Attachment as a factor in the protection of Enterobacter cloacae from chlorination

Enterobacter cloacae attached to drinking water distribution particles was subjected to chlorination. Attachment resulted in the protection of these organisms from disinfection. This effect was found to be dependent upon both the level of chlorine in the system and attachment time. The results obtained in this study indicate that attached organisms may play an important role in coliform outbreaks.     

Acid resistance of attached organisms and its implications for the pathogenicity of plasmid-bearing Escherichia coli

Organisms of Escherichia coli attached to glass beads in a model attachment system were more resistant to acid than were unattached organisms and this applied to cultures exposed to either pH 2·5 or 3·5. Attached organisms of both Col^- and Col V, I-K94⁺ strains showed the effect but with prolonged exposure to pH 2·5, the attached Col V⁺ organisms appeared more resistant than the attached Col^- ones, possibly because they formed a thicker surface layer. It is proposed that the increased resistance of attached organisms to pH 2·5 might allow the survival, in gastric acid, of organisms attached to food particles. This would be more significant for the Col V⁺ strains because the plasmid enhances enterobacterial attachment properties. The increased resistance of attached organisms to pH 3·5 might be significant for survival of particle- or surface-bound organisms in the acidic environment of the phagocyte especially since lactate (which occurs in phagocytes) enhanced the effect of exposure to pH 3·5.     

Bacteriophage resistance of attached organisms as a factor in the survival of plasmid-bearing strains of Escherichia coli

Unattached organisms of plasmid-free and plasmid-bearing strains of Escherichia coli showed marked sensitivity to phages T4, Tula and K3 on incubation in broth at 37°C but organisms attached to glass beads or sand were resistant. Phage T4 sensitivity of free organisms and resistance of glass bead-attached ones were also observed when incubation was in broth at 15° or 20°C or anaerobically at 37°C. In contrast, free and attached organisms were resistant at 37°C or lower temperature when incubation was in poor media. It seems likely that the presence of phage will be a major factor reducing survival in the intestine and in sewage and that attachment (which is more significant for strains bearing certain plasmids) will protect. In contrast, survival of either free or attached organisms in polluted water will probably not be significantly influenced by the presence of phage.     

Epimerite-host epithelium relationships among eugregarines parasitizing the damselflies Enallagma civile and Ischnura verticalis.

The host-parasite interface between 2 species of damselflies and 4 species of eugregarines was examined at the ultrastructural level. Nubenocephalus nebraskensis organisms attached to the host midgut epithelium by means of a sucker-like protomerite; the space between the epicytic folds and host epithelium was filled with electron-dense material interpreted to be adhesive in nature. Actinocephalus carrilynnae organisms attached by means of the epimerite, which had no epicytic folds, and by the fluted stalk with characteristic epicytic folds: host cell and parasite membranes appeared fused at some places on the epimerite. Hoplorhynchus acanthatholius organisms attached by means of an ovoid epimerite with backward-pointing digitations; the entire epimerite was embedded in a host cell, and host cell microvilli surrounded the stalk. Steganorhynchus dunwoodyi organisms attached by means of an ovoid stalk papilla enclosed in a retractable globular sheath; the papilla was covered with epicytic folds, but the sheath was not, and the sheath had a single membrane, whereas the epicytic folds had 2 or 3 membranes. The entire apparatus was inserted between epithelial cells, and the sheath was highly folded at its surface. The ultrastructural observations suggest that actinocephalid gregarines have evolved 2 general strategies for attaching to the host epithelium, that is, suckerlike protomerites, as in the case of N. nebraskensis, and deeply embedded epimerites inserted within or between host cells, as in the other species studied.     

