The Pharmaco –, Population and Evolutionary Dynamics of Multi-drug Therapy: Experiments with S. aureus and E. coli and Computer Simulations

There are both pharmacodynamic and evolutionary reasons to use multiple rather than single antibiotics to treat bacterial infections; in combination antibiotics can be more effective in killing target bacteria as well as in preventing the emergence of resistance. Nevertheless, with few exceptions like tuberculosis, combination therapy is rarely used for bacterial infections. One reason for this is a relative dearth of the pharmaco-, population- and evolutionary dynamic information needed for the rational design of multi-drug treatment protocols. Here, we use in vitro pharmacodynamic experiments, mathematical models and computer simulations to explore the relative efficacies of different two-drug regimens in clearing bacterial infections and the conditions under which multi-drug therapy will prevent the ascent of resistance. We estimate the parameters and explore the fit of Hill functions to compare the pharmacodynamics of antibiotics of four different classes individually and in pairs during cidal experiments with pathogenic strains of Staphylococcus aureus and Escherichia coli. We also consider the relative efficacy of these antibiotics and antibiotic pairs in reducing the level of phenotypically resistant but genetically susceptible, persister, subpopulations. Our results provide compelling support for the proposition that the nature and form of the interactions between drugs of different classes, synergy, antagonism, suppression and additivity, has to be determined empirically and cannot be inferred from what is known about the pharmacodynamics or mode of action of these drugs individually. Monte Carlo simulations of within-host treatment incorporating these pharmacodynamic results and clinically relevant refuge subpopulations of bacteria indicate that: (i) the form of drug-drug interactions can profoundly affect the rate at which infections are cleared, (ii) two-drug therapy can prevent treatment failure even when bacteria resistant to single drugs are present at the onset of therapy, and (iii) this evolutionary virtue of two-drug therapy is manifest even when the antibiotics suppress each other''s activity.     

New Approach for the Evaluation of Antimycobacterial Drug Combinations In Vitro (the Laboratory Model Man)

An attempt was made to study quantitatively the antimicrobial effect of combinations of commercially available antituberculosis drugs and antibiotics on the growth of multiple drug resistant strains of Mycobacteriunt intracellulare under simulated in vivo conditions. Combinations of erythromycin, isomiazid, methenamine, or exacillin eliminated populations of M. intracellulare when drug combinations in concentrations achievable in man were kept in contact with the organism for 10 hr daily. Although combinations of INH and rifampin failed to eliminate populations of M. intracellulare this pair seemed to be the most effective two-drug combination available. The requirement for successful treatment of drug-resistant mycobacterioses is the selection of an effective drug regimen and the maintenance of combined action of all drugs in the serum for approximately 10 hr daily. An in vitro model is described which enables the bacteriologist to design an effective combination of drugs and to measure its efficiency under simulated in vivo conditions.     

Compatibility of Evolutionary Responses to Constituent Antibiotics Drive Resistance Evolution to Drug Pairs

Antibiotic combinations are considered a relevant strategy to tackle the global antibiotic resistance crisis since they are believed to increase treatment efficacy and reduce resistance evolution (WHO treatment guidelines for drug-resistant tuberculosis: 2016 update.). However, studies of the evolution of bacterial resistance to combination therapy have focused on a limited number of drugs and have provided contradictory results (Lipsitch, Levin BR. 1997; Hegreness et al. 2008; Munck et al. 2014). To address this gap in our understanding, we performed a large-scale laboratory evolution experiment, adapting eight replicate lineages of Escherichia coli to a diverse set of 22 different antibiotics and 33 antibiotic pairs. We found that combination therapy significantly limits the evolution of de novode novo resistance in E. coli, yet different drug combinations vary substantially in their propensity to select for resistance. In contrast to current theories, the phenotypic features of drug pairs are weak predictors of resistance evolution. Instead, the resistance evolution is driven by the relationship between the evolutionary trajectories that lead to resistance to a drug combination and those that lead to resistance to the component drugs. Drug combinations requiring a novel genetic response from target bacteria compared with the individual component drugs significantly reduce resistance evolution. These data support combination therapy as a treatment option to decelerate resistance evolution and provide a novel framework for selecting optimized drug combinations based on bacterial evolutionary responses.     

