Coaggregation among nonflocculating bacteria isolated from activated sludge

Thirty-two strains of nonflocculating bacteria isolated from sewage-activated sludge were tested by a spectrophotometric assay for their ability to coaggregate with one other in two-membered systems. Among these strains, eight showed significant (74 to 99%) coaggregation with Acinetobacter johnsonii S35 while only four strains coaggregated, to a lesser extent (43 to 65%), with Acinetobacter junii S33. The extent and pattern of coaggregation as well as the aggregate size showed good correlation with cellular characteristics of the coaggregating partners. These strains were identified by sequencing of full-length 16S rRNA genes. A. johnsonii S35 could coaggregate with strains of several genera, such as Oligotropha carboxidovorans, Microbacterium esteraromaticum, and Xanthomonas spp. The role of Acinetobacter isolates as bridging organisms in multigeneric coaggregates is indicated. This investigation revealed the role of much-neglected nonflocculating bacteria in floc formation in activated sludge.     

Effect of cultivation time and growth medium on the coaggregation capability of <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> S35

The effects of different cultivation periods and growth media on the cell surface hydrophobicity and coaggregation ability of Acinetobacter johnsonii S35 with Oligotropha carboxidovorans S23 (both sewage sludge isolates) were examined. It was observed that when cultivated in polypeptone medium, A. johnsonii S35 shows high hydrophobicity and strong coaggregation (98% aggregation index) with O. carboxidovorans S23, which remain unaltered after a 1-day, 3-day or 7-day cultivation period. When A. johnsonii S35 was cultivated with different carbon substrates, large coaggregates and a high aggregation index were observed using acetate-grown cells (96%), followed by ethanol-(83%) and vegetable oil-grown cells (72%). Coaggregates were small and narrow in ethanol-grown cells and still smaller in case of the vegetable oil-grown cells. Although no direct correlation could be observed, more hydrophobic cells showed a higher aggregation index. None of the culture supernatants of A. johnsonii S35 showed emulsification of hexadecane or a significant ability to aggregate O. carboxidovorans S23 cells, indicating that the coaggregation factor of A. johnsonii S35 is cell-bound. These results demonstrate that the coaggregation ability of A. johnsonii S35 is constitutive and is not lost under diverse cultivation conditions.     

Intergeneric coaggregations among Oligotropha carboxidovorans and Acinetobacter species present in activated sludge

The coaggregation traits of two pairs of sewage sludge bacteria were tested and characterized. Oligotropha carboxidovorans S23 coaggregated with two strains of the genus Acinetobacter viz. Acinetobacter junii S33 (56%) and Acinetobacter johnsonii S35 (99%). Coaggregates of O. carboxidovorans S23 and A. junii S33 were small (20-40 microm), weak and susceptible to EDTA and a commercial protease (Actinase E). Actinase/periodate pretreatment of the partners prior to coaggregation revealed that interaction in this case was mediated by protein surface components. Coaggregates of O. carboxidovorans S23 and A. johnsonii S35 were large (above 100 microm), strong and not deflocculated by EDTA or Actinase E. Only periodate pretreatment of A. johnsonii S35 prevented this coaggregation indicating a role for a carbohydrate-containing moiety without the involvement of protein components. The potential mechanisms and strength of bacterial coaggregations seem to be pair dependent.     

Intergeneric Coaggregation among Drinking Water Bacteria: Evidence of a Role for Acinetobacter calcoaceticus as a Bridging Bacterium

Intergeneric coaggregation of drinking water bacteria was tested. Acinetobacter calcoaceticus was found not only to autoaggregate but also to coaggregate with four of the five other isolates (Burkholderia cepacia, Methylobacterium sp., Mycobacterium mucogenicum, Sphingomonas capsulata, and Staphylococcus sp.). In its absence, no coaggregation was found. Interactions were lectin-saccharide mediated. The putative bridging function of A. calcoaceticus was evidenced by multispecies biofilm studies, through a strain exclusion process.     

