Assessment of a chemostat-coupled modified Robbins device to study biofilms

The combination of a modified Robbins device (MRD) attached to the effluent line of a continuous cultivation vessel was assessed by the adhesion of planktonic bacteria maintained at a controlled growth rate. This combination of a chemostat and an MRD provides a large number of sample surfaces for monitoring both the formation and control of biofilms over extended periods of time. This apparatus was used to monitor the colonization of two soil isolates,Pseudomonas fluorescens (EX101) andPseudomonas putida (EX102) onto silastic rubber surfaces. At a similar _rel, both bacteria attached to the silastic, howeverP. fluorescens formed confluent, dense biofilms in less than 24 h, whereasP. putida adhered as single cells or microcolonies after the same period. The metabolic activity, measured by INT-formazan formation, was similar for both organisms with a peak at 6 h of colonization and a subsequent decrease after 24 h. Long term colonization studies ofP. fluorescens produced a population of greater than 9.5 log cfu cm^–2 at 28 days demonstrating the advantages of the chemostat-MRD association. This technique proved to be successful for studying bacterial adhesion and biofilm formation in tubular devices by bacterial populations at controlled and low growth rates.     

Microbial biofilms: from ecology to molecular genetics.

Biofilms are complex communities of microorganisms attached to surfaces or associated with interfaces. Despite the focus of modern microbiology research on pure culture, planktonic (free-swimming) bacteria, it is now widely recognized that most bacteria found in natural, clinical, and industrial settings persist in association with surfaces. Furthermore, these microbial communities are often composed of multiple species that interact with each other and their environment. The determination of biofilm architecture, particularly the spatial arrangement of microcolonies (clusters of cells) relative to one another, has profound implications for the function of these complex communities. Numerous new experimental approaches and methodologies have been developed in order to explore metabolic interactions, phylogenetic groupings, and competition among members of the biofilm. To complement this broad view of biofilm ecology, individual organisms have been studied using molecular genetics in order to identify the genes required for biofilm development and to dissect the regulatory pathways that control the plankton-to-biofilm transition. These molecular genetic studies have led to the emergence of the concept of biofilm formation as a novel system for the study of bacterial development. The recent explosion in the field of biofilm research has led to exciting progress in the development of new technologies for studying these communities, advanced our understanding of the ecological significance of surface-attached bacteria, and provided new insights into the molecular genetic basis of biofilm development.     

A chemostat study of ethanol inhibition

The mode of ethanol action on both the steady-state and the dynamic properties of K. aerogenes was investigated using a nitrogen-limited chemostat. Reduction in the maximum growth rate (μ_m) suggests that noncompetitive enzyme inhibition could occur, but Lineweaver–Burk analysis showed the inhibition to be more complex. A consistent mechanism of inhibition was established for 0–3% v/v ethanol. Warburg manometric experiments indicated that inhibition occurred in pathways located in the intact cell wall. Frequency response analysis, using sinusoidal variations in the dilution rate showed that ethanol increased the time constant of the metabolic parameter, Q_AC. The system was stable in the presence of ethanol and showed no evidence of oscillations following a disturbance.     

Mathematical conservation ecology: A one-predator-two-prey system as case study

A method is presented to analyse the long-term stochastic dynamics of a biological population that is at risk of extinction. From the full ecosystem the method extracts the minimal information to describe the long-term dynamics of that population by a stochastic logistic system. The method is applied to a one-predator-two-prey model. The choice of this example is motivated by a study on the near-extinction of a porcupine population by mountain lions whose presence is facilitated by mule deer taking advantage of a change in land use. The risk of extinction is quantified by the expected time of extinction of the population.     

An in vitro study of the effect of fluoridated milk on oral bacterial biofilms

Microcosmic dental plaques were grown in artificial saliva and supplemented with either milk or fluoridated milk. The presence of fluoride in the milk increased the pH of the biofilms and reduced the proportions of streptococci, demonstrating that in this model, fluoridation of milk produces biofilms with reduced cariogenic potential.     

Study of the dynamic behavior of the chemostat system

This paper deals with a theoretical study on the dynamic, character of the chemostat system. It. is primarily based on the Monod model for growth limitation, although certain more complex models are considered. Since the Monod model is described in terms of two variables, an analysis by use of a phase plane plot will show the various possible types of behavior theoretically expected for transient conditions of the system. In this paper it will be shown that the chemostat system might show an overshoot (or an underswing) with respect to changes in cell and substrate concentrations, depending on the extent to which the system might be disturbed from steady-slate conditions. Other types of transient behavior ran also be expected when one of the system parameters such as dilution rate or input substrate concentration is disturbed in a stepwise manner. The simple Monod chemostat model was found never to oscillate in either a damped or a sustained manner as has been experimentally reported. Discussion is included about the transient behavior of other chemostat models such as that involving a variable yield coefficient, i.e., including the effect of cell maintenance requirements.     

