Investigating Photoactive Antimicrobials as Alternatives (or Adjuncts) to Traditional Therapy.

Photodynamic therapy (PDT) is an established therapy used for the treatment of cutaneous skin cancers and other non-infective ailments. There has been recent interest in the opportunity to use aPDT (antimicrobial PDT) to treat skin and soft tissue infections. PDT utilizes photosensitizers that infiltrate all cells and "sensitize" them to a given wavelength of light. The photosensitizer is simply highly absorbent to a given wavelength of light and when excited will produce, in the presence of oxygen, damaging oxygen radicals and singlet oxygen. Bacterial cells are comparatively poor at combatting oxidative stress when compared with human cells therefore a degree of selective toxicity can be achieved with aPDT.In this chapter, we outline methodologies for testing aPDT in vitro using standard lab equipment.

Investigating Combination Therapy as a Means to Enhance Activity and Repurpose Antimicrobials.

Current clinical practice assumes that a single antibiotic given as a bolus or as a course will successfully treat most infections. In modern medicine, this is becoming less and less true with drug-resistant, multi-drug-resistant, extensively drug-resistant, and untreatable infections becoming more common. Where single-drug therapy (monotherapy) fails, we will turn to multi-drug therapy. Alternatively, combination therapy could be useful to prevent the emergence of resistance. Multi-drug therapy is already standard for some multi-drug resistant infections and is the standard for the treatment of some pathogens such as Mycobacterium tuberculosis.The use of combination therapy for everyday infections could be a clear course out of the current AMR crisis we are facing. With every additional drug added to a combination (n + 1) the likelihood of the pathogen evolving resistance drops exponentially.Many generic antibiotics are cheap to manufacture as they have fallen out of patent protection but are less effective at pharmacologically effective doses due to overuse in the past. Combination therapy can combine these generic compounds into cocktails that can not only treat susceptible and resistant infections but can also reduce the risk of new resistances arising and can resuscitate the use of antimicrobials once thought defunct.In this chapter, we will summarize theory behind combination therapy and standard in vitro methodologies used.

Monitoring Live Mycobacteria in Real-Time Using a Microfluidic Acoustic-Raman Platform.

Tuberculosis (TB) is the most common cause of death from an infectious disease. Although treatment has been available for more than 70 years, it still takes too long and many patients default risking relapse and the emergence of resistance. It is known that lipid-rich, phenotypically antibiotic-tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse necessitating extended therapy. Using a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis we can perform optical analysis in the form of wavelength-modulated Raman spectroscopy (WMRS) on the trapped organisms. This system can allow observations of the mycobacteria for up to 8 h. By adding antibiotics, it is possible to study the effect of antibiotics in real-time by comparing the Raman fingerprints in comparison to the unstressed condition. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to many conditions including antibiotic stress, and changes in the growth media. This opens the possibility of understanding better the stimuli that trigger the lipid-rich downregulated and phenotypically antibiotic-resistant cell state.

Using Hollow Fiber to Model Treatment of Antimicrobial-Resistant Organisms.

The use of animal models is still widespread in science but there is a movement away from this manner of experimentation. One option approved by the FDA for human-like studies is the hollow fiber bioreactor (HFS). HFSs are highly controllable, self-contained systems that allow for the modeling of individual tissues and disease phenotypes. Oxygen, drug concentration & half-life, and immune cell invasion are all scalable to human and veterinary conditions using a HFS. There are drawbacks to the systems including cost and contamination so the use of these systems must be carefully managed.With these limitations in mind, the scope of the technology is great. Antimicrobial susceptibility testing (AST) is possible with greater accuracy and clinical validity than classical in vitro techniques making minimal inhibitory concentration (MIC) data generated on the bench more translatable to the clinic.In this chapter, we will outline the background of the HFS and some typical uses.

Rapid Drug Susceptibility Testing to Preserve Antibiotics.