Immunomagnetic isolation of Xanthomonas campestris pv. pelargonii

Immunomagnetic fishing was developed as an improved procedure for increasing the bacterial target to non-target recovery ratio in suspensions containing mixtures of target and non-target organisms. A cell suspension containing the target Xanthomonas campestris pv. pelargonii and non-target organisms, is treated with rabbit polyclonal antiserum against X.c. pv. pelargonii and incubated for 1 h. The suspension is then mixed with paramagnetic iron oxide particles coated with goat anti-rabbit antibodies (immunomagnetic particles). After incubation, the polished surface of a 14 mm diameter neodymium supermagnet is placed at the air-water interace and the magnetic particles are attracted to the magnet. After all visible magnetic particles have attached to the bottom of the magnet, the magnet is dipped in sterile buffer to remove non-target organisms. The magnet with attached magnetic particles is rubbed evenly over an agar surface to dislodge the particles and attached bacteria. Conventional immunomagnetic isolation (immunomagnetic attraction) and immunomagnetic fishing were compared, for the recovery of the target organism in geranium leaf washings spiked with X.c. pv. pelargonii. With immunomagnetic attraction and immunomagnetic fishing, bacterial non-target organisms were reduced to 11.4 and 1.5% of the initial population, respectively, whereas the target was only reduced to 63.7 and 53.8%.     

Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists.

Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken. The current procedure lacks precision and accuracy and is not quantitative. Variability associated with carriers and the lack of temperature control were evaluated in this paper. The use of porcelain versus stainless steel carriers was also evaluated. When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale. The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU). The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension. Variations in drying time did not alter the number of cells attached to the carrier. When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96%. Data for B. subtilis spores were similar to those for M. bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers. It was also found that the lack of an exacting temperature control could influence the outcome of the test. Changes in temperature as little as 1 degree C could influence the rate of killing of M. bovis BCG.     

Cavity-dwelling organisms in Lower Cambrian patch reefs from southern Labrador

An intact and well-preserved biota of cavity-dwelling organisms (coelobionts) of early Cambrian age is found within growth-framework cavities in archaeocyathid patch reefs of the lower Forteau Formation (upper Bonnia-Olenellus Zone) of southern Labrador. The biota was diverse, and consisted of encrusting and vagrant organisms. Renalcis , a Renalcis-like form, Epiphyton, Girvanella , and Serligia , all lived attached to walls and roofs of the cavities; other attached forms were Bija, Archaeotrypa , and two types of Wetheredella , an agglutinated foraminifer. Other organisms, including trilobites, probable calcareous ostracodes, brachiopods, echinoderms, and worms, as well as fungi, are also found preserved within the cavities. The structure of the coelobiontic community is surprisingly modern; it indicates that as early as the Lower Cambrian coelobiontic organisms were well-established in reefs, and were an important part of the reef community as a whole.     

Attachment of Mycoplasma pulmonis to rat and mouse synovial cells cultured in vitro

The attachment of Mycoplasma pulmonis m53 organisms to mouse and rat synovial cells was examined by using the organisms and the synovial cells treated in various ways. M. pulmonis treated with trypsin attached to the synovial cells, but the organisms treated with pronase, formaldehyde, glutaraldehyde, or heat did not. These findings suggest that the sites for binding M. pulmonis to the mouse and rat synovial cells are of polypeptide nature. Treatment of M. pulmonis with sialic acid and treatment of the synovial cell sheets with neuraminidase did not affect the attachment. The synovial cell surface for receptors M. pulmonis organisms would be different from those on respiratory cells or erythrocytes for M. pneumoniae or M. gallisepticum. Even nonviable organisms and M. pulmonis membranes attached to the mouse or rat synovial cells. The nature of the receptor of mouse synovial cells would be different from that of rat cells, since rat cells were affected by treatment with formaldehyde or glutaraldehyde, but mouse cells were not.     

