Antibiotic-tolerant Bacteria Research Articles

Biguanide-Vancomycin Conjugates are Effective Broad-Spectrum Antibiotics against Actively Growing and Biofilm-Associated Gram-Positive and Gram-Negative ESKAPE Pathogens and Mycobacteria.

Strategies to increase the efficacy and/or expand the spectrum of activity of existing antibiotics provide a potentially fast path to clinically address the growing crisis of antibiotic-resistant infections. Here, we report the synthesis, antibacterial efficacy, and mechanistic activity of an unprecedented class of biguanide-antibiotic conjugates. Our lead biguanide-vancomycin conjugate, V-C6-Bg-PhCl (5e), induces highly effective cell killing with up to a 2 orders-of-magnitude improvement over its parent compound, vancomycin (V), against vancomycin-resistant enterococcus. V-C6-Bg-PhCl (5e) also exhibits improved activity against mycobacteria and each of the ESKAPE pathogens, including the Gram-negative organisms. Furthermore, we uncover broad-spectrum killing activity against biofilm-associated Gram-positive and Gram-negative bacteria as well as mycobacteria not observed for clinically used antibiotics such as oritavancin. Mode-of-action studies reveal that vancomycin-like cell wall synthesis inhibition with improved efficacy attributed to enhanced engagement at vancomycin binding sites through biguanide association with relevant cell-surface anions for Gram-positive and Gram-negative bacteria. Due to its potency, remarkably broad activity, and lack of acute mammalian cell toxicity, V-C6-Bg-PhCl (5e) is a promising candidate for treating antibiotic-resistant infections and notoriously difficult-to-treat slowly growing and antibiotic-tolerant bacteria associated with chronic and often incurable infections. More generally, this study offers a new strategy (biguanidinylation) to enhance antibiotic activity and facilitate clinical entry.

A high-throughput assay identifies molecules with antimicrobial activity against persister cells.

Introduction. Persister cells are transiently non-growing antibiotic-tolerant bacteria that cause infection relapse, and there is no effective antibiotic therapy to tackle these infections.Gap statement. High-throughput assays in drug discovery are biased towards detecting drugs that inhibit bacterial growth rather than killing non-growing bacteria. A new and simple assay to discover such drugs is needed.Aim. This study aims to develop a simple and high-throughput assay to identify compounds with antimicrobial activity against persister cells and use it to identify molecular motifs with such activity.Methodology. We quantified Staphylococcus aureus persister cells by enumeration of colony forming units after 24 h ciprofloxacin treatment. We first quantified how the cell concentration, antibiotic concentration, growth phase and presence/absence of nutrients during antibiotic exposure affected the fraction of persister cells in a population. After optimizing these parameters, we screened the antimicrobial activity of compound fragments to identify molecular structures that have activity against persister cells.Results. Exponential- and stationary-phase cultures transferred to nutrient-rich media displayed a bi-phasic time-kill curve and contained 0.001-0.07% persister cells. A short rifampicin treatment resulted in 100% persister cells for 7 h, after which cells resumed activity and became susceptible. Stationary-phase cultures displayed a low but constant death rate but ultimately resulted in similarly low survival rates as the exponential-phase cultures after 24 h ciprofloxacin treatment. The persister phenotype was only maintained in most of the population for 24 h if cells were transferred to a carbon-free minimal medium before exposure to ciprofloxacin. Keeping cells starved enabled the generation of high concentrations of S. aureus cells that tolerate 50× MIC ciprofloxacin, and we used this protocol for rapid screening for biocidal antibiotics. We identified seven compounds from four structural clusters with activity against antibiotic-tolerant S. aureus. Two compounds were moderately cytotoxic, and the rest were highly cytotoxic.Conclusion. Transferring a stationary-phase culture to a carbon-free minimal medium for antimicrobial testing is a simple strategy for high-throughput screening for new antibiotics that kill persister cells. We identified molecule fragments with such activity, but further screening is needed to identify motifs with lower general cytotoxicity.

