Effective Removal of Staphylococcal Biofilms by the Endolysin LysH5

Staphylococcal biofilms are a major concern in both clinical and food settings because they are an important source of contamination. The efficacy of established cleaning procedures is often hindered due to the ability of some antimicrobial compounds to induce biofilm formation, and to the presence of persister cells, a small bacterial subpopulation that exhibits multidrug tolerance. Phage lytic enzymes have demonstrated antimicrobial activity against planktonic and sessile bacteria. However, their ability to lyse and/or select persister cells remains largely unexplored so far. In this work, the lytic activity of the endolysin LysH5 against Staphylococcus aureus and Staphylococcus epidermidis biofilms was confirmed. LysH5 reduced staphylococcal sessile cell counts by 1–3 log units, compared with the untreated control, and sub-inhibitory concentrations of this protein did not induce biofilm formation. LysH5-surviving cells were not resistant to the lytic activity of this protein, suggesting that no persister cells were selected. Moreover, to prove the lytic ability of LysH5 against this subpopulation, both S. aureus exponential cultures and persister cells obtained after treatment with rifampicin and ciprofloxacin were subsequently treated with LysH5. The results demonstrated that besides the notable activity of endolysin LysH5 against staphylococcal biofilms, persister cells were also inhibited, which raises new opportunities as an adjuvant for some antibiotics.     

The Formation of Persister Cells in Stationary-Phase Cultures of Escherichia Coli Is Associated with the Aggregation of Endogenous Proteins

Persister cells (persisters) are transiently tolerant to antibiotics and usually constitute a small part of bacterial populations. Persisters remain dormant but are able to re-grow after antibiotic treatment. In this study we found that the frequency of persisters correlated to the level of protein aggregates accumulated in E. coli stationary-phase cultures. When 3-(N-morpholino) propanesulfonic acid or an osmolyte (trehalose, betaine, glycerol or glucose) were added to the growth medium at low concentrations, proteins were prevented from aggregation and persister formation was inhibited. On the other hand, acetate or high concentrations of osmolytes enhanced protein aggregation and the generation of persisters. We demonstrated that in the E. coli stationary-phase cultures supplemented with MOPS or a selected osmolyte, the level of protein aggregates and persister frequency were not correlated with such physiological parameters as the extent of protein oxidation, culturability, ATP level or membrane integrity. The results described here may help to understand the mechanisms underlying persister formation.     

Identification of a potent indigoid persister antimicrobial by screening dormant cells

The subpopulation of bacterial cells that survive myriad stress conditions (e.g., nutrient deprivation and antimicrobials) by ceasing metabolism, revive by activating ribosomes. These resuscitated cells can reconstitute infections; hence, it is imperative to discover compounds which eradicate persister cells. By screening 10,000 compounds directly for persister cell killing, we identified 5-nitro-3-phenyl-1H-indol-2-yl-methylamine hydrochloride (NPIMA) kills Escherichia coli persister cells more effectively than the best indigoid found to date, 5-iodoindole, and better than the DNA-crosslinker cisplatin. In addition, NPIMA eradicated Pseudomonas aeruginosa persister cells in a manner comparable to cisplatin. NPIMA also eradicated Staphylococcus aureus persister cells but was less effective than cisplatin. Critically, NPIMA kills Gram-positive and Gram-negative bacteria by damaging membranes and causing lysis as demonstrated by microscopy and release of extracellular DNA and protein. Furthermore, NPIMA was effective in reducing P. aeruginosa and S. aureus cell numbers in a wound model, and no resistance was found after 1 week. Hence, we identified a potent indigoid that kills persister cells by damaging their membranes.     

Analysis of Non-Typeable Haemophilous influenzae VapC1 Mutations Reveals Structural Features Required for Toxicity and Flexibility in the Active Site

