In-patient evolution of a high-persister Escherichia coli strain with reduced in vivo antibiotic susceptibility.

Gram-negative bacterial bloodstream infections (GNB-BSI) are common and frequently lethal. Despite appropriate antibiotic treatment, relapse of GNB-BSI with the same bacterial strain is common and associated with poor clinical outcomes and high healthcare costs. The role of persister cells, which are sub-populations of bacteria that survive for prolonged periods in the presence of bactericidal antibiotics, in relapse of GNB-BSI is unclear. Using a cohort of patients with relapsed GNB-BSI, we aimed to determine how the pathogen evolves within the patient between the initial and subsequent episodes of GNB-BSI and how these changes impact persistence. Using Escherichia coli clinical bloodstream isolate pairs (initial and relapse isolates) from patients with relapsed GNB-BSI, we found that 4/11 (36%) of the relapse isolates displayed a significant increase in persisters cells relative to the initial bloodstream infection isolate. In the relapsed E. coli strain with the greatest increase in persisters (100-fold relative to initial isolate), we determined that the increase was due to a loss-of-function mutation in the ptsI gene encoding Enzyme I of the phosphoenolpyruvate phosphotransferase system. The ptsI mutant was equally virulent in a murine bacteremia infection model but exhibited 10-fold increased survival to antibiotic treatment. This work addresses the controversy regarding the clinical relevance of persister formation by providing compelling data that not only do high-persister mutations arise during bloodstream infection in humans but also that these mutants display increased survival to antibiotic challenge in vivo.

Species Typing of Nontuberculous Mycobacteria by Use of Deoxyribozyme Sensors.

BACKGROUND: Nontuberculous mycobacteria (NTM) species are a rising threat, especially to patients living with pulmonary comorbidities. Current point-of-care diagnostics fail to adequately identify and differentiate NTM species from Mycobacterium tuberculosis (Mtb). Definitive culture- and molecular-based testing can take weeks to months and requires sending samples out to specialized diagnostic laboratories. METHODS: In this proof-of-concept study, we developed an assay based on PCR amplification of 16S ribosomal RNA (rRNA) rrs genes by using universal mycobacterial primers and interrogation of the amplified fragments with a panel of binary deoxyribozyme (BiDz) sensors to enable species-level identification of NTM (BiDz-NTMST). Each BiDz sensor consists of 2 subunits of an RNA-cleaving deoxyribozyme, which form an active deoxyribozyme catalytic core only in the presence of the complimentary target sequence. The target-activated BiDz catalyzes cleavage of a reporter substrate, thus triggering either fluorescent or colorimetric (visually observed) signal depending on the substrate used. The panel included BiDz sensors for differentiation of 6 clinically relevant NTM species (Mycobacterium abscessus, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium fortuitum, Mycobacterium kansasii, and Mycobacterium gordonae) and Mtb. RESULTS: Using the fluorescent BiDz-NTMST assay, we successfully identified the species of 38 clinical isolates. In addition, a subset of strains was tested with visual BiDz sensors, providing proof-of-concept for species typing of NTM by the naked eye. CONCLUSIONS: The BiDz-NTMST assay is a novel platform for rapid identification of NTM species. This method is highly specific and significantly faster than current tools and is easily adaptable for onsite diagnostic laboratories in hospitals or clinical laboratories.

Flagellar motility and the mucus environment influence aggregation mediated antibiotic tolerance of Pseudomonas aeruginosa in chronic lung infection.

Pseudomonas aeruginosa frequently causes chronic lung infection in individuals with muco-obstructive airway diseases (MADs). Chronic P. aeruginosa infections are difficult to treat, primarily owing to antibiotic treatment failure, which is often observed in the absence of antimicrobial resistance. In MADs, P. aeruginosa forms biofilm-like aggregates within the luminal mucus. While the contribution of mucin hyperconcentration towards antibiotic tolerance has been described, the mechanism for mucin driven antibiotic tolerance and the influence of aggregates have not been fully elucidated. In this study, we investigated the contribution of flagellar motility towards aggregate formation as it relates to the diseased mucus environment. We found that loss of flagellar motility resulted in increased P. aeruginosa aggregation and tolerance to multiple classes of antibiotics. Further, we observed differential roles in antimicrobial tolerance of the motAB and motCD stators, which power the flagellum. Additionally, we found that control of fliC expression was important for aggregate formation and antibiotic tolerance as a strain constitutively expressing fliC was unable to form aggregates and was highly susceptible to treatment. Lastly, we demonstrate that neutrophil elastase, an abundant immune mediator and biomarker of chronic lung infection, promotes aggregation and antibiotic tolerance by impairing flagellar motility. Collectively, these results highlight the key role of flagellar motility in aggregate formation and antibiotic tolerance and deepens our understanding of how the MADs lung environment promotes antibiotic tolerance of P. aeruginosa . IMPORTANCE: Antibiotic recalcitrance of chronic Pseudomonas aeruginosa infections in muco-obstructive airway diseases is a primary driver of mortality. Mechanisms that drive antibiotic tolerance are poorly understood. We investigated motility phenotypes related to P. aeruginosa adaptation and antibiotic tolerance in the diseased mucus environment. Loss of flagellar motility drives antibiotic tolerance by promoting aggregate formation. Regulation of flagellar motility appears to be a key step in aggregate formation as the inability to turn off flagellin expression resulted in poor aggregate formation and increased antibiotic susceptibility. These results deepen our understanding of the formation of antibiotic tolerant aggregates within the MADs airway and opens novel avenues and targets for treatment of chronic P. aeruginosa infections.