In vitro activity of SPR719 against Mycobacterium ulcerans,Mycobacterium marinum and Mycobacterium chimaera

Nontuberculosis mycobacterial (NTM) infections are increasing in prevalence across the world. In many cases, treatment options for these infections are limited. However, there has been progress in recent years in the development of new antimycobacterial drugs. Here, we investigate the in vitro activity of SPR719, a novel aminobenzimidazole antibiotic and the active form of the clinical-stage compound, SPR720, against several isolates of Mycobacterium ulcerans, Mycobacterium marinum and Mycobacterium chimaera. We show that SPR719 is active against these NTM species with a MIC range of 0.125–4 μg/ml and that this compares favorably with the commonly utilized antimycobacterial antibiotics, rifampicin and clarithromycin. Our findings suggest that SPR720 should be further evaluated for the treatment of NTM infections.     

Fit for consumption: zebrafish as a model for tuberculosis

Despite efforts to generate new vaccines and antibiotics for tuberculosis, the disease remains a public health problem worldwide. The zebrafish Danio rerio has emerged as a useful model to investigate mycobacterial pathogenesis and treatment. Infection of zebrafish with Mycobacterium marinum, the closest relative of the Mycobacterium tuberculosis complex, recapitulates many aspects of human tuberculosis. The zebrafish model affords optical transparency, abundant genetic tools and in vivo imaging of the progression of infection. Here, we review how the zebrafish–M. marinum system has been deployed to make novel observations about the role of innate immunity, the tuberculous granuloma, and crucial host and bacterial genes. Finally, we assess how these findings relate to human disease and provide a framework for novel strategies to treat tuberculosis.KEY WORDS: Disease models, Genetics, Mycobacterium, Pathogenesis, Tuberculosis, Zebrafish     

Synergistic interactions in two-drug and three-drug combinations (thymol,EDTA and vancomycin) against multi drug resistant bacteria including E. coli

Combinations of two or more drugs, which affect different targets, have frequently been used as a new approach against resistant bacteria. In our work we studied the antimicrobial activity (MIC, MBC) of individual drugs (the phenolic monoterpene thymol, EDTA and vancomycin), of two-drug interactions between thymol and EDTA in comparison with three-drug interactions with vancomycin against sensitive and resistant bacteria. Thymol demonstrated moderate bactericidal activity (MBC between 60 and 4000 μg/ml) while EDTA only exhibited bacteriostatic activity over a range of 60–4000 μg/ml. MICs of vancomycin were between 0.125 and 16 μg/ml against Gram-positive and between 32 and 128 μg/ml against Gram-negative bacteria. Checkerboard dilution and time-kill curve assays were performed to evaluate the mode of interaction of several combinations against Methicillin-resistant Staphylococcus aureus (MRSA NCTC 10442) and Escherichia coli (ATCC 25922). Checkerboard data indicate indifferent interaction against Gram-positive (FICI = 1–1.3) and synergy against Gram-negative bacteria (FICI ≈ 0.4), while time kill analyses suggest synergistic effect in different combinations against both types of bacteria. It is remarkable that the combinations could enhance the sensitivity of E. coli to vancomycin 16-fold to which it is normally insensitive. We have provided proof for the concept, that combinations of known antibiotics with modern phytotherapeutics can expand the spectrum of useful therapeutics.     

Whole Genome Analyses of Marine Fish Pathogenic Isolate,Mycobacterium sp. 012931

Mycobacterium is a genus within the order Actinomycetales that comprises of a large number of well-characterized species, several of which includes pathogens known to cause serious disease in human and animal. Here, we report the whole genome sequence of Mycobacterium sp. strain 012931 isolated from the marine fish, yellowtail (Seriola quinqueradiata). Mycobacterium sp. 012931 is a fish pathogen causing serious damage to aquaculture farms in Japan. DNA dot plot analysis showed that Mycobacterium sp. 012931 was more closely related to Mycobacterium marinum when compared across several Mycobacterium species. However, little conservation of the gene order was observed between Mycobacterium sp. 012931 and M. marinum genome. The annotated 5,464 genes of Mycobacterium sp. 012931 was classified into 26 subsystems. The insertion/deletion gene analysis shows Mycobacterium sp. 012931 had 643 unique genes that were not found in the M. marinum strains. In the virulence, disease, and defense subsystem, both insertion and deletion genes of Mycobacterium sp. 012931 were associated with the PPE gene cluster of Mycobacteria. Of seven plcB genes in Mycobacterium sp. 012931, plcB_2 and plcB_3 showed low identities with those of M. marinum strains. Therefore, Mycobacterium sp. 012931 has differences on genetic and virulence from M. marinum and may induce different interaction mechanisms between host and pathogen.     

