron microscope - WGA wheat germ agglutinin Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Persistence and multiplication of obligate anaerobe bacteria in amebae under aerobic conditions

After co-cultivation of Mobiluncus curtisii, an obligate non-sporeforming anaerobe, with free living amebae from the Acanthamoeba spp. under aerobic conditions, internalization, multiplication and persistence of bacterial cells were established for at least 4-6 weeks. Under the same conditions and media without viable amebae, the cells of M. curtisii did not replicate and died in 4-7 days. The infection of amebae occurred with 10 to 100 bacteria per ml of co-cultivation media. In 7-14 days the amount of bacterial cells increased to 1x10(5)-1x10(6) CFU/mL. Electron microscopic examinations revealed bacteria within vacuoles in the amebae and intracellular replication. These results suggest a previously undescribed mechanism for spread, replication and persistence of obligately anaerobe bacteria in the environment and new possible sources, reservoirs and transfer mechanisms of infections caused by obligate anaerobe bacteria.     

The cytologist and bacterioses of the vaginal-ectocervical area. Clues, commas and confusion

To evaluate the role of the cytologist in the diagnosis of bacterial vaginosis, the predominant bacterial patterns seen in 157 Papanicolaou-stained cervical smears were assessed and classified as large bacillus, anaerobic or scanty. A large bacillus pattern was found in 73 smears (46%), an anaerobic pattern in 77 (49%) and scanty bacteria in 7 (5%). Comma-shaped (curved) bacilli were found in 34 smears. The prevalence of clue cells in smears with anaerobic patterns was 73%. Gardnerella vaginalis was cultured from 89% of the cases with anaerobic-type smears containing clue cells and from 88% of those with anaerobic-type smears lacking clue cells. Mobiluncus sp. was cultured from 83% of the cases with anaerobic-type smears showing curved bacilli and 14% of those with anaerobic-type smears lacking curved bacilli. Papanicolaou-stained smears were found to be more sensitive and more specific for identifying clue cells than were wet preparations. A positive association was found between a positive KOH "whiff" test and the presence of curved bacilli on the Papanicolaou-stained smear. The clinical significance of these findings is discussed.     

Bacterial adhesion to sulcular epithelium in periodontitis

The purpose of this study was to investigate, by electron microscopy, the type of bacterial attachment to the sulcular epithelium in periodontitis. Gingiva biopsies were observed in a transmission electron microscope using cytochemical staining with ruthenium red for glycocalyx visualisation. In addition, subgingival plaque samples and biopsies from the sulcular epithelium in periodontitis from the patients were estimated microbiologically. Aerobic bacteria only were estimated in the subgingival plaque and both aerobic and anaerobic bacteria in the gingival biopsies. No bacterial internalisation could be observed. Fimbria-mediated adhesion as the only type of bacterial attachment and a large diversity of bacterial glycocalyces were detected. As the fimbrial adhesins of putative periodontal pathogens are able in vitro to induce inflammation and bone resorption via stimulation of the proinflammatory cytokine production, the demonstrated fimbrial adhesins suggest the significant role of bacterial adhesion to sulcular epithelium in periodontitis.     

Normal vaginal aerobic and anaerobic bacterial flora of the rhesus macaque (Macaca mulatta).

The most common bacterial species isolated from the vaginas of 37 healthy rhesus macaques were Streptococcus viridans, coagulase negative Staphylococcus, Mobiluncus curtisii ss. curtisii, Corynebacterium renale-like organisms, Peptostreptococcus anaerobius, Gardnerella-like organisms, and other Corynebacterium species. The vaginal flora of the rhesus macaque differs from that previously reported for five other primate species. A two-year retrospective review of clinical cases of vaginitis and metritis found Escherichia coli and coagulase positive Staphylococci to be the most common pathogens isolated.     

In situ morphology of the gut microbiota of wood-eating termites [Reticulitermes flavipes (Kollar) and Coptotermes formosanus Shiraki].