Anti-Persisters Activity of Lacticaseibacillus rhamnosus Culture Filtrates against Pseudomonas aeruginosa in Artificial Sputum Medium.

Persisters are antibiotic-tolerant bacteria, playing a role in the recalcitrance and relapse of many bacterial infections, including P. aeruginosa pulmonary infections in Cystic Fibrosis (CF) patients. Among novel antimicrobial strategies, the use of probiotics and their products is emerging as a particularly promising approach. The aim of this study was to evaluate the anti-persisters activity of culture filtrate supernatants of Lacticaseibacillus rhamnosus (LRM-CFS) against P. aeruginosa in artificial sputum medium (ASM), which resembles the CF lung environment. Planktonic persisters of two clinical strains of P. aeruginosa (PaCF1 and PaCF4) were obtained following two different procedures: (i) exposing stationary-phase cultures to cyanide m-chlorophenylhydrazone (CCCP) in LB medium; (ii) incubating stationary-phase cultures with high doses of tobramycin (128-fold MIC) in ASM. In addition, persisters from biofilm were obtained by exposing 48 h old biofilm of P. aeruginosa to 128 x MIC of ciprofloxacin. LRM-CFS at dilutions of 1:6 and 1:4 resulted in being bactericidal in ASM against both PaCF1 and PaCF4 persisters obtained after CCCP or tobramycin treatment. Moreover, LRM-CFS at dilution 1:4 caused a reduction of antibiotic-tolerant bacteria in the biofilm of both P. aeruginosa strains. Overall, LRM-CFS represents a promising adjuvant therapeutic strategy against P. aeruginosa recalcitrant infections in CF patients.

Heavy Metal Co-Resistance with Antibiotics amongst Plant Growth Promoting Bacteria Isolates from Rhizosphere of Nypa Fruticans

Heavy metal co-resistance with antibiotics has the potential to amplify antimicrobial resistance genes in the environment, and this could have an impact on clinical settings. This study aimed to investigate the presence of heavy metal co-resistance with antibiotics among plant growth promoting bacterial isolates from the rhizosphere of Nypa fruticans. The heavy metals content of the water habouring the Nypa fruticans including copper(0.07mg/L), chromium (2.3 mg/L), lead (4.22 mg/L), iron (12.4 mg/L), cadmium (0.45 mg/L), nickel (3.12 mg/L), arsenic (0.47 mg/L) and zinc (5.37 mg/L) were above acceptable limits of WHO. A total of 10 bacteria including Bacillus subtilis, Bacillus cereus, Pseudomonas fluorescens, Klebsiella oxytoca, Serratia marcescens, Achromobacter denitrificans, Bulkholderia vietnamiensis and Azospirrilum brasilense, Azobacter vinelandii and Enterobacter aerogenes were isolated and identified to specie level using 16s RNA gene sequences, which showed above 95 % similarity with reference sequences from GenBank. The isolates showed high resistance to lead, chromium and zinc. Most of the isolates were resistant to streptomycin, ciprofloxacin and augmentin. Also, most of the isolates demonstrated positive growth promoting activities of indole acetic acid production, nitrogen fixation, siderophore production and phosphate solubilization. Pot trials on the effect of heavy metal and antibiotic tolerant bacteria on growth of beans (Phaseolus vulgaris l.) in heavy metal polluted soil revealed that beans treated with consortiumof all the isolates recorded high shoot and root length. Hence these isolates can be used for bioremediation of metal polluted site and as well act as plant growth promoting bacterial in agricultural system.

