Activity of Moxifloxacin Against Biofilms Formed by Clinical Isolates of Staphylococcus aureus Differing by Their Resistant or Persister Character to Fluoroquinolones

Staphylococcus aureus biofilms are poorly responsive to antibiotics. Underlying reasons include a matrix effect preventing drug access to embedded bacteria, or the presence of dormant bacteria with reduced growth rate. Using 18 clinical isolates previously characterized for their moxifloxacin-resistant and moxifloxacin-persister character in stationary-phase culture, we studied their biofilm production and matrix composition and the anti-biofilm activity of moxifloxacin. Biofilms were grown in microtiter plates and their abundance quantified by crystal violet staining and colony counting; their content in polysaccharides, extracellular DNA and proteins was measured. Moxifloxacin activity was assessed after 24 h of incubation with a broad range of concentrations to establish full concentration-response curves. All clinical isolates produced more biofilm biomass than the reference strain ATCC 25923, the difference being more important for those with high relative persister fractions to moxifloxacin, most of which being also resistant. High biofilm producers expressed icaA to higher levels, enriching the matrix in polysaccharides. Moxifloxacin was less potent against biofilms from clinical isolates than from ATCC 25923, especially against moxifloxacin-resistant isolates with high persister fractions, which was ascribed to a lower concentration of moxifloxacin in these biofilms. Time-kill curves in biofilms revealed the presence of a moxifloxacin-tolerant subpopulation, with low multiplication capacity, whatever the persister character of the isolate. Thus, moxifloxacin activity depends on its local concentration in biofilm, which is reduced in most isolates with high-relative persister fractions due to matrix effects, and insufficient to kill resistant isolates due to their high MIC.

Nitroxoline as a promising alternative drug for the treatment of Lyme disease based on an in-vitro study

Lyme disease (LD) is the most common vector-borne disease in USA and Europe and is caused by Borrelia burgdorferi. Despite proper treatment, approximately one fifth of patients will develop post-treatment LD syndrome (PTLDS), a condition which is poorly understood. One of the possible causes is thought to be due to persister forms of B. burgdorferi that are not effectively killed by the current Lyme antibiotics. In this study, we evaluated nitroxoline, an antibiotic used to treat urinary tract infections, for its activity against a stationary-phase culture enriched with persister forms of B. burgdorferi. Nitroxoline was found to be equivalent in activity against B. burgdorferi to cefuroxime (standard Lyme antibiotic) in different experiments. Moreover, we found that the three-drug combination cefuroxime + nitroxoline + clarithromycin eradicated 98.3% of stationary phase bacteria in the drug-exposure experiment and prevented the regrowth in the subculture study after drug exposure, as well as two-drug combinations cefuroxime + nitroxoline and clarithromycin + nitroxoline. These drug combinations should be further evaluated in a LD mouse model to assess if eradication of persister forms of B. burgdorferi in-vivo is possible and if so, whether nitroxoline could be repurposed as an alternative drug for the treatment of LD.

The Persister Character of Clinical Isolates of Staphylococcus aureus Contributes to Faster Evolution to Resistance and Higher Survival in THP-1 Monocytes: A Study With Moxifloxacin

Staphylococcus aureus may cause relapsing infections. We previously showed that S. aureus SH1000 surviving intracellularly to bactericidal antibiotics are persisters. Here, we used 54 non-duplicate clinical isolates to assess links between persistence, resistance evolution, and intracellular survival, using moxifloxacin throughout as test bactericidal antibiotic. The relative persister fraction (RPF: percentage of inoculum surviving to 100× MIC moxifloxacin in stationary phase culture for each isolate relative to ATCC 25923) was determined to categorize isolates with low (≤10) or high (>10) RPF. Evolution to resistance (moxifloxacin MIC ≥ 0.5 mg/L) was triggered by serial passages at 0.5× MIC (with daily concentration readjustments). Intracellular moxifloxacin maximal efficacy (Emax) was determined by 24 h concentration-response experiments [pharmacodynamic model (Hill-Langmuir)] with infected THP-1 monocytes exposed to moxifloxacin (0.01 to 100× MIC) after phagocytosis. Division of intracellular survivors was followed by green fluorescence protein dilution (FACS). Most (30/36) moxifloxacin-susceptible isolates showed low RPF but all moxifloxacin-resistant (n = 18) isolates harbored high RPF. Evolution to resistance of susceptible isolates was faster for those with high vs. low RPF (with SOS response and topoisomerase-encoding genes overexpression). Intracellularly, moxifloxacin Emax was decreased (less negative) for isolates with high vs. low RPF, independently from resistance. Moxifloxacin intracellular survivors were non-dividing. The data demonstrate and quantitate persisters in clinical isolates of S. aureus, and show that this phenotype accelerates resistance evolution and is associated with intracellular survival in spite of high antibiotic concentrations. Isolates with high RPF may represent a possible cause of treatment failure not directly related to resistance in patients receiving active antibiotics.