Pair-dependent co-aggregation behavior of non-flocculating sludge bacteria

Two strains of non-flocculating sewage sludge bacteria (Xanthomonassp. S53 and Microbacterium esteraromaticum S51) showed 91% and 77% co-aggregation, respectively, with Acinetobacter johnsonii S35 using a spectrophometric assay. The co-aggregates in case of Xanthomonas sp. S53 and A. johnsonii S35 were above 100 m and stable against EDTA (2 mM) and a commercial protease (0.2 mg ml^–1). Protease/periodate pretreatment of the partners did not affect this co-aggregation. On the other hand, co-aggregates of M. esteraromaticum S51 and A. johnsonii S35 (50–70 m) were deflocculated by EDTA or protease. Protease pretreatment of M. esteraromaticum S51 and periodate pretreatment of A. johnsonii S35 prevented their co-aggregation with respective untreated partners. The potential co-aggregation mechanisms of A. johnsonii S35 varied depending upon the other partner involved.     

Diverse Responses to UV-B Radiation and Repair Mechanisms of Bacteria Isolated from High-Altitude Aquatic Environments

Acinetobacter johnsonii A2 isolated from the natural community of Laguna Azul (Andean Mountains at 4,560 m above sea level), Serratia marcescens MF42, Pseudomonas sp. strain MF8 isolated from the planktonic community, and Cytophaga sp. strain MF7 isolated from the benthic community from Laguna Pozuelos (Andean Puna at 3,600 m above sea level) were subjected to UV-B (3,931 J m⁻²) irradiation. In addition, a marine Pseudomonas putida strain, 2IDINH, and a second Acinetobacter johnsonii strain, ATCC 17909, were used as external controls. Resistance to UV-B and kinetic rates of light-dependent (UV-A [315 to 400 nm] and cool white light [400 to 700 nm]) and -independent reactivation following exposure were determined by measuring the survival (expressed as CFU) and accumulation of cyclobutane pyrimidine dimers (CPD). Significant differences in survival after UV-B irradiation were observed: Acinetobacter johnsonii A2, 48%; Acinetobacter johnsonii ATCC 17909, 20%; Pseudomonas sp. strain MF8, 40%; marine Pseudomonas putida strain 2IDINH, 12%; Cytophaga sp. strain MF7, 20%; and Serratia marcescens, 21%. Most bacteria exhibited little DNA damage (between 40 and 80 CPD/Mb), except for the benthic isolate Cytophaga sp. strain MF7 (400 CPD/Mb) and Acinetobacter johnsonii ATCC 17909 (160 CPD/Mb). The recovery strategies through dark and light repair were different in all strains. The most efficient in recovering were both Acinetobacter johnsonii A2 and Cytophaga sp. strain MF7; Serratia marcescens MF42 showed intermediate recovery, and in both Pseudomonas strains, recovery was essentially zero. The UV-B responses and recovery abilities of the different bacteria were consistent with the irradiation levels in their native environment.     

Involvement of Acinetobacter sp. in the floc-formation in activated sludge process

The coaggregation behavior of Acinetobacter johnsonii S35 isolate with sewage bacteria was assessed by a spectrophotometric assay using different samples from a municipal wastewater treatment plant and a community plant. A. johnsonii S35 coaggregated well with other free bacteria and microflocs at the mixing ratios of 0.2:1-0.6:1 of A. johnsonii S35 and sewage samples. In addition, the size of coaggregates became larger (100 μm or more) under the same conditions. A. johnsonii S35 cells were highly adsorbed (adsorption=93-99%) onto sludge samples. Microbial adhesion to hydrocarbon (MATH) test and adsorption to octyl-Sepharose CL-4B showed that A. johnsonii S35 cells and sludge samples had a hydrophobic character. The population of Acinetobacter spp. in sewage treatment plants was 2-7% and its role in bioflocculation was discussed. The present study revealed that A. johnsonii S35 isolate can play as a bridging organism and contribute in floc-formation in activated sludge process.     