Light microscopy and scanning and transmission electron microscopy were used to examine the in situ morphology of the gut microbiota of Reticulitermes flavipes and Caoptotermes formosanus. Laboratory-maintained termites were used and, for R. flavipes, specimens were also prepared immediately after collection from a natural infestation. The latter endeavor enabled a study of different castes and developmental stages of R. flavipes and revealed differences in the microbiota of field versus laboratory specimens. The termite paunch microbiota consisted of an abundance of morphologically diverse bacteria and protozoa. Thirteen bacterial morphotypes in the paunch were described in detail: seven were observed only in R. flavipes, three were observed only in C. formosanus, and three were common to both termite species. The paunch epithelium was densely colonized by bacteria, many of which possessed holdfast elements that secured them tightly to this tissue and to other bacterial cells. Besides bacteria, the protozoan Pyrsonympha vertens adhered to the paunch epithelium of R. flavipes by means of an attachment organelle. Cuplike indentations were present on the paunch epithelial surface and were sites of bacterial aggregation. Ultrastructural features of cups suggested their involvement in ion absorption. In addition to the paunch, the midgut was also colonized by bacteria that were situated between epithelial microvilli. Results suggest that bacteria are an integral part of the gut ecosystem.     

Electron microscopic examination of wastewater biofilm formation and structural components

This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties.     

Electron microscopic examination of wastewater biofilm formation and structural components.

This research documents in situ wastewater biofilm formation, structure, and physiochemical properties as revealed by scanning and transmission electron microscopy. Cationized ferritin was used to label anionic sites of the biofilm glycocalyx for viewing in thin section. Wastewater biofilm formation paralleled the processes involved in marine biofilm formation. Scanning electron microscopy revealed a dramatic increase in cell colonization and growth over a 144-h period. Constituents included a variety of actively dividing morphological types. Many of the colonizing bacteria were flagellated. Filaments were seen after primary colonization of the surface. Transmission electron microscopy revealed a dominant gram-negative cell wall structure in the biofilm constituents. At least three types of glycocalyces were observed. The predominant glycocalyx possessed interstices and was densely labeled with cationized ferritin. Two of the glycocalyces appeared to mediate biofilm adhesion to the substratum. The results suggest that the predominant glycocalyx of this thin wastewater biofilm serves, in part, to: (i) enclose the bacteria in a matrix and anchor the biofilm to the substratum and (ii) provide an extensive surface area with polyanionic properties.     

Leech mycetome endosymbionts are a new lineage of alphaproteobacteria related to the Rhizobiaceae

Mycetomal organs attached to the esophagus of hematophagous leeches which are known to harbor endosymbiotic bacteria were removed from three species in the leech family Glossiphoniidae. Anatomical observations indicated that placobdellid mycetomes are paired and caecate, inserting into the esophagus posterior to the proboscis. Light and electron microscopy demonstrated that there is a single layer of mycetome epithelial cells harboring gram-negative rods and that these epithelial cells are ultrastructurally distinct from neighboring esophageal epithelial cells. Fluorescent in situ hybridization with eubacterial and alphaproteobacterial probes localized the bacteria solely to the mycetomes both in adult and in unfed juvenile leeches whereas a gammaproteobacterial probe did not yield a bound fluorescencent signal. DNA was isolated from these tissues and subjected to PCR amplification using bacteria-specific primers for 16S and 23S rDNA. Results from sequencing the amplification products and phylogenetic analysis with other Alphaproteobacteria revealed that the bacteria resident in these organs comprise a new genus of Alphaproteobacteria, Reichenowia n. gen., closely related to the nitrogen-fixing, nodule-forming Rhizobiaceae. The three bacterial strains, though different from each other were each other's closest relatives, suggesting a history of close coevolution with their leech hosts.     