Identification of a Small Molecule Compound Active against Antibiotic-Tolerant Staphylococcus aureus by Boosting ATP Synthesis

Antibiotic tolerance poses a threat to current antimicrobial armamentarium. Bacteria at a tolerant state survive in the presence of antibiotic treatment and account for persistence, relapse and recalcitrance of infections. Antibiotic treatment failure may occur due to antibiotic tolerance. Persistent infections are difficult to treat and are often associated with poor prognosis, imposing an enormous burden on the healthcare system. Effective strategies targeting antibiotic-tolerant bacteria are therefore highly warranted. In this study, small molecule compound SA-558 was identified to be effective against Staphylococcus aureus that are tolerant to being killed by conventional antibiotics. SA-558 mediated electroneutral transport across the membrane and led to increased ATP and ROS generation, resulting in a reduction of the population of antibiotic-tolerant bacteria. In a murine chronic infection model, of which vancomycin treatment failed, we demonstrated that SA-558 alone and in combination with vancomycin caused significant reduction of MRSA abundance. Our results indicate that SA-558 monotherapy or combinatorial therapy with vancomycin is an option for managing persistent S. aureus bacteremia infection and corroborate that bacterial metabolism is an important target for counteracting antibiotic tolerance.

Stimulating Transcription in Antibiotic-Tolerant Escherichia coli Sensitizes It to Fluoroquinolone and Nonfluoroquinolone Topoisomerase Inhibitors.

Antibiotic tolerant bacteria and persistent cells that remain alive after a course of antibiotic treatment can foster the chronicity of infections and the development of antibiotic resistance. Elucidating how bacteria overcome antibiotic action and devising strategies to bolster a new drug's activity can allow us to preserve our antibiotic arsenal. Here, we investigate strategies to potentiate the activities of topoisomerase inhibitors against nongrowing Escherichia coli that are often recalcitrant to existing antibiotics. We focus on sensitizing bacteria to the fluoroquinolone (FQ) levofloxacin (Levo) and to the spiropyrimidinetrione zoliflodacin (Zoli)-the first antibiotic in its class of compounds in clinical development. We found that metabolic stimulation either alone or in combination with inhibiting the AcrAB-TolC efflux pump sensitized stationary-phase E. coli to Levo and Zoli. We demonstrate that the added metabolites increased proton motive force generation and ATP production in stationary-phase cultures without restarting growth. Instead, the stimulated bacteria increased transcription and translation, which rendered the populations more susceptible to topoisomerase inhibitors. Our findings illuminate potential vulnerabilities of antibiotic-tolerant bacteria that can be leveraged to sensitize them to new and existing classes of topoisomerase inhibitors. These approaches enable us to stay one step ahead of adaptive bacteria and safeguard the efficacy of our existing antibiotics.

Single-cell patterning and characterisation of antibiotic persistent bacteria using bio-sCAPA.

In microbiology, accessing single-cell information within large populations is pivotal. Here we introduce bio-sCAPA, a technique for patterning bacterial cells in defined geometric arrangements and monitoring their growth in various nutrient environments. We demonstrate bio-sCAPA with a study of subpopulations of antibiotic-tolerant bacteria, known as persister cells, which can survive exposure to high doses of antibiotics despite lacking any genetic resistance to the drug. Persister cells are associated with chronic and relapsing infections, yet are difficult to study due in part to a lack of scalable, single-cell characterisation methods. As >105 cells can be patterned on each template, and multiple templates can be patterned in parallel, bio-sCAPA allows for very rare population phenotypes to be monitored with single-cell precision across various environmental conditions. Using bio-sCAPA, we analysed the phenotypic characteristics of single Staphylococcus aureus cells tolerant to flucloxacillin and rifampicin killing. We find that antibiotic-tolerant S. aureus cells do not display significant heterogeneity in growth rate and are instead characterised by prolonged lag-time phenotypes alone.