Isolation and Characterization of Broad Spectrum Coaggregating Bacteria from Different Water Systems for Potential Use in Bioaugmentation

The bridging bacteria with broad-spectrum coaggregation ability play an important role during multispecies-biofilm development. In this study, through a visual and semi-quantitative assay, twenty-two bacterial strains with aggregation ability were obtained from 8 different water environments, and these strains were assigned to 7 genera according to their 16S rDNA and they were Aeromonas, Bacillus, Comamonas, Exiguobacterium, Pseudomonas, Shewanella and Comamonas. Furthermore, all possible 231 pairwise combinations among these 22 strains were explored for coaggregation ability by spectrophotometric assay. Among all these strains, it was found that Bacillus cereus G5 and Bacillus megaterium T1 coaggregated with themajority of assayed other strains, 90.5% (19 of 21 strains) and 76.2% respectively (17 of 21 strains) at a higher coaggregation rates (A.I. greater than 50%), indicating they have a broad-spectrum coaggregation property. The images of coaggregates also confirmed the coexistence of G5 and T1 with their partner strains. Biofilm biomass development of G5 cocultured with each of its partner strains were further evaluateded. The results showed that 15 of 21 strains, when paired with G5, developed greater biofilm biomass than the monocultures. Furthermore, the images from both fluorescence microscopy and scanning electron microscopy (SEM) demonstrated that G5 and A3-GFP (a 3,5-dinitrobenzoic acid-degrading strain, staining with gfp),could develop a typical spatial structure of dual-species biofilm when cocultured. These results suggested that bridging-bacteria with a broad spectrum coaggregating ability, such as G5,could mediate the integration of exogenous degrading bacteria into biofilms and contribute to the bioaugmentation treatment.     

Novel coaggregating microbial consortium: testing strength for field applications

Acinetobacter johnsonii S35 has an ability to coaggregate with Oligotropha carboxidovorans S23 when grown with favorable carbon substrate and nutrient-rich growth media. However, nutrient-deficient situations or poorly accessible carbon substrates in waste streams could trigger changes in the cell surface properties and thus modify the coaggregation behavior. Thus, in the present study, we test the stability of these novel interactions under various stresses that might transpire in the waste streams. The effect of mineral-component deficiency and shortage of substrate on the growth, cell surface hydrophobicity and coaggregation ability of S35 and O. carboxidovorans S23 has been investigated. As compared to the control, growth of S35 was adversely affected in mineral-deficient medium; the cells had lower hydrophobicity but displayed only slightly lower aggregation index (AI, 75-84%) as compared to control (92%). S35 grown on marginally supplemented Sterile Filtrate of Activated Sludge (SFAS) could also grow well and form larger stable coaggregates with S23. S35 grown with mineral-deficient medium and with the supplemented SFAS still posses the ability to coaggregate with S23. Hence the coaggregation ability of S35 strain is constitutive and is not significantly affected by environmental factors. It would be desirable to work out suitable formulations of such robust and environmentally relevant microbes.     

An Outbreak of Nonflocculating Catabolic Populations Caused the Breakdown of a Phenol-Digesting Activated-Sludge Process

Activated sludge was fed phenol as the sole carbon source, and the phenol-loading rate was increased stepwise from 0.5 to 1.0 g liter⁻¹ day⁻¹ and then to 1.5 g liter⁻¹ day⁻¹. After the loading rate was increased to 1.5 g liter⁻¹ day⁻¹, nonflocculating bacteria outgrew the sludge, and the activated-sludge process broke down within 1 week. The bacterial population structure of the activated sludge was analyzed by temperature gradient gel electrophoresis (TGGE) of PCR-amplified 16S ribosomal DNA (rDNA) fragments. We found that the population diversity decreased as the phenol-loading rate increased and that two populations (designated populations R6 and R10) predominated in the sludge during the last several days before breakdown. The R6 population was present under the low-phenol-loading-rate conditions, while the R10 population was present only after the loading rate was increased to 1.5 g liter⁻¹ day⁻¹. A total of 41 bacterial strains with different repetitive extragenic palindromic sequence PCR patterns were isolated from the activated sludge under different phenol-loading conditions, and the 16S rDNA and gyrB fragments of these strains were PCR amplified and sequenced. Some bacterial isolates could be associated with major TGGE bands by comparing the 16S rDNA sequences. All of the bacterial strains affiliated with the R6 population had almost identical 16S rDNA sequences, while the gyrB phylogenetic analysis divided these strains into two physiologically divergent groups; both of these groups of strains could grow on phenol, while one group (designated the R6F group) flocculated in laboratory media and the other group (the R6T group) did not. A competitive PCR analysis in which specific gyrB sequences were used as the primers showed that a population shift from R6F to R6T occurred following the increase in the phenol-loading rate to 1.5 g liter⁻¹ day⁻¹. The R10 population corresponded to nonflocculating phenol-degrading bacteria. Our results suggest that an outbreak of nonflocculating catabolic populations caused the breakdown of the activated-sludge process. This study also demonstrated the usefulness of gyrB-targeted fine population analyses in microbial ecology.     