Cultural characteristics of the marine ciliated protozoan, Uronema marinum Dujardin

The marine hymenostome ciliate Uronema marinum Dujardin was isolated from sediment from Robin Hood's Bay, Yorkshire. Experiments on the growth of U. marinum were carried out in batch cultures in which the ciliate was maintained on a diet of Vibrio sp. The duration of the lag phase varied directly with the age of the inoculum. Factorially designed experiments were used to investigate the effects of salinity and temperature on the rate of logarithmic growth of the ciliate. A response surface analysis of the data indicated that there was interaction between salinity and temperature. Regression equations predicted a minimum doubling time of 2.5 h at 32‰ and 25 °C. The cell volume of U. marinum varied during growth; it reached a maximum at the end of the lag phase and decreased during the logarithmic phase to become minimal in the stationary phase.     

Comparative Genome Analysis of Fish and Human Isolates of Mycobacterium marinum

Mycobacterium marinum is a major causative agent of mycobacteriosis in fish that has a broad range of hosts, including in human isolates. So far, genomic analyses have focused on the human isolate. Here, we compared the draft genome sequences of two strains of M. marinum isolated from fish (MB2 and Europe) with the M. marinum M isolated from humans. M. marinum MB2 and Europe have single, circular chromosomes of 6,134,389 and 6,029,340 bp, and average G + C contents of 65.7 and 65.5 %, respectively. A total of 5,464 coding DNA sequences were annotated in both M. marinum MB2 and Europe genome. Dot plot analyses showed that M. marinum MB2 and Europe were closer to M. marinum M when compared to three other Mycobacterium species. The insertion/deletion gene analysis showed that M. marinum MB2 and Europe contained 342 and 487 genes that were not found in M. marinum M, and lacked 625 and 776 genes found in M. marinum M, respectively. Most of the inserted and deleted genes were classified in the fatty acid, lipid, and isoprenoid subsystem and the virulence, disease, and defense subsystem. Therefore, these results provide insights into the genomic diversity associated with variable hosts and pathogens.     

The role of the N-terminal amphipathic helix in bacterial YidC: Insights from functional studies,the crystal structure and molecular dynamics simulations

The evolutionary conserved YidC is a unique dual-function membrane protein that adopts insertase and chaperone conformations. The N-terminal helix of Escherichia coli YidC functions as an uncleaved signal sequence and is important for membrane insertion and interaction with the Sec translocon. Here, we report the first crystal structure of Thermotoga maritima YidC (TmYidC) including the N-terminal amphipathic helix (N-AH) (PDB ID: 6Y86). Molecular dynamics simulations show that N-AH lies on the periplasmic side of the membrane bilayer forming an angle of about 15° with the membrane surface. Our functional studies suggest a role of N-AH for the species-specific interaction with the Sec translocon. The reconstitution data and the superimposition of TmYidC with known YidC structures suggest an active insertase conformation for YidC. Molecular dynamics (MD) simulations of TmYidC provide evidence that N-AH acts as a membrane recognition helix for the YidC insertase and highlight the flexibility of the C1 region underlining its ability to switch between insertase and chaperone conformations. A structure-based model is proposed to rationalize how YidC performs the insertase and chaperone functions by re-positioning of N-AH and the other structural elements.     

