Persistent bacterial infections

Persister cells are difficult to study owing to their transient nature and their usually small number in bacterial populations. In the past, numerous attempts have been made to elucidate persistence mechanisms. However, because of the challenges involved in studying persisters and the clear redundancy in mechanisms underlying their generation, our knowledge of molecular pathways to persistence remains incomplete. Here, we describe how to use experimental evolution with cyclic antibiotic treatments to generate mutants with an increased persister level in stationary phase, ranging from the initial ancestral level up to 100 %. This method will help to unravel molecular pathways to persistence, and opens up a myriad of new possibilities in persister research, such as the convenient study of nearly pure persister cultures and the possibility to investigate the role of time and environmental aspects in the evolution of persistence.

Bacterial and viral infections are associated with many chronic illnesses as causative agents, cofactors or more
likely as opportunistic infections in immune suppressed individuals. The prevalence of invasive pathogenic
Mycoplasma species infections (and possibly other bacterial infections, such as Chlamydia, Borrelia, etc.) in
patients with Chronic Fatigue Syndrome, Fibromyalgia Syndrome, Gulf War Illness, Rheumatoid Arthritis and
other chronic illnesses was significantly higher than in healthy controls. When we examined chronic illness
patients for multiple Mycoplasma species infections, we found that almost all patients had multiple
intracellular infections, suggesting that multiple bacterial infections commonly occur in certain chronic
illness patients. These patients generally respond to particular antibiotics if administered long-term, but an
important part of their recovery involves nutritional supplementation with appropriate vitamins, minerals,
immune enhancement and other supplements. Nutraceuticals appear to be necessary for recovery and
maintenance of a strong immune system. In addition, patients should be removed from potentially immunedepressing
drugs, such as some antidepressants, to allow recovery of their immune systems. Other chronic
infections (viral), may also be involved in various chronic fatigue illnesses with or without mycoplasmal and
other bacterial infections, and these multiple infections could be important in causing patient morbidity and
resulting difficulties in treating these illnesses.
INTRODUCTION
Most if not all debilitating chronic illnesses
are characterized by the presence of chronic fatigue
(1), the most commonly reported medical complaint
of all patients seeking medical care (2). The fatigue
syndromes, such as Chronic Fatigue Syndrome (CFS
or Myalgic Encephalomyelitis [ME]), Fibromyalgia
Syndrome (FMS) and Gulf War Illnesses (GWI),
share many complex, multi-organ signs and
symptoms (3-6), including immune system
abnormalities (7), but are distinguishable as separate
syndromes that have muscle and overall fatigue as
major signs. These syndromes usually have
overlapping signs and symptoms, including muscle
pain, chronic fatigue, headaches, memory loss,
nausea, gastrointestinal problems, joint pain, vision
problems, breathing problems, depression, low grade
fevers, skin disorders, tissue swelling, chemical
sensitivities, among others (5, 6, 8). Because of the
complex nature of these illnesses, many patients are
often diagnosed with multiple syndromes, and their
potential to recover from their chronic illness is
usually poor

A hallmark of bacterial populations cultured in vitro is their homogeneity of growth, where the majority of cells display identical growth rate, cell size and content. Recent insights, however, have revealed that even cells growing in exponential growth phase can be heterogeneous with respect to variables typically used to measure cell growth. Bacterial heterogeneity has important implications for how bacteria respond to environmental stresses, such as antibiotics. The phenomenon of antimicrobial persistence, for example, has been linked to a small subpopulation of cells that have entered into a state of dormancy where antibiotics are no longer effective. While methods have been developed for identifying individual non-growing cells in bacterial cultures, there has been less attention paid to how these cells may influence growth in colonies on a solid surface. In response, we have developed a low-cost, open-source platform to perform automated image capture and image analysis of bacterial colony growth on multiple nutrient agar plates simultaneously. The descriptions of the hardware and software are included, along with details about the temperature-controlled growth chamber, high-resolution scanner, and graphical interface to extract and plot the colony lag time and growth kinetics. Experiments were conducted using a wild type strain of Escherichia coli K12 to demonstrate the feasibility and operation
of our setup. By automated tracking of bacterial growth kinetics in colonies, the system holds the potential to reveal new insights into understanding the impact of microbial heterogeneity on antibiotic resistance and persistence.

Bacterial and viral infections are associated with many chronic illnesses as causative agents, cofactors or more likely as opportunistic infections in immune suppressed individuals. The prevalence of invasive pathogenic Mycoplasma species infections (and possibly other bacterial infections, such as Chlamydia, Borrelia, etc.) in patients with Chronic Fatigue Syndrome, Fibromyalgia Syndrome, Gulf War Illness, Rheumatoid Arthritis and other chronic illnesses was significantly higher than in healthy controls. When we examined chronic illness patients for multiple Mycoplasma species infections, we found that almost all patients had multiple intracellular infections, suggesting that multiple bacterial infections commonly occur in certain chronic illness patients. These patients generally respond to particular antibiotics if administered long-term, but an important part of their recovery involves nutritional supplementation with appropriate vitamins, minerals, immune enhancement and other ...

Persistence is a prime example of phenotypic heterogeneity, where a microbial population splits into two distinct subpopulations with different growth and survival properties as a result of reversible phenotype switching. Specifically, persister cells grow more slowly than normal cells under unstressed growth conditions, but survive longer under stress conditions such as the treatment with bactericidal antibiotics. We analyze the population dynamics of such a population for several typical experimental scenarios, namely a constant environment, shifts between growth and stress conditions, and periodically switching environments. We use an approximation scheme that allows us to map the dynamics to a logistic equation for the subpopulation ratio and derive explicit analytical expressions for observable quantities that can be used to extract underlying dynamic parameters from experimental data. Our results provide a theoretical underpinning for the study of phenotypic switching, in particular for organisms where detailed mechanistic knowledge is scarce.

This communication reports preliminary data towards the development of a live ex vivo model of persistent infection that is based on the chick embryo chorioallantoic membrane (CAM), which can be used for pre-screening biomaterials with antimicrobial properties for their antimicrobial and angiogenic potential. Our results showed that it was possible to infect chicken embryos with Staphylococcus aureus, one of the main types of bacteria found in the persistent infection associated with chronic wounds, and maintain the embryos' survival for up to 48 h. Survival of the embryos varied with the dose of bacteria inoculum and with the use and time of streptomycin application after infection. In infected yet viable embryos, the blood vessels network of the CAM was maintained with minimal disruption. Microbiological tests could confirm embryo infection, but quantification was difficult. By publishing these preliminary results, we hope that not only our group but others within the scientific community further this research towards the establishment of biomimetic and reproducible ex vivo models of persistent infection.