Studies on the surface coat (glycocalyx) of the dauer larva of Anguina agrostis

Imprints of the surface coat (glycocalyx) from the cuticles of living second stage dauer larvae (DL2) of Anguina agrostis (syn. A. funesta) have been examined using incident light fluorescence microscopy and scanning electron microscopy. These surface coats contain residues of N-acetyl-D-glucosamine which were detected by treatment with wheat germ agglutinin labelled with either fluorescein or rhodamine. They also contain protein which was demonstrated by treatment with either pepsin or trypsin. These enzymes inhibited the attachment of the coryneform bacterium Clavibacter sp. to the surface coat, indicating that proteins play a crucial role in the adhesion of these bacteria to the nematode. This inhibition of attachment was reversed within 18 h after removal of the DL2 from the enzymes, indicating that the nematode was capable of renewing its surface proteins.     

Effect of Motility on Surface Colonization and Reproductive Success of Pseudomonas fluorescens in Dual-Dilution Continuous Culture and Batch Culture Systems

The colonization of glass surfaces by motile and nonmotile strains of Pseudomonas fluorescens was evaluated by using dual-dilution continuous culture (DDCC), competitive and noncompetitive attachment assays, and continuous-flow slide culture. Both strains possessed identical growth rates whether in the attached or planktonic state. Results of attachment assays using radiolabeled bacteria indicated that both strains obeyed first-order (monolayer) adsorption kinetics in pure culture. However, the motile strain attached about four times more rapidly and achieved higher final cell densities on surfaces than did the nonmotile strain (2.03 × 10⁸ versus 5.57 × 10⁷ cells vial^-1) whether evaluated alone or in cocultures containing motile and nonmotile P. fluorescens. These kinetics were attributed to the increased transport of motile cells from the bulk aqueous phase to the hydrodynamic boundary layer where bacterial attachment, growth, and recolonization could occur. First-order attachment kinetics were also observed for both strains by using continuous-flow slide culture assays analyzed by image analysis. The DDCC system contained both aqueous and particulate phases which could be diluted independently. DDCC results indicated that when cocultures containing motile and nonmotile P. fluorescens colonized solid particles, the motile strain replaced the nonmotile strain in the system over time. Increasing the aqueous-phase rates of dilution decreased the time required for extinction of the nonmotile strain while concurrently decreasing the overall carrying capacity of the DDCC system for both strains. These results confirmed that bacterial motility conveyed a selective advantage during surface colonization even in aqueous-phase systems not dominated by laminar flow.     

High motility reduces grazing mortality of planktonic bacteria

We tested the impact of bacterial swimming speed on the survival of planktonic bacteria in the presence of protozoan grazers. Grazing experiments with three common bacterivorous nanoflagellates revealed low clearance rates for highly motile bacteria. High-resolution video microscopy demonstrated that the number of predator-prey contacts increased with bacterial swimming speed, but ingestion rates dropped at speeds of >25 microm s(-1) as a result of handling problems with highly motile cells. Comparative studies of a moderately motile strain (<25 microm s(-1)) and a highly motile strain (>45 microm s(-1)) further revealed changes in the bacterial swimming speed distribution due to speed-selective flagellate grazing. Better long-term survival of the highly motile strain was indicated by fourfold-higher bacterial numbers in the presence of grazing compared to the moderately motile strain. Putative constraints of maintaining high swimming speeds were tested at high growth rates and under starvation with the following results: (i) for two out of three strains increased growth rate resulted in larger and slower bacterial cells, and (ii) starved cells became smaller but maintained their swimming speeds. Combined data sets for bacterial swimming speed and cell size revealed highest grazing losses for moderately motile bacteria with a cell size between 0.2 and 0.4 microm(3). Grazing mortality was lowest for cells of >0.5 microm(3) and small, highly motile bacteria. Survival efficiencies of >95% for the ultramicrobacterial isolate CP-1 (< or =0.1 microm(3), >50 microm s(-1)) illustrated the combined protective action of small cell size and high motility. Our findings suggest that motility has an important adaptive function in the survival of planktonic bacteria during protozoan grazing.     