Effect of surfactants on stability of Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 coaggregates

The effect of anionic (sodium dodecyl sulphate or SDS) and cationic (cetyltrimethylammonium bromide or CTAB) surfactants on the stability of binary bacterial coaggregates comprising Acinetobacter johnsonii S35 and Oligotropha carboxidovorans S23 (both sewage sludge isolates) was studied and compared with that on the complex sewage sludge flocs. Both SDS and CTAB enhanced the bacterial coaggregation at their lower concentrations of 0.2 and 0.07 mg ml(-1), respectively. However, complete deflocculation of coaggregates was observed at 1 mg ml(-1) SDS and 0.3 mg l(-1) CTAB concentrations. Further, sewage sludge flocs did not deflocculate in the presence of CTAB, although a concentration-dependent deflocculation was observed in the presence of SDS. A. johnsonii S35 and O. carboxidovorans S23 cells were separately pretreated (prior to coaggregation) with the surfactants. In spite of the partial (complete) loss of viability during SDS (CTAB) pretreatment, washed cells still retained hydrophobic character and displayed significant coaggregation (aggregation index ranging from 84% to 97% in comparison to 96% in the case of non-treated cells), demonstrating reversibility of the surfactant induced deflocculation. Further, when exposed to lower concentration of surfactants (0.2 mg ml(-1) SDS), coaggregates were more resistant (76% viability) as compared to the individual partner (S35: 52%; S23: 39% viability). Since the coaggregates are stable and provide protection from surfactants at lower concentrations (those normally expected in the sewage treatment plants), their presence as well as a sustained role in the sewage sludge bioflocculation is evident.     

Characterization of novel diesel-degrading strains <Emphasis Type="Italic">Acinetobacter haemolyticus</Emphasis> MJ01 and <Emphasis Type="Italic">Acinetobacter johnsonii</Emphasis> MJ4 isolated from oil-contaminated soil

The diesel-degrading strains, designated as MJ01 and MJ4, were isolated from oil-contaminated soil in Daejeon (South Korea) and were taxonomically characterized using a polyphasic approach and their diesel oil degradation abilities were analyzed. The isolates MJ01 and MJ4 were identified as Acinetobacter haemolyticus and Acinetobacter johnsonii, respectively, based on their 16S rDNA gene sequences, DNA–DNA relatedness, fatty acid profiles and various physiological characteristics. Strains MJ01 and MJ4 were able to use diesel oil as the sole carbon and energy source. Both strains could degrade over 90% of diesel oil with an initial concentration of 20,000 mg/l after incubation for 7 days, the most significant degradation occurred during the first 3 days. To our knowledge, this is the first report on diesel oil-degrading microorganisms among bacterial strains belonging to A. haemolyticus and A. johnsonii.     