Severe inhibition of lipooligosaccharide synthesis induces TLR2-dependent elimination of <Emphasis Type="Italic">Mycobacterium marinum</Emphasis> from THP1-derived macrophages

Background

Although mycobacterial glycolipids are among the first-line molecules involved in host–pathogen interactions, their contribution in virulence remains incomplete. Mycobacterium marinum is a waterborne pathogen of fish and other ectotherms, closely related to Mycobacterium tuberculosis. Since it causes tuberculosis-like systemic infection it is widely used as a model organism for studying the pathogenesis of tuberculosis. It is also an occasional opportunistic human pathogen. The M. marinum surface-exposed lipooligosaccharides (LOS) are immunogenic molecules that participate in the early interactions with macrophages and modulate the host immune system. Four major LOS species, designated LOS-I to LOS-IV, have been identified and characterized in M. marinum. Herein, we investigated the interactions between a panel of defined M. marinum LOS mutants that exhibited various degrees of truncation in the LOS structure, and human-derived THP-1 macrophages to address the potential of LOSs to act as pro- or avirulence factors.

Results

A moderately truncated LOS structure did not interfere with M. marinum invasion. However, a deeper shortening of the LOS structure was associated with increased entry of M. marinum into host cells and increased elimination of the bacilli by the macrophages. These effects were dependent on Toll-like receptor 2.

Conclusion

We provide the first evidence that LOSs inhibit the interaction between mycobacterial cell wall ligands and appropriate macrophage pattern recognition receptors, affecting uptake and elimination of the bacteria by host phagocytes.

     

Oxidative Stress Response and Characterization of the oxyR-ahpC and furA-katG Loci in Mycobacterium marinum

Oxidative stress response in pathogenic mycobacteria is believed to be of significance for host-pathogen interactions at various stages of infection. It also plays a role in determining the intrinsic susceptibility to isoniazid in mycobacterial species. In this work, we characterized the oxyR-ahpC and furA-katG loci in the nontuberculous pathogen Mycobacterium marinum. In contrast to Mycobacterium smegmatis and like Mycobacterium tuberculosis and Mycobacterium leprae, M. marinum was shown to possess a closely linked and divergently oriented equivalents of the regulator of peroxide stress response oxyR and its subordinate gene ahpC, encoding a homolog of alkyl hydroperoxide reductase. Purified mycobacterial OxyR was found to bind to the oxyR-ahpC promoter region from M. marinum and additional mycobacterial species. Mobility shift DNA binding analyses using OxyR binding sites from several mycobacteria and a panel of in vitro-generated mutants validated the proposed consensus mycobacterial recognition sequence. M. marinum AhpC levels detected by immunoblotting, were increased upon treatment with H₂O₂, in keeping with the presence of a functional OxyR and its binding site within the promoter region of ahpC. In contrast, OxyR did not bind to the sequences upstream of the katG structural gene, and katG expression did not follow the pattern seen with ahpC. Instead, a new open reading frame encoding a homolog of the ferric uptake regulator Fur was identified immediately upstream of katG in M. marinum. The furA-katG linkage and arrangement are ubiquitous in mycobacteria, suggesting the presence of additional regulators of oxidative stress response and potentially explaining the observed differences in ahpC and katG expression. Collectively, these findings broaden our understanding of oxidative stress response in mycobacteria. They also suggest that M. marinum will be useful as a model system for studying the role of oxidative stress response in mycobacterial physiology, intracellular survival, and other host-pathogen interactions associated with mycobacterial diseases.     

Bacterial Classification of Fish-Pathogenic Mycobacterium Species by Multigene Phylogenetic Analyses and MALDI Biotyper Identification System

Mycobacterium marinum is difficult to distinguish from other species of Mycobacterium isolated from fish using biochemical methods. Here, we used genetic and proteomic analyses to distinguish three Mycobacterium strains: M. marinum strains MB2 and Europe were isolated from tropical and marine fish in Thailand and Europe, and Mycobacterium sp. 012931 strain was isolated from yellowtail in Japan. In phylogenetic trees based on gyrB, rpoB, and Ag85B genes, Mycobacterium sp. 012931 clustered with M. marinum strains MB2 and Europe, but in trees based on 16S rRNA, hsp65, and Ag85A genes Mycobacterium sp. 012931 did not cluster with the other strains. In proteomic analyses using a Bruker matrix-assisted laser desorption ionization Biotyper, the mass profile of Mycobacterium sp. 012931 differed from the mass profiles of the other two fish M. marinum strains. Therefore, Mycobacterium sp. 012931 is similar to M. marinum but is not the same, suggesting that it could be a subspecies of M. marinum.     