Multi-roles of nanoscale bismuth metal-organic frameworks: Infectious photoacoustic probe and inhibitor of antibiotics tolerant bacteria via targeting endogenous H2S

Increasing evidence has emphasized the pivotal role of antibiotic tolerance in the evolution of antibiotic resistance, refractory infection and infection recurrence. Moreover, satisfactory treatment of infection relies highly on early, precise detection and subsequent timely therapeutic interventions. In this study, the phenotype of increased production of H2S in antibiotic-tolerant Staphylococcus aureus (S. aureus) was identified and proven to be essential to the maintenance of antibiotic tolerance. Hence, a photoacoustic probe for detecting infection as well as an antibiotic enhancer targeting bacterial endogenous H2S to eradicate antibiotic-tolerant bacteria was successfully developed based on a bismuth metal-organic framework (Bi-MOF). Bi-MOF achieved early in situ and precise photoacoustic imaging of infection by generating Bi2S3 inside bacteria. Moreover, Bi-MOF enhanced the eradication efficacy of methicillin against tolerant S. aureus by downregulating the intracellular H2S levels. Overall, Bi-MOF offers a promising diagnostic tool for early, precise infection diagnosis and a therapeutic strategy for tolerant bacteria-related refractory infection.

Transient Antibiotic Tolerance Triggered by Nutrient Shifts From Gluconeogenic Carbon Sources to Fatty Acid.

Nutrient shifts from glycolytic-to-gluconeogenic carbon sources can create large sub-populations of extremely antibiotic tolerant bacteria, called persisters. Positive feedback in Escherichia coli central metabolism was believed to play a key role in the formation of persister cells. To examine whether positive feedback in nutrient transport can also support high persistence to β-lactams, we performed nutrient shifts for E. coli from gluconeogenic carbon sources to fatty acid (FA). We observed tri-phasic antibiotic killing kinetics characterized by a transient period of high antibiotic tolerance, followed by rapid killing then a slower persister-killing phase. The duration of transient tolerance (3–44 h) varies with pre-shift carbon source and correlates strongly with the time needed to accumulate the FA degradation enzyme FadD after the shift. Additionally, FadD accumulation time and thus transient tolerance time can be reduced by induction of the glyoxylate bypass prior to switching, highlighting that two interacting feedback loops simultaneously control the length of transient tolerance. Our results demonstrate that nutrient switches along with positive feedback are not sufficient to trigger persistence in a majority of the population but instead triggers only a temporary tolerance. Additionally, our results demonstrate that the pre-shift metabolic state determines the duration of transient tolerance and that supplying glyoxylate can facilitate antibiotic killing of bacteria.

Modular Synthetic Routes to Fluorine-Containing Halogenated Phenazine and Acridine Agents That Induce Rapid Iron Starvation in Methicillin-Resistant Staphylococcus aureus Biofilms.

During infection, bacteria use an arsenal of resistance mechanisms to negate antibiotic therapies. In addition, pathogenic bacteria form surface-attached biofilms bearing enriched populations of metabolically dormant persister cells. Bacteria develop resistance in response to antibiotic insults; however, nonreplicating biofilms are innately tolerant to all classes of antibiotics. As such, molecules that can eradicate antibiotic-resistant and antibiotic-tolerant bacteria are of importance. Here, we report modular synthetic routes to fluorine-containing halogenated phenazine (HP) and halogenated acridine (HA) agents with potent antibacterial and biofilm-killing activities. Nine fluorinated phenazines were rapidly accessed through a synthetic strategy involving (1) oxidation of fluorinated anilines to azobenzene intermediates, (2) SNAr with 2-methoxyaniline, and (3) cyclization to phenazines upon treatment with trifluoroacetic acid. Five structurally related acridine heterocycles were synthesized using SNAr and Buchwald-Hartwig approaches. From this focused collection, phenazines 5g, 5h, 5i, and acridine 9c demonstrated potent antibacterial activities against Gram-positive pathogens (MIC = 0.04-0.78 μM). Additionally, 5g and 9c eradicated Staphylococcus aureus, Staphylococcus epidermidis and Enterococcus faecalis biofilms with excellent potency (5g, MBEC = 4.69-6.25 μM; 9c, MBEC = 4.69-50 μM). Using real-time quantitative polymerase chain reaction (RT-qPCR), 5g, 5h, 5i, and 9c rapidly induce the transcription of iron uptake biomarkers isdB and sbnC in methicillin-resistant S. aureus (MRSA) biofilms, and we conclude that these agents operate through iron starvation. Overall, fluorinated phenazine and acridine agents could lead to ground-breaking advances in the treatment of challenging bacterial infections.