High-resolution yeast quiescence profiling in human-like media reveals interacting influences of auxotrophy and nutrient availability

Yeast cells survive in stationary phase culture by entering quiescence, which is measured by colony forming capacity upon nutrient re-exposure. Yeast chronological lifespan (CLS) studies, employing the comprehensive collection of gene knockout strains, have correlated weakly between independent laboratories, which is hypothesized to reflect differential interaction between the deleted genes, auxotrophy, media composition and other assay conditions influencing quiescence. This hypothesis was investigated by high-throughput quiescence profiling of the parental prototrophic strain, from which the gene deletion strain libraries were constructed, and all possible auxotrophic allele combinations in that background. Defined media resembling human cell culture media promoted long-term quiescence, and was used to assess effects of glucose, ammonium sulfate, auxotrophic nutrient availability, Target of Rapamycin signaling, and replication stress. Frequent, high-replicate measurements of colony forming capacity from cultures aged past 60 days provided profiles of quiescence phenomena such as gasping and hormesis. Media acidification was assayed in parallel to assess correlation. Influences of leucine, methionine, glucose, and ammonium sulfate metabolism were clarified, and a role for lysine metabolism newly characterized, while histidine and uracil perturbations had less impact. Interactions occurred between glucose, ammonium sulfate, auxotrophy, auxotrophic nutrient limitation, aeration, TOR signaling, and/or replication stress. Weak correlation existed between media acidification and maintenance of quiescence. In summary, experimental factors, uncontrolled across previous genome-wide yeast CLS studies, influence quiescence and interact extensively, revealing quiescence as a complex metabolic and developmental process that should be studied in a prototrophic context, omitting ammonium sulfate from defined media, and employing highly replicable protocols.

Transcriptome profiling of Variovorax paradoxus EPS under different growth conditions reveals regulatory and structural novelty in biofilm formation

ABSTRACTWe used transcriptome analysis by paired-end strand specific RNA-seq to evaluate the specific changes in gene expression associated with the transition to static biofilm growth in the rhizosphere plant growth promoting bacterium Variovorax paradoxus EPS. Triplicate biological samples of exponential growth, stationary phase, and static biofilm samples were examined. DESeq2 and Rockhopper were used to identify robust and widespread shifts in gene expression the transcriptomic signals specific to each growth phase. Weidentified 1711 protein coding genes (28%) using DESeq2 that had altered expression greater than 2-fold specifically in biofilms compared to exponential growth. Fewer genes were specifically differentially expressed in stationary phase culture (757, 12%). A small set of genes (103/6020) were differentially expressed in opposing fashions in biofilm and stationary phase, indicating potentially substantial shifts in phenotype. Gene Ontology analysis showed that the only class of genes specifically upregulated in biofilms were associated with nutrient transport, highlighting the importance of nutrient uptake in the biofilm. The biofilm specific genes did not overlap substantially with the loci identified by mutagenesis studies, although some were present in both sets. The most highly upregulated biofilm specific gene is predicted to be a part of the RNA degradosome, which indicates that RNA stability is used to regulate the biofilm phenotype. Two small putative proteins, Varpa_0407 and Varpa_3832, are highly expressed specifically in biofilms and are predicted to be secreted DNA binding proteins, that may stabilize extracellular DNA as a component of the biofilm matrix. An flp/tad type IV pilus locus (Varpa_5148-60) is strongly downregulated in specifically in biofilms, in contrast with results from other systems for these pili. Mutagenesis confirms that this locus is important in surface motility rather than biofilm formation. These experimental results suggest that V. paradoxus EPS biofilms have substantial regulatory and structural novelty.