Evaluation of carriers used in the test methods of the Association of Official Analytical Chemists

Revision of the official test method for the determination of the tuberculocidal activity of disinfectants is being undertaken. The current procedure lacks precision and accuracy and is not quantitative. Variability associated with carriers and the lack of temperature control were evaluated in this paper. The use of porcelain versus stainless steel carriers was also evaluated. When carriers of either type were contaminated with Mycobacterium bovis BCG, the number of organisms on the carriers varied by as much as 1.0 on the log10 scale. The average number of organisms attached to each porcelain carried was 1.10 x 10(5) CFU (range, 2.7 x 10(4) to 2.7 x 10(5) CFU), whereas the average number of organisms attached to each stainless steel carrier was 1.38 x 10(5) CFU (range, 2.9 x 10(4) to 4.0 x 10(5) CFU). The average number of cells attached to the carrier was directly proportional to the number of cells in the contaminating cell suspension. Variations in drying time did not alter the number of cells attached to the carrier. When porcelain carriers were placed in a test solution, the average number of organisms washed from the carriers was 55% of the total, with a range of 19 to 80%, whereas for stainless steel carriers, the average number was 82% of the total, with a range of 52 to 96%. Data for B. subtilis spores were similar to those for M. bovis BCG, suggesting that there may be similar problems with the Association of Official Analytical Chemists sporicidal test, which uses carriers. It was also found that the lack of an exacting temperature control could influence the outcome of the test. Changes in temperature as little as 1 degree C could influence the rate of killing of M. bovis BCG.     

Modulation by centrifugation of cell susceptibility to chlamydial infection.

Enhancement of chlamydial infection of cell monolayers by centrifugation was shown to depend on induced cell surface changes. Evidence for this came from analysis of two forms of organism attachment which take place during centrifugation. In 'productive binding', organisms attached to cells and then entered and infected them. In 'unproductive binding', organisms became attached to cells but were not ingested. These organisms could be stripped from the cells by treatment with trypsin and could then infect fresh monolayers. Measurement of attachment kinetics during centrifugation showed that cells passed through three different susceptibility states. Only productive binding occurred in the first 20 min; cells then entered a refractory state during which no attachment took place At about 45 min, attachment recommenced but this allowed only unproductive binding. Induced movement of cell surface structures may enhance infection by promoting specific or non-specific interactions. Failure of ingestion may result from insufficient cell 'receptors' for circumferential binding of the whole chlamydial surface so that engulfment cannot take place.     

The relationship between instability of H2 production and compositions of bacterial communities within a dark fermentation fluidized-bed bioreactor

Microbial community composition dynamics was studied during H(2) fermentation from glucose in a fluidized-bed bioreactor (FBR) aiming at obtaining insight into the H(2) fermentation microbiology and factors resulting in the instability of biofilm processes. FBR H(2) production performance was characterised by an instable pattern of prompt onset of H(2) production followed by rapid decrease. Gradual enrichment of organisms increased the diversity of FBR attached and suspended-growth phase bacterial communities during the operation. FBR bacteria included potential H(2) producers, H(2) consumers and neither H(2) producers nor consumers, and those distantly related to any known organisms. The prompt onset of H(2) production was due to rapid growth of Clostridium butyricum (99-100%) affiliated strains after starting continuous feed. The proportion trend of C. butyricum in FBR attached and suspended-growth phase communities coincided with H(2) and butyrate production. High glucose loading rate favoured the H(2) production by Escherichia coli (100%) affiliated strain. Decrease in H(2) production, associated with a shift from acetate-butyrate to acetate-propionate production, was due to changes in FBR attached and suspended-growth phase bacterial community compositions. During the shift, organisms, including potential propionate producers, were enriched in the communities while the proportion trend of C. butyricum decreased. We suggest that the instability of H(2) fermentation in biofilm reactors is due to enrichment and efficient adhesion of H(2) consumers on the carrier and, therefore, biofilm reactors may not favour mesophilic H(2) fermentation.     

Tumoricidal adsorption of Staphylococcus aureus organisms on Ehrlich ascites tumor cells sensitized with rabbit antibody

A tumoricidal effect was observed when protein A-bearing Staphylococcus aureus organisms were adsorbed on Ehrlich ascites tumor cells previously sensitized with antiserum from a rabbit immunized with Ehrlich ascites tumor cells. Electron micrographs showed that staphylococci were firmly attached to the tumor cells, which might explain how effectively the attached cocci killed the tumor cells. The tumoricidal effect was confirmed not only by an in vitro experiment but also by an in vivo one. The possible applications of the tumoricidal adsorption as an indicator for staphylococcal virulence or for selective anti-tumor action was discussed.     