Behavior ofPseudomonas fluorescens within the hydrodynamic boundary layers of surface microenvironments

Phase, darkfield, and computer-enhanced microscopy were used to observe the surface microenvironment of flow cells during bacterial colonization. Microbial behavior was consistent with the assumptions used previously to derive surface colonization kinetics and to calculate surface growth and attachment rates from cell number and distribution. Surface microcolonies consisted of closely packed cells. Each colony contained 2ⁿ cells, where n is the number of cell divisions following attachment. Initially, cells were freely motile while attached, performing circular looping movements within the plane of the solid-liquid interface. Subsequently, cells attached apically, maintained a fixed position on the surface, and rotated. This type of attachment was reversible and did not necessarily lead to the formation of microcolonies. Cells became irreversibly attached by progressing from apical to longitudinal attachment. Longitudinally attached cells increased in length, then divided, separated, moved apart laterally, and slid next to one another. This resulted in tight cell packing and permitted simultaneous growth and adherence. After approximately 4 generations, individual cells emigrated from developing microcolonies to recolonize the surface at new locations. Surface colonization byPseudomonas fluorescens can thus be subdivided into the following sequential colonization phases: motile attachment phase, reversible attachment phase, irreversible attachment phase, growth phase, and recolonization phase.     

Ellipsometric Measurement of Bacterial Films at Metal-Electrolyte Interfaces

Ellipsometric measurements were used to monitor the formation of a bacterial cell film on polarized metal surfaces (Al-brass and Ti). Under cathodic polarization bacterial attachment was measured from changes in the ellipsometric angles. These were fitted to an effective medium model for a nonabsorbing bacterial film with an effective refractive index (n_f) of 1.38 and a thickness (d_f) of 160 ± 10 nm. From the optical measurements a surface coverage of 17% was estimated, in agreement with direct microscopic observations. The influence of bacteria on the formation of oxide films was monitored by ellipsometry following the film growth in situ. A strong inhibition of metal oxide film formation was observed, which was assigned to the decrease in oxygen concentration due to the presence of bacteria. It is shown that the irreversible adhesion of bacteria to the surface can be monitored ellipsometrically. Electrophoretic mobility is proposed as one of the factors determining bacterial attachment. The high sensitivity of ellipsometry and its usefulness for the determination of growth of interfacial bacterial films is demonstrated.     

Characterization of anaerobic heterotrophic bacteria isolated from freshwater lake sediments.

Strict anaerobic culture techniques were used to quantitatively and qualitatively evaluate the anaerobic heterotrophic bacteria present at the sediment-water interface of hyperutrophic Wintergreen Lake (Augusta, Mich.). Anaerobic plate counts remained constant from March through December, 1973, ranging from 2.4 X 10(6) to 5.7 X 10(6) organisms/g (dry weight) of sediment. The isolatable bacteria represented a small percentage of the total microbial community, which was shown by direct microscopic counts to be 2.0 X 10' organisms/g (dry weight) of sediment during June and July. Bacteria of the genus Clostridium dominated the isolates obtained, accounting for 71.8% of the 960 isolates examined. A single species, Clostridium bifermentens, comprised 47.7% of the total. Additional bacterial groups and the percentage in which they were isolated included: Streptococcus sp. (10.8%), unidentified curved rods (9.5%y, gram-positive nonsporing rods (5.6%), and motile gram-negative rods (1.9%). Temperature growth studies demonstrated the ability of all the isolates to grow at in situ sediment temperatures. Gas-liqid radiochromatography was used to determine the soluble metabolic end products from [U-14C]glucose and a U-14C-labeled amino acid mixture by representative sedimentary clostridial isolates and by natural sediment microbial communities. At in situ temperatures the natural sediment microflora produced soluble fermentative end products characteristic of those elaborated by the clostridial isolates tested. These results are considered strong presumptive evidence that clostridia are actively metabolizing in the sediments of Wintergreen Lake.