Econazole as adjuvant to conventional antibiotics is able to eradicate starvation-induced tolerant bacteria by causing proton motive force dissipation.

Bacterial antibiotic tolerance is responsible for the recalcitrance of chronic infections. This study aims to investigate a potential drug that can effectively kill antibiotic-tolerant bacteria and evaluate the ability of this drug on the eradication of tolerant cells both in vitro and in vivo. The in vitro effect of econazole on eradicating starvation-induced tolerant bacterial populations was studied by testing the amount of survival bacteria in the presence of econazole combining conventional antibiotics. Proton motive force (PMF) was determined after econazole treatment by DiOC2(3). Finally, mouse infection models were used to detect the ability of econazole on killing the tolerant populations in vivo. Econazole eradicated starvation-induced tolerant cells of various bacterial species within 24 or 96 h when used in combination with conventional antibiotics. Moreover, mouse survival rate drastically increased along with the decrease of in vivo bacterial count after treatment of infected mice with the econazole and ceftazidime combination for 72 h. PMF was found to have dissipated almost completely in econazole-treated cells. Econazole could act in combination with conventional antibiotics to effectively eradicate bacterial tolerant cells. The combined use of econazole and ceftazidime was shown to be effective for eradicating tolerant cells in a mouse infection model. The ability of econazole to eradicate tolerant cells was due to its ability to cause dissipation of bacterial transmembrane PMF. Econazole-mediated PMF disruption is a feasible strategy for the treatment of chronic and recurrent bacterial infections.

Genetic Determinants of Intrinsic Antibiotic Tolerance in Mycobacterium avium.

ABSTRACTThe Mycobacterium avium complex (MAC) is one of the most prevalent causes of nontuberculous mycobacteria pulmonary infection in the United States, and yet it remains understudied. Current MAC treatment requires more than a year of intermittent to daily combination antibiotic therapy, depending on disease severity. In order to shorten and simplify curative regimens, it is important to identify the innate bacterial factors contributing to reduced antibiotic susceptibility, namely, antibiotic tolerance genes. In this study, we performed a genome-wide transposon screen to elucidate M. avium genes that play a role in the bacterium’s tolerance to first- and second-line antibiotics. We identified a total of 193 unique M. avium mutants with significantly altered susceptibility to at least one of the four clinically used antibiotics we tested, including two mutants (in DFS55_00905 and DFS55_12730) with panhypersusceptibility. The products of the antibiotic tolerance genes we have identified may represent novel targets for future drug development studies aimed at shortening the duration of therapy for MAC infections.IMPORTANCE The prolonged treatment required to eradicate Mycobacterium avium complex (MAC) infection is likely due to the presence of subpopulations of antibiotic-tolerant bacteria with reduced susceptibility to currently available drugs. However, little is known about the genes and pathways responsible for antibiotic tolerance in MAC. In this study, we performed a forward genetic screen to identify M. avium antibiotic tolerance genes, whose products may represent attractive targets for the development of novel adjunctive drugs capable of shortening the curative treatment for MAC infections.

RNA antitoxin SprF1 binds ribosomes to attenuate translation and promote persister cell formation in Staphylococcus aureus.