Antibiotic resistance is a global challenge likely to cost trillions of dollars in excess costs in the health system and more importantly, millions of lives every year. A major driver of resistance is the absence of susceptibility testing at the time a healthcare worker needs to prescribe an antimicrobial. The effect is that many prescriptions are unintentionally wasted and expose mutable organisms to antibiotics increasing the risk of resistance emerging. Often simplistic solutions are applied to this growing issue, such as a naïve drive to increase the speed of drug susceptibility testing. This puts a spotlight on a technological solution and there is a multiplicity of such candidate DST tests in development. Yet, if we do not define the necessary information and the speed at which it needs to be available in the clinical decision-making progress as well as the necessary integration into clinical pathways, then little progress will be made. In this chapter, we place the technological challenge in a clinical and systems context. Further, we will review the landscape of some promising technologies that are emerging and attempt to place them in the clinic where they will have to succeed.

A simple label-free method reveals bacterial growth dynamics and antibiotic action in real-time.

Understanding the response of bacteria to environmental stress is hampered by the relative insensitivity of methods to detect growth. This means studies of antibiotic resistance and other physiological methods often take 24 h or longer. We developed and tested a scattered light and detection system (SLIC) to address this challenge, establishing the limit of detection, and time to positive detection of the growth of small inocula. We compared the light-scattering of bacteria grown in varying high and low nutrient liquid medium and the growth dynamics of two closely related organisms. Scattering data was modelled using Gompertz and Broken Stick equations. Bacteria were also exposed meropenem, gentamicin and cefoxitin at a range of concentrations and light scattering of the liquid culture was captured in real-time. We established the limit of detection for SLIC to be between 10 and 100 cfu mL-1 in a volume of 1-2 mL. Quantitative measurement of the different nutrient effects on bacteria were obtained in less than four hours and it was possible to distinguish differences in the growth dynamics of Klebsiella pneumoniae 1705 possessing the BlaKPC betalactamase vs. strain 1706 very rapidly. There was a dose dependent difference in the speed of action of each antibiotic tested at supra-MIC concentrations. The lethal effect of gentamicin and lytic effect of meropenem, and slow bactericidal effect of cefoxitin were demonstrated in real time. Significantly, strains that were sensitive to antibiotics could be identified in seconds. This research demonstrates the critical importance of improving the sensitivity of bacterial detection. This results in more rapid assessment of susceptibility and the ability to capture a wealth of data on the growth dynamics of bacteria. The rapid rate at which killing occurs at supra-MIC concentrations, an important finding that needs to be incorporated into pharmacokinetic and pharmacodynamic models. Importantly, enhanced sensitivity of bacterial detection opens the possibility of susceptibility results being reportable clinically in a few minutes, as we have demonstrated.

Real-time monitoring of live mycobacteria with a microfluidic acoustic-Raman platform.

Tuberculosis (TB) remains a leading cause of death worldwide. Lipid rich, phenotypically antibiotic tolerant, bacteria are more resistant to antibiotics and may be responsible for relapse and the need for long-term TB treatment. We present a microfluidic system that acoustically traps live mycobacteria, M. smegmatis, a model organism for M. tuberculosis. We then perform optical analysis in the form of wavelength modulated Raman spectroscopy (WMRS) on the trapped M. smegmatis for up to eight hours, and also in the presence of isoniazid (INH). The Raman fingerprints of M. smegmatis exposed to INH change substantially in comparison to the unstressed condition. Our work provides a real-time assessment of the impact of INH on the increase of lipids in these mycobacteria, which could render the cells more tolerant to antibiotics. This microfluidic platform may be used to study any microorganism and to dynamically monitor its response to different conditions and stimuli.

Centrifugation and decontamination procedures selectively impair recovery of important populations in Mycobacterium smegmatis.