Using phenotype microarrays in the assessment of the antibiotic susceptibility profile of bacteria isolated from wastewater in on-site treatment facilities

The scope of the study was to apply Phenotype Biolog MicroArray (PM) technology to test the antibiotic sensitivity of the bacterial strains isolated from on-site wastewater treatment facilities. In the first step of the study, the percentage values of resistant bacteria from total heterotrophic bacteria growing on solid media supplemented with various antibiotics were determined. In the untreated wastewater, the average shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria were 53, 56, and 42%, respectively. Meanwhile, the shares of kanamycin-, streptomycin-, and tetracycline-resistant bacteria in the treated wastewater were 39, 33, and 29%, respectively. To evaluate the antibiotic susceptibility of the bacteria present in the wastewater, using the phenotype microarrays (PMs), the most common isolates from the treated wastewater were chosen: Serratia marcescens ss marcescens, Pseudomonas fluorescens, Stenotrophomonas maltophilia, Stenotrophomonas rhizophila, Microbacterium flavescens, Alcaligenes faecalis ss faecalis, Flavobacterium hydatis, Variovorax paradoxus, Acinetobacter johnsonii, and Aeromonas bestiarum. The strains were classified as multi-antibiotic-resistant bacteria. Most of them were resistant to more than 30 antibiotics from various chemical classes. Phenotype microarrays could be successfully used as an additional tool for evaluation of the multi-antibiotic resistance of environmental bacteria and in preliminary determination of the range of inhibition concentration.     

Isolation and characterization of coaggregation-defective (Cog−) mutants ofStreptococcus gordonii DL1 (Challis)

Streptococcus gordonii DL1 (Challis) bears coaggregation-mediating surface adhesins which recognize galactoside-containing surface polysaccharides onStreptococcus oralis 34,Streptococcus oralis C104, andStreptococcus SM PK509. Fifty-nine spontaneously-occurring coaggregation-defective (Cog^–) mutants ofS. gordonii DL1 unable to coaggregate with partner streptococci were isolated. Six representative Cog^– mutants were characterized by their coaggregation properties with fourActinomyces naeslundii strains (T14V, PK947, PK606, PK984),Veillonella atypica PK1910, andPropionibacterium acnes PK93. The six representative Cog^– mutants showed altered coaggregation with their streptococcal partners,A. naeslundii PK947, andP. acnes PK93. Based on the coaggregation phenotypes of these mutants, a model for the lactose-inhibitable coaggregation betweenS. gordonii DL1 and its partner bacteria is proposed. The potential use of these mutants in studies of oral biofilms is discussed.     

Characterization of a Streptococcus sp.-Veillonella sp. community micromanipulated from dental plaque

Streptococci and veillonellae occur in mixed-species colonies during formation of early dental plaque. One factor hypothesized to be important in assembly of these initial communities is coaggregation (cell-cell recognition by genetically distinct bacteria). Intrageneric coaggregation of streptococci occurs when a lectin-like adhesin on one streptococcal species recognizes a receptor polysaccharide (RPS) on the partner species. Veillonellae also coaggregate with streptococci. These genera interact metabolically; lactic acid produced by streptococci is a carbon source for veillonellae. To transpose these interactions from undisturbed dental plaque to an experimentally tractable in vitro biofilm model, a community consisting of RPS-bearing streptococci juxtaposed with veillonellae was targeted by quantum dot-based immunofluorescence and then micromanipulated off the enamel surface and cultured. Besides the expected antibody-reactive cell types, a non-antibody-reactive streptococcus invisible during micromanipulation was obtained. The streptococci were identified as Streptococcus oralis (RPS bearing) and Streptococcus gordonii (adhesin bearing). The veillonellae could not be cultivated; however, a veillonella 16S rRNA gene sequence was amplified from the original isolation mixture, and this sequence was identical to the sequence of the previously studied organism Veillonella sp. strain PK1910, an oral isolate in our culture collection. S. oralis coaggregated with S. gordonii by an RPS-dependent mechanism, and both streptococci coaggregated with PK1910, which was used as a surrogate during in vitro community reconstruction. The streptococci and strain PK1910 formed interdigitated three-species clusters when grown as a biofilm using saliva as the nutritional source. PK1910 grew only when streptococci were present. This study confirms that RPS-mediated intrageneric coaggregation occurs in the earliest stages of plaque formation by bringing bacteria together to create a functional community.     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Acinetobacter bohemicus sp. nov. widespread in natural soil and water ecosystems in the Czech Republic