Antibiotic-induced accumulation of lipid II synergizes with antimicrobial fatty acids to eradicate bacterial populations.

Antibiotic tolerance and antibiotic resistance are the two major obstacles to the efficient and reliable treatment of bacterial infections. Identifying antibiotic adjuvants that sensitize resistant and tolerant bacteria to antibiotic killing may lead to the development of superior treatments with improved outcomes. Vancomycin, a lipid II inhibitor, is a frontline antibiotic for treating methicillin-resistant Staphylococcus aureus and other Gram-positive bacterial infections. However, vancomycin use has led to the increasing prevalence of bacterial strains with reduced susceptibility to vancomycin. Here, we show that unsaturated fatty acids act as potent vancomycin adjuvants to rapidly kill a range of Gram-positive bacteria, including vancomycin-tolerant and resistant populations. The synergistic bactericidal activity relies on the accumulation of membrane-bound cell wall intermediates that generate large fluid patches in the membrane leading to protein delocalization, aberrant septal formation, and loss of membrane integrity. Our findings provide a natural therapeutic option that enhances vancomycin activity against difficult-to-treat pathogens, and the underlying mechanism may be further exploited to develop antimicrobials that target recalcitrant infection.

Overcoming biological barriers to improve treatment of a Staphylococcus aureus wound infection.

Chronic wounds frequently become infected with bacterial biofilms which respond poorly to antibiotic therapy. Aminoglycoside antibiotics are ineffective at treating deep-seated wound infections due to poor drug penetration, poor drug uptake into persister cells, and widespread antibiotic resistance. In this study, we combat the two major barriers to successful aminoglycoside treatment against a biofilm-infected wound: limited antibiotic uptake and limited biofilm penetration. To combat the limited antibiotic uptake, we employ palmitoleic acid, a host-produced monounsaturated fatty acid that perturbs the membrane of gram-positive pathogens and induces gentamicin uptake. This novel drug combination overcomes gentamicin tolerance and resistance in multiple gram-positive wound pathogens. To combat biofilm penetration, we examined the ability of sonobactericide, a non-invasive ultrasound-mediated-drug delivery technology to improve antibiotic efficacy using an in vivo biofilm model. This dual approach dramatically improved antibiotic efficacy against a methicillin-resistant Staphylococcus aureus (MRSA) wound infection in diabetic mice.

Stimulating Aminoglycoside Uptake to Kill Staphylococcus aureus Persisters.

Aminoglycosides are bactericidal drugs which require a proton motive force (PMF) for uptake into the bacterial cell. Low energy cells, such as persisters, maintain a PMF below the threshold for drug uptake and are tolerant to aminoglycosides. In this chapter, we discuss mechanisms to target the bacterial membrane and stimulate aminoglycoside uptake to kill Staphylococcus aureus persisters.

Harnessing ultrasound-stimulated phase change contrast agents to improve antibiotic efficacy against methicillin-resistant Staphylococcus aureus biofilms.

Bacterial biofilms, often associated with chronic infections, respond poorly to antibiotic therapy and frequently require surgical intervention. Biofilms harbor persister cells, metabolically indolent cells, which are tolerant to most conventional antibiotics. In addition, the biofilm matrix can act as a physical barrier, impeding diffusion of antibiotics. Novel therapeutic approaches frequently improve biofilm killing, but usually fail to achieve eradication. Failure to eradicate the biofilm leads to chronic and relapsing infection, is associated with major financial healthcare costs and significant morbidity and mortality. We address this problem with a two-pronged strategy using 1) antibiotics that target persister cells and 2) ultrasound-stimulated phase-change contrast agents (US-PCCA), which improve antibiotic penetration. We previously demonstrated that rhamnolipids, produced by Pseudomonas aeruginosa, could induce aminoglycoside uptake in gram-positive organisms, leading to persister cell death. We have also shown that US-PCCA can transiently disrupt biological barriers to improve penetration of therapeutic macromolecules. We hypothesized that combining antibiotics which target persister cells with US-PCCA to improve drug penetration could improve treatment of methicillin resistant S. aureus (MRSA) biofilms. Aminoglycosides alone or in combination with US-PCCA displayed limited efficacy against MRSA biofilms. In contrast, the anti-persister combination of rhamnolipids and aminoglycosides combined with US-PCCA dramatically improved biofilm killing. This novel treatment strategy has the potential for rapid clinical translation as the PCCA formulation is a variant of FDA-approved ultrasound contrast agents that are already in clinical practice and the low-pressure ultrasound settings used in our study can be achieved with existing ultrasound hardware at pressures below the FDA set limits for diagnostic imaging.