It More than Adds Up: Interaction of Antibiotic Mixing and Temperature

Use of antibiotics for the treatment and prevention of bacterial infections in humans, agri- and aquaculture as well as livestock rearing leads to antibiotic pollution of fresh water and these antibiotics have an impact on free-living bacteria. While we know which antibiotics are most common in natural environments such as rivers and streams, there is considerable uncertainty regarding antibiotics’ interactions with one another and the effect of abiotic factors such as temperature. Here, we used an experimental approach to explore the effects of antibiotic identity, concentration, mixing and water temperature on the growth of Pseudomonas fluorescens, a common, ubiquitous bacterium. We exposed P. fluorescens to the four antibiotics most commonly found in surface waters (ciprofloxacin, ofloxacin, sulfamethoxazole and sulfapyridine) and investigated antibiotic interactions for single and mixed treatments at different, field-realistic temperatures. We observed an overall dependence of antibiotic potency on temperature, as temperature increased efficacy of ciprofloxacin and ofloxacin with their EC50 lowered by >75% with a 10 °C temperature increase. Further, we show that mixtures of ciprofloxacin and ofloxacin, despite both belonging to the fluoroquinolone class, exhibit low-temperature-dependent synergistic effects in inhibiting bacterial growth. These findings highlight the context dependency of antibiotic efficacy. They further suggest antibiotic-specific off-target effects that only affect the bacteria once they enter a certain temperature range. This has important implications as freshwater systems already contain multi-drug antibiotic cocktails and are changing temperature due to environmental warming. These factors will interact and affect aquatic food webs, and hence this creates an urgent need to adapt and improve laboratory testing conditions to closer reflect natural environments.

Visualizing pyrazinamide action by live single cell imaging of phagosome acidification and Mycobacterium tuberculosis pH homeostasis

Mycobacterium tuberculosis (Mtb) segregates within multiple subcellular niches with different biochemical and biophysical properties that, upon treatment, may impact antibiotic distribution, accumulation, and efficacy. However, it remains unclear whether fluctuating intracellular microenvironments alter mycobacterial homeostasis and contribute to antibiotic enrichment and efficacy. Here, we describe a live dual-imaging approach to monitor host subcellular acidification and Mtb intrabacterial pH. By combining this approach with pharmacological and genetic perturbations, we show that Mtb can maintain its intracellular pH independently of the surrounding pH in human macrophages. Importantly, unlike bedaquiline (BDQ), isoniazid (INH) or rifampicin (RIF), the drug pyrazinamide (PZA) displays antibacterial efficacy by acting as protonophore which disrupts intrabacterial pH homeostasis in cellulo. By using Mtb mutants, we confirmed that intracellular acidification is a prerequisite for PZA efficacy in cellulo. We anticipate this imaging approach will be useful to identify host cellular environments that affect antibiotic efficacy against intracellular pathogens.

Combining Ivacaftor and Intensive Antibiotics Achieves Limited Clearance of Cystic Fibrosis Infections

Recent work shows that people with cystic fibrosis (CF) and chronic lung infections generally remain persistently infected after treatment with drugs that target the CF physiological defect (called CFTR modulators). However, changes produced by modulators could increase antibiotic efficacy.

The utilization of Blaptica dubia cockroaches as an in vivo model to test antibiotic efficacy

Insects are now well recognized as biologically relevant alternative hosts for dozens of mammalian pathogens and they are routinely used in microbial pathogenesis studies. Unfortunately, these models have yet to be incorporated into the drug development pipeline. The purpose of this work was to begin to evaluate the utility of orange spotted (Blaptica dubia) cockroaches in early antibiotic characterization. To determine whether these model hosts could exhibit mortality when infected with bacteria that are pathogenic to humans, we subjected B. dubia roaches to a range of infectious doses of Klebsiella pneumoniae, Escherichia coli, Staphylococcus aureus, and Acinetobacter baumannii to identify the medial lethal dose. These results showed that lethal disease did not develop following infection of high doses of S. aureus, and A. baumannii. However, cockroaches infected with E. coli and K. pneumoniae succumbed to infection (LD50s of 5.82 × 106 and 2.58 × 106 respectively) suggesting that this model may have limitations based on pathogen specificity. However, because these cockroaches were susceptible to infection from E. coli and K. pneumoniae, we used these bacterial strains for subsequent antibiotic characterization studies. These studies suggested that β-lactam antibiotic persistence and dose was associated with reduction of hemolymph bacterial burden. Moreover, our data indicated that the reduction of bacterial CFU was directly due to the drug activity. Altogether, this work suggests that the orange-spotted cockroach infection model provides an alternative in vivo setting from which antibiotic efficacy can be evaluated.

Finafloxacin, a Novel Fluoroquinolone, Reduces the Clinical Signs of Infection and Pathology in a Mouse Model of Q Fever

Finafloxacin is a novel fluoroquinolone with optimal antibacterial activity in low pH environments, therefore offering a therapeutic advantage over some traditional antibiotics, in treating bacterial infections associated with acidic foci. Coxiella burnetii, the causative agent of Q fever, is a bacterium which resides and replicates in acidic intracellular parasitic vacuoles. The efficacy of finafloxacin was evaluated in vivo using the A/J mouse model of inhalational Q fever and was compared to doxycycline, the standard treatment for this infection and ciprofloxacin, a comparator fluoroquinolone. Finafloxacin reduced the severity of the clinical signs of infection and weight loss associated with Q fever, but did not reduce the level of bacterial colonization in tissues compared to doxycycline or ciprofloxacin. However, histopathological analysis suggested that treatment with finafloxacin reduced tissue damage associated with C. burnetii infection. In addition, we report for the first time, the use of viable counts on axenic media to evaluate antibiotic efficacy in vivo.