Variation in pre-therapy levels of selected Mycobacterium tuberculosis transcripts in sputum and their relationship with 2-month culture conversion

Background:The abundance of transcripts arising fromMycobacterium tuberculosis(MTB) in sputum pre-chemotherapy may enhance our understanding of factors influencing treatment response. We hypothesized that differences in the prevalence of pre-existing slowly metabolizing MTB in sputum may be partially responsible for differences in the rate of sputum clearance during treatment.Methods:Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to characterize a selected limited transcription profile of MTB in sputum pre-chemotherapy and assess inter-individual variation. The difference in cycle threshold (Ct) per gene, normalized to 16S, between exponential/stationary phase culture and sputum was calculated and stratified by 2-month culture converter status.Results:HIV-1 uninfected patients with rifampicin-susceptible tuberculosis provided sputum pre-chemotherapy; 11 patients were negative for MTB culture after two months of therapy and 8 remained culture-positive.Increasedicl1andprpDandrpsN2:rpsN1in sputum relative to culture suggested cholesterol utilization and a low-zinc environment respectively. IncreasedhspXand decreasedatpAandnuoGrelative to exponential culture suggested a slowly metabolizing subpopulation of MTB. While the thehspXhiatpAlonuoGlosignal varied, we did not observe statistically significant enrichment of this phenotype in the non-converter population nor an association with MTB-lineage.Conclusion:Differential abundance of selected informative transcripts suggested a metabolically less-active subpopulation with a prevalence that varied between individual untreated patients.

Identifying Vancomycin as an Effective Antibiotic for KillingBorrelia burgdorferi

ABSTRACTBorrelia burgdorferiis the causative agent of Lyme borreliosis. Antibiotic therapy of early acute infection is effective for most patients, but 10 to 20% go on to develop posttreatment Lyme disease syndrome (PTLDS). The nature of PTLDS remains unknown, but currently approved antibiotics for the treatment of Lyme disease do not appear to impact these symptoms after they have developed. We reason that minimizing the time the pathogen interacts with the host will diminish the probability of developing PTLDS, irrespective of its nature. This calls for an efficient eradication of the pathogen during acute infection. In search of a superior killing antibiotic, we examined approved antibiotics for their ability to killB. burgdorferi. Vancomycin proved more effective in killing the pathogenin vitrothan ceftriaxone, the standard of care for disseminatedB. burgdorferiinfection. Both compounds were also the most effective in killing stationary-phase cells. This is surprising, given that inhibitors of cell wall biosynthesis are known to only kill growing bacteria. We found that peptidoglycan synthesis continues in stationary-phase cells ofB. burgdorferi, explaining this paradox. A combination of vancomycin and gemifloxacin sterilized a stationary-phase culture ofB. burgdorferi. Examination of the action of antibiotics in severe combined immunodeficient (SCID) mice showed that doxycycline, a standard of care for uncomplicated acute infection, did not clear the pathogen. In contrast, both ceftriaxone and vancomycin cleared the infection. A trial examining the early use of more potent antibiotics on the development of PTLDS may be warranted.

Additional Essential Oils with High Activity against Stationary PhaseBorrelia burgdorferi

ABSTRACTLyme disease is the most common vector borne-disease in the US. While the majority of the Lyme disease patients can be cured with 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from persisting symptoms. While the cause of this condition is unclear, persistent infection was proposed as one possibility. It has recently been shown thatB. burgdorferidevelops dormant persisters in stationary phase cultures that are not killed by the current Lyme antibiotics, and there is interest to identify novel drug candidates that more effectively kill such forms. We previously evaluated 34 essential oils and identified some highly active candidates with excellent activity against biofilm and stationary phaseB. burgdorferi.Here we screened another 35 essential oils and found 10 essential oils (garlic, allspice, cumin, palmarosa, myrrh, hedycheim, amyris, thyme white, litsea cubeba, lemon eucalyptus) and the active component of cinnamon bark cinnamaldehyde (CA) at a low concentration of 0.1% to have high activity against stationary phaseB. burgdorferi.At a very low 0.05% concentration, garlic, allspice, palmarosa and CA still exhibited strong activity against the stationary phaseB. burgdorferi. CA also showed strong activity against replicatingB. burgdorferi, with a MIC of 0.02% (or 0.2 μg/mL). In subculture studies, the top 5 hits garlic, allspice, myrrh, hedycheim, and litsea cubeba completely eradicated allB. burgdorferistationary phase cells at 0.1%, while palmarosa, lemon eucalyptus, amyris, cumin, and thyme white failed to do so as shown by visible spirochetal growth after 21-day subculture. At 0.05% concentration, only garlic essential oil and CA sterilized theB. burgdorferistationary phase culture as shown by no regrowth during subculture, while allspice, myrrh, hedycheim and litsea cubeba all had visible growth during subculture. Future studies are needed to determine if these highly active essential oils could eradicate persistentB. burgdorferiinfection in vivo.