Simplified preparation of mycoplasmas, an acholeplasma, and a spiroplasma for scanning electron microscopy.

A simple, effective procedure was developed for scanning electron microscopic examination of mycoplasmas and similar organisms. Cultivation of several mycoplasmal species, an acholeplasma, and a spiroplasma in broth media in Leighton tubes with cover slips resulted in attachment of the organisms to the cover slips. The attached cells were easily processed for either scanning electron microscopy or light microscopy. By eliminating the need for centrifugation, which was used in previously described techniques, physical stress on the cell is minimized. The effects of different preparative procedures on the morphology of Mycoplasma gallisepticum are described.     

Prediction of Substrate Removal Rates of Attached Microorganisms and of Relative Contributions of Attached and Suspended Communities at Field Sites

A mathematical model composed of a direct proportionality relationship between bulk water velocities and field-determined second-order microbial transformation rate coefficients, and the relative rate coefficient of a benchmark chemical, was developed for estimating the substrate removal rates of rapidly degraded chemicals by attached organisms in shallow (<1 m deep) aquatic ecosystems. Data from 31 field experiments involving the addition of 2,4-dichlorophenoxyacetic acid methyl ester (2,4-DME) in nine field areas were used to determine a field-derived second-order rate coefficient for microbial transformation of the ester. By using 2,4-DME as a benchmark chemical, the model was used to predict microbial transformation rates of the butoxyethyl ester of 2,4-dichlorophenoxyacetic acid (2,4-DBE) at five other field sites. The predicted half-lives of 2,4-DBE varied 1,500-fold and were within about a threefold range or less of the measured half-lives. Under conditions of mass transport limitation, the contributions of attached microorganisms relative to total microbial activities at various field sites were related to the ratio of water velocity, U, and depth, D, showing that historical definitions of ecosystems according to flow and depth characteristics are also valid for describing the process-related structure of ecosystems. An equation was developed for predicting the relative contributions of attached and suspended communities with values of U and D for lotic and lentic ecosystems. On the basis of this equation, attached microorganisms were expected to be insignificant in deep lentic ecosystems and suspended microorganisms were expected to be insignificant in shallow lotic systems for the same process carried out by both populations. Neglecting epiphytic microorganisms, both suspended and attached organisms were expected to be significant in wetlands.     

Scanning electron microscopy of the gut microflora of two earthworms: Lumbricus terrestris and Octolasion cyaneum

Scanning electron microscopy was used to investigate the presence of microorganisms, probably bacteria, on the gut surface of earthworms. The washed surfaces of the intestines of two earthworms, Lumbricus terrestris and Octolasion cyaneum, were examined. Numerous organisms resembling bacteria were observed throughout the gut, some in situations suggesting attachment. Compared with similar investigations in other invertebrates, there were fewer bacteria, showing less morphological diversity, on the earthworm gut surface. The majority of organisms viewed were coccoid, some were filamentous, and a few rod-shaped cells were observed. Cocci, often in chains, were seen in the foregut of both species. Although cocci were also numerous in the midgut region, particularly in the typhlosole, in O. cyaneum tufts of segmented, filamentous organisms were also seen with some segments resembling spores. Fewer organisms were found in the hindgut, but in L. terrestris there were segmented, filamentous organisms, attached to the epithelium by way of a socket-like structure, similar to that by which segmented, filamentous bacteria (SFBs) are attached to the ileum of rats and mice. Transmission electron microscopy of the hindgut of L. terrestris was undertaken to explore the structure and attachment of SFBs to the gut epithelium. However, although a few rod-shaped bacteria were observed, no SFBs were located. The observations reported here provide evidence that earthworms have an attached gut microflora of filamentous microorganisms which are probably indigenous, and as far as we are aware this is the first published report of such findings in these invertebrates. Offprint requests to: J.M. Jolly.     