Using a Sequential Regimen to Eliminate Bacteria at Sublethal Antibiotic Dosages

We need to find ways of enhancing the potency of existing antibiotics, and, with this in mind, we begin with an unusual question: how low can antibiotic dosages be and yet bacterial clearance still be observed? Seeking to optimise the simultaneous use of two antibiotics, we use the minimal dose at which clearance is observed in an in vitro experimental model of antibiotic treatment as a criterion to distinguish the best and worst treatments of a bacterium, Escherichia coli. Our aim is to compare a combination treatment consisting of two synergistic antibiotics to so-called sequential treatments in which the choice of antibiotic to administer can change with each round of treatment. Using mathematical predictions validated by the E. coli treatment model, we show that clearance of the bacterium can be achieved using sequential treatments at antibiotic dosages so low that the equivalent two-drug combination treatments are ineffective. Seeking to treat the bacterium in testing circumstances, we purposefully study an E. coli strain that has a multidrug pump encoded in its chromosome that effluxes both antibiotics. Genomic amplifications that increase the number of pumps expressed per cell can cause the failure of high-dose combination treatments, yet, as we show, sequentially treated populations can still collapse. However, dual resistance due to the pump means that the antibiotics must be carefully deployed and not all sublethal sequential treatments succeed. A screen of 136 96-h-long sequential treatments determined five of these that could clear the bacterium at sublethal dosages in all replicate populations, even though none had done so by 24 h. These successes can be attributed to a collateral sensitivity whereby cross-resistance due to the duplicated pump proves insufficient to stop a reduction in E. coli growth rate following drug exchanges, a reduction that proves large enough for appropriately chosen drug switches to clear the bacterium.     

Mycobacterium Ulcerans Treatment – Can Antibiotic Duration Be Reduced in Selected Patients?

Introduction

Mycobacterium ulcerans (M. ulcerans) is a necrotizing skin infection endemic to the Bellarine Peninsula, Australia. Current treatment recommendations include 8 weeks of combination antibiotics, with adjuvant surgery if necessary. However, antibiotic toxicity often results in early treatment cessation and local experience suggests that shorter antibiotic courses may be effective with concurrent surgery. We report the outcomes of patients in the Barwon Health M. ulcerans cohort who received shorter courses of antibiotic therapy than 8 weeks.

Methodology / Principal findings

A retrospective analysis was performed of all M. ulcerans infections treated at Barwon Health from March 1, 1998 to July 31, 2013. Sixty-two patients, with a median age of 65 years, received < 56 days of antibiotics and 51 (82%) of these patients underwent concurrent surgical excision. Most received a two-drug regimen of rifampicin combined with either ciprofloxacin or clarithromycin for a median 29 days (IQR 21–41days). Cessation rates were 55% for adverse events and 36% based on clinician decision. The overall success rate was 95% (98% with concurrent surgery; 82% with antibiotics alone) with a 50% success rate for those who received < 14 days of antibiotics increasing to 94% if they received 14–27 days and 100% for 28–55 days (p<0.01). A 100% success rate was seen for concurrent surgery and 14–27 days of antibiotics versus 67% for concurrent surgery and < 14 days of antibiotics (p = 0.12). No previously identified risk factors for treatment failure with surgery alone were associated with reduced treatment success rates with < 56 days of antibiotics.

Conclusion

In selected patients, antibiotic treatment durations for M. ulcerans shorter than the current WHO recommended 8 weeks duration may be associated with successful outcomes.     

Transient binding patches: a plausible concept for drug binding

Dose–response curves for inhibitors (drugs) generally are analyzed by means of four-parameter fits, yielding IC₅₀, background, amplitude, and Hill coefficient. Hill coefficients ≠1 contradict 1:1 competition. If binding of substrates to proteins is a stepwise process where initial binding to initial locations (patches) leads to strong binding on defined sites, then drugs (non-endogenous inhibitors) may bind to those presumably larger patches and need not follow a 1:1 stoichiometry for specific inhibition. This concept was translated into three computable models and successfully fitted to 1,282 phosphatase dose–response curves. The models only required four parameters, namely, the equilibrium dissociation constant K _D(1) of the first inhibitor binding step, background, amplitude, and a compound interaction factor to quantify the interaction of inhibitors on those patches. Binding of one established inhibitor to the vaccinia virus VH1-related (VHR) phosphatase was directly measured with microcalorimetry, confirming multiple inhibitor binding with equilibrium constants obtained from corresponding inhibition curves.     