We investigated the taxonomic status of a phenetically unique group of 25 Acinetobacter strains which were isolated from multiple soil and water samples collected in natural ecosystems in the Czech Republic. Based on the comparative sequence analyses of the rpoB, gyrB, and 16S rRNA genes, the strains formed a coherent and well separated branch within the genus Acinetobacter. The genomic uniqueness of the group at the species level was supported by the low average nucleotide identity values (≤77.37%) between the whole genome sequences of strain ANC 3994^T (NCBI accession no. APOH00000000) and the representatives of the known Acinetobacter species. Moreover, all 25 strains created a tight cluster clearly separated from all hitherto described species based on whole-cell protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and shared a unique combination of metabolic and physiological properties. The capacity to assimilate l-histidine and the inability to grow at 35 °C differentiated them from their phenotypically closest neighbor, Acinetobacter johnsonii. We conclude that the 25 strains represent a novel Acinetobacter species, for which the name Acinetobacter bohemicus sp. nov. is proposed. The type strain of A. bohemicus is ANC 3994^T (=CIP 110496^T = CCUG 63842^T = CCM 8462^T).     

Coaggregation properties of human oral Veillonella spp.: relationship to colonization site and oral ecology.

The primary habitats of oral veillonellae are the tongue, dental plaque, and the buccal mucosa. Isolates were obtained from each habitat and tested for coaggregation with a battery of other oral bacterial strains. All 59 tongue isolates tested for coaggregation were Veillonella atypica or Veillonella dispar. All but one of them coaggregated with strains of Streptococcus salivarius, a predominant inhabitant of the tongue surface but not subgingival dental plaque. These tongue isolates were unable to coaggregate with most normal members of the subgingival flora such as Actinomyces viscosus, Actinomyces naeslundii, Actinomyces israelii, and Streptococcus sanguis. In contrast, 24 of 29 Veillonella isolates, of which 20 were Veillonella parvula from subgingival dental plaque samples, coaggregated strongly with the three species of Actinomyces, S. sanguis, and other bacteria usually present in subgingival plaque, but they did not coaggregate with S. salivarius. The majority of isolates from the buccal mucosa (42 of 55) has coaggregation properties like those from the tongue. These results indicate that the three human oral Veillonella species are distributed on oral surfaces that are also occupied by their coaggregation partners and thus provide strong evidence that coaggregation plays a critical role in the bacterial ecology of the oral cavity.     

Microbial manganese reduction mediated by bacterial strains isolated from aquifer sediments

One hundred and five strains isolated from aquifer sediments andEscherichia coli ML30S were tested for their ability to reduce manganese oxides. Eighty-two strains, includingE. coli, reduced manganese. In most cases the bacterial activity decreased the pH and Eh below 6.75 and 350 mV, respectively, enhancing a spontaneous and nonspecific reduction of manganese. However, for 12 strains the reduction was specifically catalyzed by bacteria; the high pH and Eh values would not permit a spontaneous reduction of manganese. Some of the most active strains were identified as genera common in soils and waters, i.e.,Pseudomonas, Bacillus, Corynebacterium, andAcinetobacter. Two strains were studied in detail. One of the strains, identified asPseudomonas fluorescens, required contact between the cells and the manganese oxides for reduction to occur. The reduction was inhibited by 15 mM of sodium azide. The other strain, identified asAcinetobacter johnsonii, catalyzed manganese reduction by an inductive and dialyzable substance which was excreted by the bacteria. The mechanism involved has not been previously demonstrated.     

Coaggregation between Aquatic Bacteria Is Mediated by Specific-Growth-Phase-Dependent Lectin-Saccharide Interactions

Coaggregating strains of aquatic bacteria were identified by partial 16S rRNA gene sequencing. The coaggregation abilities of four strains of Blastomonas natatoria and one strain of Micrococcus luteus varied with culture age but were always maximum in the stationary phase of growth. Each member of a coaggregating pair carried either a heat- and protease-sensitive protein (lectin) adhesin or a saccharide receptor, as coaggregation was reversed by sugars.