Cryptosporidium cuniculus in the rabbit (Oryctolagus cuniculus)

Cryptosporidium cuniculus was identified by light and electron microscopy in two apparently healthy rabbits. Organisms were firmly attached at the brush borders of intestinal epithelial cells. Cellular alterations were minimal, consisting of elongation and shortening of microvilli adjacent to the attachment sites of organisms. Various developmental stages of cryptosporidium were identified by electron microscopy.     

Amino Acid Assimilation and Electron Transport System Activity in Attached and Free-Living Marine Bacteria

Amino acid assimilation and electron transport system activity of a marine Pseudomonas sp. was evaluated to determine whether the activity of bacteria attached to solid surfaces differed from that of free-living bacteria or bacteria which had been attached but subsequently desorbed from the substratum (detached bacteria). Bacteria were allowed to attach to glass and to a range of plastic surfaces (Thermanox, polyvinylidene fluoride, polyethylene, polytetrafluoroethylene). Microautoradiography and staining with a tetrazolium salt to demonstrate electron transport system activity were used to compare the activity of these organisms with that of free-living or detached cells. The water-wettability of the surfaces was evaluated by measuring the advancing contact angle (θ_A) of water on each surface, to determine whether there was a relationship between activity and substratum hydrophilicity. There was an increase in the proportion of leucine-assimilating attached bacteria and in the proportion of attached cells demonstrating electron transport system activity with an increase in substratum θ_A, but the relationship between activity of attached and free-living cells depended on the substratum. Activity appeared to promote firm attachment, and detached bacteria assimilated fewer amino acids than did attached cells. There was no general effect of surfaces on attached bacterial activity, and attached cells may be more, or less, active than free-living cells, depending on the amino acid, its concentration, and substratum properties.     

Lytic Action of Inducible Bacteriocin Clostocin O

Clostocin O had a remarkable lytic action toward the exponentially growing organisms of Clostridium saccharoperbutylacetonicum No. 8. The cellular lysis was inhibited by addition of heavy metal cations such as Cu²⁺, Ni²⁺, and Cd²⁺, or fradiomycin and RNase, which had been reported to be the inhibitors for lytic enzymes such as some of clostridial phage-endolysin and clostocin O-endolysin. The formalin-treated organisms and antibiotic-treated organisms, of which autolysin activity was inhibited, were also lysed by clostocin O. The induced cellular lysis by clostocin O was thought to be due to the lytic enzyme attached to clostocin O.     

Development of engineered biofilms on poly- L-lysine patterned surfaces

A technique has been developed to selectively attach bacteria to solid supports using poly-l-lysine. The patterned biofilms were labeled with green fluorescent protein (GFP) or a nucleic acid stain and imaged using both confocal microscopy and GFP stereomicroscopy. E. coli DH10B, E. coli MC1061, and Pseudomonas sp. GJ1 were selectively attached to regions coated with poly-l-lysine but not to uncoated regions. In contrast, E. coli DH5, W3110 and 33456 attached indiscriminately to the coated and uncoated regions of the surface. Those organisms that selectively attached to the poly-l-lysine coated regions formed biofilms twice as thick as the organisms that attached indiscriminately to the surface. This technique can be used for selectively patterning surfaces with genetically engineered microorganisms for biosynthesis of secondary metabolites and biodegradation or for developing a bacterial-based microscale medical diagnostic tool.     

Geometry of cell division in Staphylococcus aureus.

The process of division in Staphylococcus aureus was examined by phase-contrast microscopy. The organisms appeared to divide in three alternating perpendicular planes, with sister cells remaining attached to each other after division. The resulting point of attachment was usually not exactly at the point corresponding to the center of the previous septal disk. Moreover, sister cells often changed position with respect to one another while still remaining attached. These factors are apparently responsible for the irregularity of staphylococcal clumps. Studies with penicillin and the examination of thin sections in the electron microscope confirm the conclusion, based upon light microscopy, that successive divisions in S. aureus occur in perpendicular planes.