In water microcosm experiments, the survival times ofCampylobacter isolates differed by up to twofold, as determined by culturing; this difference increased to fourfold when particular combinations of temperature and oxygenation were used. The mean survival times were much longer at 4 and 10°C (202 and 176 h, respectively) than at 22 and 37°C (43 and 22 h, respectively). The influence of anaerobiosis on survival time was less dramatic and differed considerably between isolates. In a two-stage water distribution model preparation containing a biofilm consisting of standardized autochthonous water microflora, Campylobacterisolates continued to differ in survival time. However, the survival times of cultures were considerably longer in the presence of the autochthonous water microflora (strains CH1 and 9752 survived 700 and 360 h, respectively, at 4°C) than in the sterile microcosms (strains CH1 and 9752 survived 230 and 157 h, respectively). Although increased temperature and oxygenation were generally detrimental to culturability, the interaction of these two factors influenced the two strains examined differently. When the organisms were grown aerobically at 30°C, the survival of the two strains was reversed; aerobiosis decreased the survival time of strain CH1 by 30%, but unexpectedly improved the persistence time of strain 9752 by more than threefold. Persistence times within biofilms were much longer when they were determined by detection methods not involving culturing. Immunofluorescent-antibody staining demonstrated that the pathogen persisted up to the termination of the experiments after 28 and 42 days of incubation at 30 and 4°C, respectively. The specificity of detection within intact biofilms was reduced because of high background fluorescence. However, preliminary studies with aCampylobacter-specific rRNA probe revealed the same extended persistence of the pathogen within the biofilms.
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.Coaggregation is the cell-to-cell recognition of genetically distinct partner cell types (13) and was first demonstrated for bacteria from dental plaque (6). Coaggregation between oral bacteria is mediated by lectin-saccharide interactions between cell surface molecules on the partner organisms (1,5,8,10). Coaggregation also occurs between members of the urogenital flora (16) and between strains of Lactobacillus from chicken crops (19). Most recently, coaggregation between aquatic biofilm-forming bacteria was described and found to be reversed by simple sugars (2), although involvement of surface proteins was not investigated. In addition, most of the aquatic strains were unidentified, and the coaggregation scores for some pairs of aquatic bacteria showed variation between different batch cultures. This study describes the identification of five aquatic coaggregating bacteria by using 16S rRNA gene sequencing and investigates the role of surface proteins in the coaggregation process. In addition, the relationship between coaggregation ability and phase of growth in batch culture is presented.Five coaggregating bacteria isolated from biofilm samples and previously designated as strains 2.1, 2.3, 2.6, 2.8, and 2.13 (2) were grown on R2A agar at 25°C (Difco) (15). Batch cultures were grown in 100 ml of liquid R2A broth, with shaking at 200 rpm at 25°C. All five strains were characterized by a combination of biochemical tests and light microscopy and by sequencing approximately 650 bases of the 16S rRNA gene. Bacterial genomic DNA from each strain was obtained by boiling a single bacterial colony, and the primers used for amplification and sequencing of 16S rRNA gene fragments were 8FPL (20) and 806R (22). The nucleotide sequence of each PCR product was compared to known sequences in the EMBL database, and the organism with the closest sequence similarity was identified.All five strains could be identified to the species level, as all had greater than 98.5% similarity with the closest sequence in the database. Four strains were identified as Blastomonas natatoria (strains 2.1, 2.3, 2.6, and 2.8) and one strain was identified as Micrococcus luteus (strain 2.13). B. natatoria strains were gram-negative, obligately aerobic, oxidase-and catalasepositive rods giving highly pigmented yellow colonies on R2A agar. All four strains divided asymmetrically to give daughter cells with a single polar flagellum. Comparison of the partial 16S rRNA gene sequence of each of the B. natatoria strains showed that they had 97.9 to 99.7% identity, indicating that they were very closely rela...
Nineteen numerically dominant heterotrophic bacteria from a freshwater biofilm were identified by 16S ribosomal DNA gene sequencing, and their coaggregation partnerships were determined. Phylogenetic trees showed that both distantly related and closely related strains coaggregated at intergeneric, intrageneric, and intraspecies levels. One strain, Blastomonas natatoria 2.1, coaggregated with all 18 other strains and may function as a bridging organism in biofilm development.Coaggregation between bacteria occurs when two or more genetically distinct strains interact by specific cell-cell recognition (12). The phenomenon was first recognized between different oral plaque-forming bacteria, where both intergeneric and intrageneric coaggregation occurs (11). Coaggregation also occurs between bacteria isolated from a freshwater biofilm (3,19), and it has been suggested that coaggregation may also mediate in the sequential integration of species of bacteria into freshwater biofilms (8,20). Recently, Rickard et al. (19) used partial 16S rRNA gene sequencing to identify four coaggregating strains of Blastomonas natatoria and one strain of Micrococcus luteus from an established freshwater biofilm community (3). Six coaggregation partnerships between these five strains were found and shown to be mediated by growth-phasedependent lectin-saccharide interactions (19). These five coaggregating strains of B. natatoria and M. luteus were part of a larger community of 19 coaggregating strains that were all isolated simultaneously from a biofilm formed on glass in a chemostat (3). The identities of the other members of the consortium are unknown, and the extent of intergeneric and intrageneric and intraspecies coaggregation between all members of the community has not been analyzed previously. Since coaggregation may be an adhesion mechanism involved with integrating and establishing bacteria in the biofilm community, it is important to know the extent of this specific adhesion mechanism in the freshwater biofilm. It is also relevant to know how closely related coaggregating strains are, since this has implications for understanding the biodiversity of the biofilm community. Therefore, the work reported here had three main objectives: (i) identification by 16S rRNA gene sequencing of all strains in the freshwater biofilm community; (ii) construction of phylogenetic trees by the computation of evolutionary distance matrices and maximum-likelihood rooted dendrograms; and (iii) analysis of intergeneric and intrageneric and intraspecies (interstrain) coaggregation partnerships between members of the biofilm community deduced using the phylogenetic trees.All strains used in this study were isolated from a 14-day-old biofilm on a glass coupon in a two-stage chemostat kept at 4°C, which was initially inoculated with water from a borehole water source (Porton borehole, Salisbury, United Kingdom) (3). The ionic composition of the water and temperature within the chemostat were very similar to the conditions found in the source borehole wa...
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