Diagnosis and treatment monitoring of patients with tuberculosis (TB) requires detection of all viable mycobacteria in clinical samples. Quantitation of Mycobacterium tuberculosis (Mtb) in sputum is commonly performed by culture after sample decontamination to prevent overgrowth by contaminant organisms. Exponentially growing cultures have cells that predominately lack non-polar lipid bodies whereas stationary cultures have a predominance of cells with non-polar lipid bodies. This may reflect rapidly growing 'active' and non-replicating 'persister' sub-populations respectively in sputum from TB patients. We investigated the effect of decontamination on culture-based quantitation of exponential and stationary phase cultures of Mycobacterium smegmatis in an artificial sputum model. Exponentially growing populations were between 89 and 50 times more susceptible to decontamination than stationary phase cultures when quantified by most probable number and colony forming units. These findings suggest that decontamination selectively eliminates the 'active' population. This may impair diagnostic sensitivity, treatment monitoring, and compromise clinical trials designed to identify new antibiotic combinations with activity against all mycobacterial cell states.

Enhanced Methodologies for Detecting Phenotypic Resistance in Mycobacteria.

Lipid droplets found in algae and other microscopic organisms have become of interest to many researchers partially because they carry the capacity to produce bio-oil for the mass market. They are of importance in biology and clinical practice because their presence can be a phenotypic marker of an altered metabolism, including reversible resistance to antibiotics, prompting intense research.A useful stain for detecting lipid bodies in the lab is Nile red. It is a dye that exhibits solvatochromism; its absorption band varies in spectral position, shape and intensity with the nature of its solvent environment, it will fluoresce intensely red in polar environment and blue shift with the changing polarity of its solvent. This makes it ideal for the detection of lipid bodies within Mycobacterium spp. This is because mycobacterial lipid bodies' primary constituents are nonpolar lipids such as triacylglycerols but bacterial cell membranes are primarily polar lipid species. In this chapter we describe an optimal method for using Nile red to distinguish lipid containing (Lipid rich or LR cells) from those without lipid bodies (Lipid Poor or LP). As part of the process we have optimized a method for separating LP and LR cells that does not require the use of an ultracentrifuge or complex separation media. We believe that these methods will facilitate further research in these enigmatic, transient and important cell states.

Label-free optical vibrational spectroscopy to detect the metabolic state of M. tuberculosis cells at the site of disease.

Tuberculosis relapse is a barrier to shorter treatment. It is thought that lipid rich cells, phenotypically resistant to antibiotics, may play a major role. Most studies investigating relapse use sputum samples although tissue bacteria may play an important role. We developed a non-destructive, label-free technique combining wavelength modulated Raman (WMR) spectroscopy and fluorescence detection (Nile Red staining) to interrogate Mycobacterium tuberculosis cell state. This approach could differentiate single "dormant" (lipid rich, LR) and "non-dormant" (lipid poor, LP) cells with high sensitivity and specificity. We applied this to experimentally infected guinea pig lung sections and were able to distinguish both cell types showing that the LR phenotype dominates in infected tissue. Both in-vitro and ex-vivo spectra correlated well, showing for the first time that Mycobacterium tuberculosis, likely to be phenotypically resistant to antibiotics, are present in large numbers in tissue. This is an important step in understanding the pathology of relapse supporting the idea that they may be caused by M. tuberculosis cells with lipid inclusions.

Phenotypic resistance in mycobacteria: is it because I am old or fat that I resist you?

OBJECTIVES: We aimed to explore the phenomenon of phenotypic resistance to antimycobacterial antibiotics and to determine whether this was associated with cell age or the presence of lipid bodies. METHODS: The accumulation of lipid-body-positive [lipid-rich (LR)] cells was followed using cell staining and flow cytometry. LR cells of Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium fortuitum and Mycobacterium bovis (BCG) were separated from non-lipid-body-containing [lipid-poor (LP)] cells and their MBCs determined. We also compared the MBCs for LR and LP cells from 'old' and 'young' cultures. RESULTS: The LR cells of all species were more resistant to antibiotics than LP cells. For BCG, the susceptibility ratios were as follows: rifampicin, 5×; isoniazid, 16.7×; ethambutol, 5×; and ciprofloxacin, 5×. Phenotypic resistance was found in LR cells irrespective of cell age. CONCLUSIONS: We have shown that phenotypic antibiotic resistance is associated with the presence of lipid bodies irrespective of cell age. These data have important implications for our understanding of relapse in mycobacterial infections.