Translation of Pharmacodynamic Biomarkers of Antibiotic Efficacy in Specific Populations to Optimize Doses

Antibiotic efficacy determination in clinical trials often relies on non-inferiority designs because they afford smaller study sample sizes. These efficacy studies tend to exclude patients within specific populations or include too few patients to discern potential differences in their clinical outcomes. As a result, dosing guidance in patients with abnormal liver and kidney function, age across the lifespan, and other specific populations relies on drug exposure-matching. The underlying assumption for exposure-matching is that the disease course and the response to the antibiotic are similar in patients with and without the specific condition. While this may not be the case, clinical efficacy studies are underpowered to ensure this is true. The current paper provides an integrative review of the current approach to dose selection in specific populations. We review existing clinical trial endpoints that could be measured on a more continuous rather than a discrete scale to better inform exposure–response relationships. The inclusion of newer systemic biomarkers of efficacy can help overcome the current limitations. We use a modeling and simulation exercise to illustrate how an efficacy biomarker can inform dose selection better. Studies that inform response-matching rather than exposure-matching only are needed to improve dose selection in specific populations.

Potentiating antibiotic efficacy via perturbation of non-essential gene expression

Proliferation of multidrug-resistant (MDR) bacteria poses a threat to human health, requiring new strategies. Here we propose using fitness neutral gene expression perturbations to potentiate antibiotics. We systematically explored 270 gene knockout-antibiotic combinations in Escherichia coli, identifying 90 synergistic interactions. Identified gene targets were subsequently tested for antibiotic synergy on the transcriptomic level via multiplexed CRISPR-dCas9 and showed successful sensitization of E. coli without a separate fitness cost. These fitness neutral gene perturbations worked as co-therapies in reducing a Salmonella enterica intracellular infection in HeLa. Finally, these results informed the design of four antisense peptide nucleic acid (PNA) co-therapies, csgD, fnr, recA and acrA, against four MDR, clinically isolated bacteria. PNA combined with sub-minimal inhibitory concentrations of trimethoprim against two isolates of Klebsiella pneumoniae and E. coli showed three cases of re-sensitization with minimal fitness impacts. Our results highlight a promising approach for extending the utility of current antibiotics.

Inter-species interactions alter antibiotic efficacy in bacterial communities

The efficacy of antibiotic treatments targeting polymicrobial communities is not well predicted by conventional in vitro susceptibility testing based on determining minimum inhibitory concentration (MIC) in monocultures. One reason for this is that inter-species interactions can alter the community members’ susceptibility to antibiotics. Here we quantify, and identify mechanisms for, community-modulated changes of efficacy for clinically relevant antibiotics against the pathogen Pseudomonas aeruginosa in model cystic fibrosis (CF) lung communities derived from clinical samples. We demonstrate that multi-drug resistant Stenotrophomonas maltophilia can provide high levels of antibiotic protection to otherwise sensitive P. aeruginosa. Exposure protection to imipenem was provided by chromosomally encoded metallo-β-lactamase that detoxified the environment; protection was dependent upon S. maltophilia cell density and was provided by S. maltophilia strains isolated from CF sputum, increasing the MIC of P. aeruginosa by up to 16-fold. In contrast, the presence of S. maltophilia provided no protection against meropenem, another routinely used carbapenem. Mathematical ordinary differential equation modelling shows that the level of exposure protection provided against different carbapenems can be explained by differences in antibiotic efficacy and inactivation rate. Together, these findings reveal that exploitation of pre-occurring antimicrobial resistance, and inter-specific competition, can have large impacts on pathogen antibiotic susceptibility, highlighting the importance of microbial ecology for designing successful antibiotic treatments for multispecies communities.

Differences in characteristics, aetiologies, isolated pathogens, and the efficacy of antibiotics in adult patients with preseptal cellulitis and orbital cellulitis between 2000–2009 and 2010–2019

Background/aimsTo understand whether the epidemiology, aetiologies, common pathogens and the antibiotic efficacy against the identified bacteria of periorbital cellulitis in adults have changed recently (2010–2019) compared with the past decade (2000–2009).MethodsAdult patients (n=224) diagnosed with preseptal cellulitis and orbital cellulitis admitted to Kaohsiung Veterans General Hospital during 2000–2019 were retrospectively reviewed. Demographic and clinical characteristics, isolated pathogens and antibiotic susceptibility tests against the commonly cultured bacteria were analysed.ResultsPreseptal cellulitis showed a tendency of female predominance. Patients in their 60s showed an incidence peak; more cases were observed during winter. The most common predisposing factor was dacryocystitis (15.5%–30.5%), followed by hordeolum (15.5%–24.8%). Aetiology of sinusitis (p=0.001) decreased and that of conjunctivitis (p=0.007) increased significantly with time. Culture results of nasopharyngeal swabs and local abscess showed higher positivity rate than conjunctival swab. The most common isolates were methicillin-susceptible Staphylococcus aureus, methicillin-resistant S. aureus, coagulase-negative staphylococci and Pseudomonas aeruginosa. Antibiotics including fluoroquinolones and vancomycin were effective; in contrast, ampicillin/sulbactam and oxacillin showed decreasing efficacy against gram-positive bacteria. For antibiotic treatment against P. aeruginosa, fluoroquinolones, ceftazidime, piperacillin and imipenem were ideal choices.ConclusionIn isolated pathogens, the increasing trend of methicillin-resistant S. aureus detection was compatible with reducing oxacillin efficacy against periorbital infection. In our study, the report of antibiotic efficacy against the most common identified bacteria offered empirical choices for hospitalised patients with periorbital infection before obtaining culture results.