Growth Dynamics and Survival of Liberibacter crescens BT-1, an Important Model Organism for the Citrus Huanglongbing Pathogen "Candidatus Liberibacter asiaticus".

Liberibacter crescens is the only cultured member of its genus, which includes the devastating plant pathogen "Candidatus Liberibacter asiaticus," associated with citrus greening/Huanglongbing (HLB). L. crescens has a larger genome and greater metabolic flexibility than "Ca Liberibacter asiaticus" and the other uncultured plant-pathogenic Liberibacter species, and it is currently the best model organism available for these pathogens. L. crescens grows slowly and dies rapidly under current culture protocols and this extreme fastidiousness makes it challenging to study. We have determined that a major cause of rapid death of L. crescens in batch culture is its alkalinization of the medium (to pH 8.5 by the end of logarithmic phase). The majority of this alkalinization is due to consumption of alpha-ketoglutaric acid as its primary carbon source, with a smaller proportion of the pH rise due to NH3 production. Controlling the pH rise with higher buffering capacity and lower starting pH improved recoverability of cells from 10-day cultures by >1,000-fold. We have also performed a detailed analysis of L. crescens growth with total cell numbers calibrated to the optical density and the percentage of live and recoverable bacteria determined over 10-day time courses. We modified L. crescens culture conditions to greatly enhance survival and increase maximum culture density. The similarities between L. crescens and the pathogenic liberibacters make this work relevant to efforts to culture the latter organisms. Our results also suggest that growth-dependent pH alteration that overcomes medium buffering should always be considered when growing fastidious bacteria.IMPORTANCELiberibacter crescens is a bacterium that is closely related to plant pathogens that have caused billions of dollars in crop losses in recent years. Particularly devastating are citrus losses due to citrus greening disease, also known as Huanglongbing, which is caused by "Candidatus Liberibacter asiaticus" and carried by the Asian citrus psyllid. L. crescens is the only close relative of "Ca Liberibacter asiaticus" that can currently be grown in culture, and it therefore serves as an important model organism for the growth, genetic manipulation, and biological control of the pathogenic species. Here, we show that one of the greatest limitations to L. crescens growth is the sharp increase in alkaline conditions it produces as a consequence of consumption of its preferred nutrient source. In addition to new information about L. crescens growth and metabolism, we provide new guidelines for culture conditions that improve the survival and yield of L. crescens.

Enhanced disinfection of bacterial populations by nutrient and antibiotic challenge timing.

Several difficult to treat illnesses like tuberculosis, chronic pneumonia, and inner ear infections are caused by tolerant bacteria enmeshed in a biofilm. Bacterial tolerance can be genotypic (resistance-i.e. MRSA), phenotypic (non-heritable) or environmental (e.g. nutrient gradients). Persister formation is a phenotypic expression and this phenotype is highly tolerant of disinfection. Constant dosing is typically ineffective and to generate an effective treatment protocol, we need to understand the dynamics of persister cells. In this study, we investigate how manipulating the application of antibiotics and addition of nutrients enhances the disinfection of a bacterial population in batch culture. Eliminating persister bacteria is considered as a challenge for the food industry or wastewater treatment, since the failure may result in food contamination and disease transmission. Previous studies focused on the antimicrobial agent as a control variable to eliminate the bacterial population. In addition to antibiotic, we consider the significance of the nutrient in eradicating the susceptible and persister cells since the disinfection of susceptible population depends on the nutrient intake. We present a mathematical model that captures the dynamics between susceptible and persister bacteria with antibiotic and nutrient control variables. We investigate the optimal dose-withdrawal of antibiotic timing in several cases including constant nutrient in time, dynamic nutrient in time and piecewise constant nutrient in time.

Predictive Computer Models for Biofilm Detachment Properties in Pseudomonas aeruginosa.

Microbial biofilm communities are protected against environmental extremes or clearance by antimicrobial agents or the host immune response. They also serve as a site from which microbial populations search for new niches by dispersion via single planktonic cells or by detachment by protected biofilm aggregates that, until recently, were thought to become single cells ready for attachment. Mathematically modeling these events has provided investigators with testable hypotheses for further study. Such was the case in the recent article by Kragh et al. (K. N. Kragh, J. B. Hutchison, G. Melaugh, C. Rodesney, A. E. Roberts, Y. Irie, P. Ø. Jensen, S. P. Diggle, R. J. Allen, V. Gordon, and T. Bjarnsholt, mBio 7:e00237-16, 2016, http://dx.doi.org/10.1128/mBio.00237-16), in which investigators were able to identify the differential competitive advantage of biofilm aggregates to directly attach to surfaces compared to the single-celled planktonic populations. Therefore, as we delve deeper into the properties of the biofilm mode of growth, not only do we need to understand the complexity of biofilms, but we must also account for the properties of the dispersed and detached populations and their effect on reseeding.

Biofilms and infectious diseases: biology to mathematics and back again.

There has been tremendous growth in biofilm research in the past three decades. This growth has been reflected in development of a wide variety of experimental, clinical, and theoretical techniques fostered by our increased knowledge. Keeping the theoretical developments abreast of the experimental advancements and ensuring that the theoretical results are disseminated to the experimental and clinical community is a major challenge. This manuscript provides an overview of recent developments in each scientific domain. More importantly, this manuscript aims to identify areas where the theory lags behind the experimental understanding (and vice versa). The major themes of the manuscript derive from discussions and presentations at a recent interdisciplinary workshop that brought together a variety of scientists whose underlying studies focus on biofilm processes.