A study of the efficacy of ultrasonic waves in removing biofilms

doi:10.1111/j.1741‐2358.2009.00325.x
A study of the efficacy of ultrasonic waves in removing biofilms Objective: The removal of adherent biofilms was assessed using ultrasonic waves in a non‐contact mode. Materials and Methods: In in vitro experiments, Streptococcus mutans (S. mutans) biofilms were exposed to ultrasonic waves at various frequencies (280 kHz, 1 MHz, or 2 MHz), duty ratios (0–90%), and exposure times (1–3 minutes), and the optimal conditions for biofilm removal were identified. Furthermore, the effect of adding a contrast medium, such as micro bubbles (Sonazoid^®), was examined. The spatial distribution and architecture of S. mutans biofilms before and after ultrasonic wave exposure were examined via scanning electron microscopy. The biofilm removal effect was also examined in in vivo experiments, using a custom‐made oral cleaning device. Results: When a 280 kHz probe was used, the biofilm‐removing effect increased significantly compared to 1 and 2 MHz probes; more than 80% of the adherent biofilm was removed with a duty cycle of 50–90% and a 3 minutes exposure time. The maximum biofilm‐removing effect was observed with a duty cycle of 80%. Furthermore, the addition of micro bubbles enhanced this biofilm‐removing effect. In in vivo experiments, moderate biofilm removal was observed when a 280 kHz probe was used for 5 minutes. Conclusions: This study demonstrated that ultrasonic wave exposure in a non‐contact mode effectively removed adherent biofilms composed of S. mutans in vitro.     

Characterization of antibiotic resistance determinants in oral biofilms

Oral biofilms contain numerous antibiotic resistance determinants that can be transferred within or outside of the oral cavity. The aim of this study was to evaluate the prevalence and the relative level of antibiotic resistance determinants from oral biofilms. Oral biofilm samples that were collected from healthy subjects and periodontitis patients were subjected to qualitative and quantitative analyses for selected antibiotic resistance determinants using PCR. The prevalence of tet(Q), tet(M), cfxA, and bla _TEM was very high both in the patient and the healthy subject group, with a tendency toward higher values in the patient group, with the exception of erm(F), which was more prevalent in the healthy group. The two extended spectrum β-lactam (ESBL) resistance determinants bla _SHV and bla _TEM showed a dramatic difference, as bla _TEM was present in all of the samples and bla _SHV was not found at all. The aacA-aphD, vanA, and mecA genes were rarely detected, suggesting that they are not common in oral bacteria. A quantitative PCR analysis showed that the relative amount of resistance determinants present in oral biofilms of the patient group was much greater than that of the healthy group, exhibiting 17-, 13-, 145-, and 3-fold increases for tet(Q), tet(M), erm(F), and cfxA, respectively. The results of this study suggest that the oral antibiotic resistome is more diverse and abundant in periodontitis patients than in healthy subjects, suggesting that there is a difference in the diversity and distribution of antibiotic resistance in oral biofilms associated with health and disease.     

A multiple chemostat system for consortia studies on microbially influenced corrosion

A multiple chemostat system has been developed in which metal specimens can be exposed to a consortium of bacteria. The system comprises a single test chemostat containing the test specimen operated at a high dilution rate to facilitate the wash out of planktonic bacteria, selecting for attached or biofilm growth. This chemostat is fed at a steady low rate by a number of separate chemostats each of which contains a pure axenic culture of one member of the consortium being tested. This system has the advantage of providing a continual inoculum of the test species to the test specimen allowing both aerobic and anaerobic bacteria to be grown in the same system. Constant levels of three bacterial types were maintained in the system: Pseudomonas aeruginosa, Thiobacillus ferrooxidans and Desulfovibrio vulgaris. Exposure of 316L stainless steel electrodes to this system resulted in increased corrosion of coupons exposed biotically, as compared to those exposed abiotically. A current monitoring technique and electrochemical impedance spectroscopy were used to evaluate effects of bacteria on metallic corrosion.     