Persister cells are a subpopulation of transiently antibiotic-tolerant bacteria associated with chronic infection and antibiotic treatment failure. Toxin-antitoxin systems have been linked to persister cell formation but the molecular mechanisms leading to bacterial persistence are mostly unknown. Here, we show that SprF1, a type I antitoxin, associates with translating ribosomes from the major human pathogen Staphylococcus aureus to reduce the pathogen's overall protein synthesis during growth. Under hyperosmotic stress, SprF1 levels increase due to enhanced stability, accumulate on polysomes and attenuate protein synthesis. Using an internal 6-nucleotide sequence on its 5'-end, SprF1 binds ribosomes and interferes with initiator transfer RNA binding, thus reducing translation initiation. An excess of messenger RNA displaces the ribosome-bound antitoxin, freeing the ribosomes for new translation cycles; however, this RNA antitoxin can also displace ribosome-bound mRNA. This translation attenuation mechanism, mediated by an RNA antitoxin, promotes antibiotic persister cell formation. The untranslated SprF1 is a dual-function RNA antitoxin that represses toxin expression by its 3'-end and fine-tunes overall bacterial translation via its 5'-end. These findings demonstrate a general function for a bacterial RNA antitoxin beyond protection from toxicity. They also highlight an RNA-guided molecular process that influences antibiotic persister cell formation.

The Crohn's disease-associated Escherichia coli strain LF82 relies on SOS and stringent responses to survive, multiply and tolerate antibiotics within macrophages.

Adherent Invasive Escherichia coli (AIEC) strains recovered from Crohn's disease lesions survive and multiply within macrophages. A reference strain for this pathovar, AIEC LF82, forms microcolonies within phagolysosomes, an environment that prevents commensal E. coli multiplication. Little is known about the LF82 intracellular growth status, and signals leading to macrophage intra-vacuolar multiplication. We used single-cell analysis, genetic dissection and mathematical models to monitor the growth status and cell cycle regulation of intracellular LF82. We found that within macrophages, bacteria may replicate or undergo non-growing phenotypic switches. This switch results from stringent response firing immediately after uptake by macrophages or at later stages, following genotoxic damage and SOS induction during intracellular replication. Importantly, non-growers resist treatment with various antibiotics. Thus, intracellular challenges induce AIEC LF82 phenotypic heterogeneity and non-growing bacteria that could provide a reservoir for antibiotic-tolerant bacteria responsible for relapsing infections.

Bacterial Persisters and Infection: Past, Present, and Progressing.

Persisters are nongrowing, transiently antibiotic-tolerant bacteria within a clonal population of otherwise susceptible cells. Their formation is triggered by environmental cues and involves the main bacterial stress response pathways that allow persisters to survive many harsh conditions, including antibiotic exposure. During infection, bacterial pathogens are exposed to a vast array of stresses in the host and form nongrowing persisters that survive both antibiotics and host immune responses, thereby most likely contributing to the relapse of many infections. While antibiotic persisters have been extensively studied over the last decade, the bulk of the work has focused on how these bacteria survive exposure to drugs in vitro. The ability of persisters to survive their interaction with a host is important yet underinvestigated. In order to tackle the problem of persistence of infections that contribute to the worldwide antibiotic resistance crisis, efforts should be made by scientific communities to understand and merge these two fields of research: antibiotic persisters and host-pathogen interactions. Here we give an overview of the history of the field of antibiotic persistence, report evidence for the importance of persisters in infection, and highlight studies that bridge the two areas.

Optimizing Cell-Free Protein Synthesis for Increased Yield and Activity of Colicins.

Colicins are antimicrobial proteins produced by Escherichia coli that hold great promise as viable complements or alternatives to antibiotics. Cell-free protein synthesis (CFPS) is a useful production platform for toxic proteins because it eliminates the need to maintain cell viability, a common problem in cell-based production. Previously, we demonstrated that colicins produced by CFPS based on crude Escherichia coli lysates are effective in eradicating antibiotic-tolerant bacteria known as persisters. However, we also found that some colicins have poor solubility or low cell-killing activity. In this study, we improved the solubility of colicin M from 16% to nearly 100% by producing it in chaperone-enriched E. coli extracts, resulting in enhanced cell-killing activity. We also improved the cytotoxicity of colicin E3 by adding or co-expressing the E3 immunity protein during the CFPS reaction, suggesting that the E3 immunity protein enhances colicin E3 activity in addition to protecting the host strain. Finally, we confirmed our previous finding that active colicins can be rapidly synthesized by observing colicin E1 production over time in CFPS. Within three hours of CFPS incubation, colicin E1 reached its maximum production yield and maintained high cytotoxicity during longer incubations up to 20 h. Taken together, our findings indicate that colicin production can be easily optimized for improved solubility and activity using the CFPS platform.