Mathematical modeling of pulmonary tuberculosis therapy: Insights from a prototype model with rifampin

There is a critical need for improved and shorter tuberculosis (TB) treatment. Current in vitro models of TB, while valuable, are poor predictors of the antibacterial effect of drugs in vivo. Mathematical models may be useful to overcome the limitations of traditional approaches in TB research. The objective of this study was to set up a prototype mathematical model of TB treatment by rifampin, based on pharmacokinetic, pharmacodynamic and disease submodels.The full mathematical model can simulate the time-course of tuberculous disease from the first day of infection to the last day of therapy. Therapeutic simulations were performed with the full model to study the antibacterial effect of various dosage regimens of rifampin in lungs.The model reproduced some qualitative and quantitative properties of the bactericidal activity of rifampin observed in clinical data. The kill curves simulated with the model showed a typical biphasic decline in the number of extracellular bacteria consistent with observations in TB patients. Simulations performed with more simple pharmacokinetic/pharmacodynamic models indicated a possible role of a protected intracellular bacterial compartment in such a biphasic decline.This modeling effort strongly suggests that current dosage regimens of RIF may be further optimized. In addition, it suggests a new hypothesis for bacterial persistence during TB treatment.     

A Novel ESX-1 Locus Reveals that Surface-Associated ESX-1 Substrates Mediate Virulence in Mycobacterium marinum

EsxA (ESAT-6) and EsxB (CFP-10) are virulence factors exported by the ESX-1 system in mycobacterial pathogens. In Mycobacterium marinum, an established model for ESX-1 secretion in Mycobacterium tuberculosis, genes required for ESX-1 export reside at the extended region of difference 1 (RD1) locus. In this study, a novel locus required for ESX-1 export in M. marinum was identified outside the RD1 locus. An M. marinum strain bearing a transposon-insertion between the MMAR_1663 and MMAR_1664 genes exhibited smooth-colony morphology, was deficient for ESX-1 export, was nonhemolytic, and was attenuated for virulence. Genetic complementation revealed a restoration of colony morphology and a partial restoration of virulence in cell culture models. Yet hemolysis and the export of ESX-1 substrates into the bacteriological medium in vitro as measured by both immunoblotting and quantitative proteomics were not restored. We show that genetic complementation of the transposon insertion strain partially restored the translocation of EsxA and EsxB to the mycobacterial cell surface. Our findings indicate that the export of EsxA and EsxB to the cell surface, rather than secretion into the bacteriological medium, correlates with virulence in M. marinum. Together, these findings not only expand the known genetic loci required for ESX-1 secretion in M. marinum but also provide an explanation for the observed disparity between in vitro ESX-1 export and virulence.     

Fish Tank Granuloma Caused by Mycobacterium marinum

Introduction

Mycobacterium marinum causes skin and soft tissue, bone and joint, and rare disseminated infections. In this study, we aimed to investigate the relationship between treatment outcome and antimicrobial susceptibility patterns. A total of 27 patients with M. marinum infections were enrolled.

Methods

Data on clinical characteristics and therapeutic methods were collected and analyzed. We also determined the minimum inhibitory concentrations of 7 antibiotics against 30 isolates from these patients.

Results

Twenty-seven patients received antimycobacterial agents with or without surgical debridement. Eighteen patients were cured, 8 failed to respond to treatment, and one was lost to follow-up. The duration of clarithromycin (147 vs. 28; p = 0.0297), and rifampicin (201 vs. 91; p = 0.0266) treatment in the cured patients was longer than that in the others. Surgical debridement was performed in 10 out of the 18 cured patients, and in 1 of another group (p = 0.0417). All the 30 isolates were susceptible to clarithromycin, amikacin, and linezolid; 29 (96.7%) were susceptible to ethambutol; 28 (93.3%) were susceptible to sulfamethoxazole; and 26 (86.7%) were susceptible to rifampicin. However, only 1 (3.3%) isolate was susceptible to doxycycline.

Discussion

Early diagnosis of the infection and appropriate antimicrobial therapy with surgical debridement are the mainstays of successful treatment. Clarithromycin and rifampin are supposed to be more effective agents.