A dynamic mathematical model of the chemostat

A number of experimental studies on the dynamic, behavior of the chemostat have shown that the specific growth rate does not, instantaneously adjust to changes in the concentration of limiting substrate in the chemostat following disturbances in the steady state input limiting substrate concentration or in the steady state dilution rate. Instead of an instantaneous response, as would be predicted by the Monod equation, experimental studies have shown that the specific growth rate experiences a dynamic lag in responding to the changes in the concentration of limiting substrate in the culture vessel. The observed dynamic lag has been recognized by researchers in such terms as an inertial phenomenon and as a hysteresis effect, but as yet a systems engineering approach has not been applied to the observed data. The present paper criticizes the use of the Monod equation as a dynamic relationship and offers as an alternative a dynamic equation relating specific growth rate to the limiting substrate concentration in the chemostat. Following the development of equations, experimental methods of evaluating parameters are discussed. Dynamic responses of analog simulations (incorporating the newly derived equations) are compared with the dynamic responses predicted by the Monod equation and with the dynamic responses of experimental chemostats.     

A chemostat study on proline uptake and metabolism of Leishmania donovani.

Leishmania donovani grew in the chemostat on proline as its sole carbon and energy source at a maximum growth rate of 1.39 divisions per day. The efficiency of proline metabolism decreased with increasing external proline concentration. The internal concentration of proline and its intracellular metabolites was low when proline was the growth rate limiting substrate and high when proline was available in excess. In time-course experiments proline uptake leveled off after 30 min, independent of the culture conditions prior to the experiment. Proline uptake depended on the external proline concentration in a manner that is best described as the combination of an enzymatic and a diffusion component. Adaptation to different proline concentrations did not occur and no evidence was found that proline is actively transported by L. donovani.     

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.     

The effect of chlorhexidine on defined, mixed culture oral biofilms grown in a novel model system

In order to develop an improved method to evaluate antimicrobial agents for use in clinical dentistry, a constant-depth film fermenter (CDFF) has been used to generate biofilms of fixed depth comprising nine species of bacteria commonly found in dental plaque in health and disease. These bacteria were grown together initially in a conventional chemostat which was used to inoculate the CDFF over an 8 h period. Medium was then supplied directly to the CDFF and biofilms allowed to develop. The biofilms were then challenged with eight short pulses of two concentrations of chlorhexidine (0·0125 and 0·125% w/v). The lower concentration had a limited effect on the composition of the biofilms while a differential and substantial inhibition was obtained with a higher concentration. Actinomyces naeslundii was lost from the biofilm, and the viable counts of streptococci, Fusobacterium nucleatum and Porphyromonas gingivalis were inhibited by over three orders of magnitude by 0·125% chlorhexidine, whereas Veillonella dispar was only transiently affected. The findings were consistent with those from clinical studies of dental plaque, suggesting that this model would have a predictive value when evaluating novel antiplaque or antimicrobial inhibitors.     

A modified bait for oral delivery of biological agents to raccoons and feral swine

A field study was conducted on Ossabaw Island (Georgia, USA) in March 1994 to evaluate four different types of bait for delivering orally effective biological agents to raccoons (Procyon lotor) and feral swine (Sus scrofa). A deep-fried corndog batter bait, which was previously shown to be ingested by both captive and free-ranging raccoons, and a polymer fishmeal bait which had been shown effective for both raccoons and feral swine were compared with a grain-based dog food meal polymer bait topically coated with corn oil and cornmeal or with fish oil and fishmeal. Tracking stations were used to determine the number of each bait type visited and removed by animals visiting stations. We found no significant differences in the numbers of different baits removed by either species. These data support the results of earlier studies which also indicated that an inexpensive grain-based matrix bait surface-coated with attractive flavors can be used to deliver oral biologics to problem species.     

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.     

Examination and characterization of distribution system biofilms

Investigations concerning the role of distribution system biofilms on water quality were conducted at a drinking water utility in New Jersey. The utility experienced long-term bacteriological problems in the distribution system, while treatment plant effluents were uniformly negative for coliform bacteria. Results of a monitoring program showed increased coliform levels as the water moved from the treatment plant through the distribution system. Increased coliform densities could not be accounted for by growth of the cells in the water column alone. Identification of coliform bacteria showed that species diversity increased as water flowed through the study area. All materials in the distribution system had high densities of heterotrophic plate count bacteria, while high levels of coliforms were detected only in iron tubercles. Coliform bacteria with the same biochemical profile were found both in distribution system biofilms and in the water column. Assimilable organic carbon determinations showed that carbon levels declined as water flowed through the study area. Maintenance of a 1.0-mg/liter free chlorine residual was insufficient to control coliform occurrences. Flushing and pigging the study area was not an effective control for coliform occurrences in that section. Because coliform bacteria growing in distribution system biofilms may mask the presence of indicator organisms resulting from a true breakdown of treatment barriers, the report recommends that efforts continue to find methods to control growth of coliform bacteria in pipeline biofilms.