A multiplexable assay for screening antibiotic lethality against drug-tolerant bacteria.

Antibiotic screens typically rely on growth inhibition to characterize compound bioactivity-an approach that cannot be used to assess the bactericidal activity of antibiotics against bacteria in drug-tolerant states. To address this limitation, we developed a multiplexed assay that uses metabolism-sensitive staining to report on the killing of antibiotic-tolerant bacteria. This method can be used with diverse bacterial species and applied to genome-scale investigations to identify therapeutic targets against tolerant pathogens.

In-host evolution of Staphylococcus epidermidis in a pacemaker-associated endocarditis resulting in increased antibiotic tolerance

Treatment failure in biofilm-associated bacterial infections is an important healthcare issue. In vitro studies and mouse models suggest that bacteria enter a slow-growing/non-growing state that results in transient tolerance to antibiotics in the absence of a specific resistance mechanism. However, little clinical confirmation of antibiotic tolerant bacteria in patients exists. In this study we investigate a Staphylococcus epidermidis pacemaker-associated endocarditis, in a patient who developed a break-through bacteremia despite taking antibiotics to which the S. epidermidis isolate is fully susceptible in vitro. Characterization of the clinical S. epidermidis isolates reveals in-host evolution over the 16-week infection period, resulting in increased antibiotic tolerance of the entire population due to a prolonged lag time until growth resumption and a reduced growth rate. Furthermore, we observe adaptation towards an increased biofilm formation capacity and genetic diversification of the S. epidermidis isolates within the patient.

Biomarker discovery in the biofilm-forming process of Burkholderia pseudomallei by mass-spectrometry

A serious human infectious disease called Melioidosis is a result of Burkholderia pseudomallei infection. Treatment for infected individuals is difficult due to a wide range of ineffective antibiotics including a high level of antibiotic tolerance which has been known to be caused by biofilm production. However, biofilm forming processes of this bacterium are not well documented despite multiple-methodologies being applied. In this study, we utilized a proteomics strategy called whole cell matrix-assisted laser desorption ionization-time of flight mass spectrometry (whole cell MALDI-TOF MS) to discover a potential biomarker relating biofilm forming in B. pseudomallei. The results presented a novel specific type of enzyme amylo-alpha-1, 6-glucosidase, which was demonstrated by a higher level of gene expression during the biofilm development. Our results also suggested a list of candidate markers that might be involved in this scenario. Eventually, this knowledge may expand valuable data to the biofilm study that may increase effective treatments for people infected with B. pseudomallei and possibly other antibiotic tolerant bacteria.

Current therapies in treatment and prevention of fracture wound biofilms: why a multifaceted approach is essential for resolving persistent infections.

Traumatic orthopedic injuries, particularly extremity wounds, are a significant cause of morbidity. Despite prophylactic antibiotic treatment and surgical intervention, persistent infectious complications can and do occur. Persistent bacterial infections are often caused by biofilms, communities of antibiotic tolerant bacteria encased within a matrix. The structural and metabolic differences in this mode of growth make treatment difficult. Herein, we describe both established and novel, experimental treatments targeted at various stages of wound healing that are specifically aimed at reducing and eliminating biofilm bacteria. Importantly, the highly tolerant nature of these bacterial communities suggests that most singular approaches could be circumvented and a multifaceted, combinatorial approach will be the most effective strategy for treating these complicated infections.