High Activity of Selective Essential Oils against Stationary PhaseBorrelia burgdorferi

ABSTRACTAlthough the majority of patients with Lyme disease can be cured with the standard 2-4 week antibiotic treatment, about 10-20% of patients continue to suffer from post-treatment Lyme disease syndrome (PTLDS). While the cause for this is debated, one possibility is due to persisters not killed by the current Lyme antibiotics. It has been reported that essential oils have antimicrobial activities and some have been used by patients with persisting Lyme disease symptoms. However, the activity of essential oils against the causative agentBorrelia burgdorferi (B. burgdorferi)has not been carefully studied. Here, we evaluated the activity of 34 essential oils againstB. burgdorferistationary phase culture as a model for persisters. We found that many essential oils had varying degrees of activity againstB. burgdorferi, with top 5 essential oils (oregano, cinnamon bark, clove bud, citronella, and wintergreen) at a low concentration of 0.25% showing more activity than the persister drug daptomycin. Interestingly, some highly active essential oils were found to have excellent anti-biofilm ability as shown by their ability to dissolve the aggregated biofilm-like structures. The top 3 hits, oregano, cinnamon bark and clove bud, completely eradicated all viable cells without regrowth in subculture. Carvacrol was found to be the most active ingredient of oregano oil showing excellent activity againstB. burgdorferistationary phase cells, while p-cymene and α-terpinene had no apparent activity. Future studies are needed to characterize and optimize the active essential oils in drug combinations in vitro and in vivo for improved treatment of persistent Lyme disease.IMPORTANCEThere is a huge need for effective treatment of patients with Lyme disease who suffer from PTLDS. Recent in vitro and in vivo studies suggest thatB. burgdorferidevelops persisters that are not killed by the current Lyme antibiotics as a possible contributor to this condition. Although essential oils are used by patients with Lyme disease with variable improvement in symptoms, their anti-borrelia activity has not been carefully studied. Here we found that not all essential oils have adequate anti-borrelia activity and identified some highly potent essential oils (oregano, cinnamon bark, clove bud) that have even higher anti-persister and anti-biofilm activity than the persister drug daptomycin. Carvacrol was found to be the most active ingredient of oregano oil and have the potential to serve as a promising oral persister drug. Our findings may have implications for developing improved treatment of persisting Lyme disease.

Possible odour-mediated attraction of flies to Bacillus subtilis NRS-762 stationary phase culture

Signalling helps connects different organisms in the biosphere. This research note describes the possible concentration-dependent odour mediated attraction of flies to stationary phase aerobic liquid cultures of Bacillus subtilis NRS-762 cultivated on an open orbital shaker at 25 oC. Greater odour pungency correlated with more intense “foraging” attempts, which suggested the compounds’ possible behaviour modifying effects. Additionally, co-occurrence of optical density decline and increase in odour pungency suggested volatile compound(s) secretion might be a cell survival response mediated by a cell density-based signalling mechanism. Flies were not attracted to odourous stationary phase cultures of other common bacteria (Escherichia coli DH5α, Pseudomonas aeruginosa PRD-10, and Pseudomonas protegens Pf-5, which suggested species-specificity of the volatile compound(s)). Altogether, volatile compound(s) might serve as interkingdom messengers to enlist flies for dispersing B. subtilis to habitats with more favourable conditions in coping with possible irreversible decline in habitability, and operate in parallel with other in situ mechanisms such as cannibalism and sporulation that help B. subtilis ride out short- and long-term environmental fluctuations, respectively. Interested researchers are invited to build upon the preliminary findings.