Bacteria have evolved mechanisms that allow them to survive in the face of a variety of stresses including nutrient deprivation, antibiotic challenge and engulfment by predator cells. A switch to dormancy represents one strategy that reduces energy utilization and can render cells resistant to compounds that kill growing bacteria. These persister cells pose a problem during treatment of infections with antibiotics, and dormancy mechanisms may contribute to latent infections. Many bacteria encode toxin-antitoxin (TA) gene pairs that play an important role in dormancy and the formation of persisters. VapBC gene pairs comprise the largest of the Type II TA systems in bacteria and they produce a VapC ribonuclease toxin whose activity is inhibited by the VapB antitoxin. Despite the importance of VapBC TA pairs in dormancy and persister formation, little information exists on the structural features of VapC proteins required for their toxic function in vivo. Studies reported here identified 17 single mutations that disrupt the function of VapC1 from non-typeable H. influenzae in vivo. 3-D modeling suggests that side chains affected by many of these mutations sit near the active site of the toxin protein. Phylogenetic comparisons and secondary mutagenesis indicate that VapC1 toxicity requires an alternative active site motif found in many proteobacteria. Expression of the antitoxin VapB1 counteracts the activity of VapC1 mutants partially defective for toxicity, indicating that the antitoxin binds these mutant proteins in vivo. These findings identify critical chemical features required for the biological function of VapC toxins and PIN-domain proteins.     

Reverting Antibiotic Tolerance of Pseudomonas aeruginosa PAO1 Persister Cells by (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one

Background

Bacteria are well known to form dormant persister cells that are tolerant to most antibiotics. Such intrinsic tolerance also facilitates the development of multidrug resistance through acquired mechanisms. Thus persister cells are a promising target for developing more effective methods to control chronic infections and help prevent the development of multidrug-resistant bacteria. However, control of persister cells is still an unmet challenge.

Methodology/Principal Findings

We show in this report that (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8) can restore the antibiotic susceptibility of Pseudomonas aeruginosa PAO1 persister cells at growth non-inhibitory concentrations. Persister control by BF8 was found to be effective against both planktonic and biofilm cells of P. aeruginosa PAO1. Interestingly, although BF8 is an inhibitor of quorum sensing (QS) in Gram-negative bacteria, the data in this study suggest that the activities of BF8 to revert antibiotic tolerance of P. aeruginosa PAO1 persister cells is not through QS inhibition and may involve other targets.

Conclusion

BF8 can sensitize P. aeruginosa persister cells to antibiotics.     

Mannitol Enhances Antibiotic Sensitivity of Persister Bacteria in Pseudomonas aeruginosa Biofilms

The failure of antibiotic therapies to clear Pseudomonas aeruginosa lung infection, the key mortality factor for cystic fibrosis (CF) patients, is partly attributed to the high tolerance of P. aeruginosa biofilms. Mannitol has previously been found to restore aminoglycoside sensitivity in Escherichia coli by generating a proton-motive force (PMF), suggesting a potential new strategy to improve antibiotic therapy and reduce disease progression in CF. Here, we used the commonly prescribed aminoglycoside tobramycin to select for P. aeruginosa persister cells during biofilm growth. Incubation with mannitol (10–40 mM) increased tobramycin sensitivity of persister cells up to 1,000-fold. Addition of mannitol to pre-grown biofilms was able to revert the persister phenotype and improve the efficacy of tobramycin. This effect was blocked by the addition of a PMF inhibitor or in a P. aeruginosa mutant strain unable to metabolise mannitol. Addition of glucose and NaCl at high osmolarity also improved the efficacy of tobramycin although to a lesser extent compared to mannitol. Therefore, the primary effect of mannitol in reverting biofilm associated persister cells appears to be an active, physiological response, associated with a minor contribution of osmotic stress. Mannitol was tested against clinically relevant strains, showing that biofilms containing a subpopulation of persister cells are better killed in the presence of mannitol, but a clinical strain with a high resistance to tobramycin was not affected by mannitol. Overall, these results suggest that in addition to improvements in lung function by facilitating mucus clearance in CF, mannitol also affects antibiotic sensitivity in biofilms and does so through an active, physiological response.     

Structural effects on persister control by brominated furanones

Bacterial persister cells are a small population of dormant cells that are tolerant to essentially all antibiotics. Recently, we reported that a quorum sensing (QS) inhibitor, (Z)-4-bromo-5-(bromomethylene)-3-methylfuran-2(5H)-one (BF8), can revert antibiotic tolerance of Pseudomonas aeruginosa persister cells. To better understand this phenomenon, several synthetic brominated furanones with similar structures were compared for their activities in persister control and inhibition of acyl-homoserine lactone (AHL) mediated QS. The results show that some other furanones in addition to BF8 are also AHL QS inhibitors and can revert antibiotic tolerance of P. aeruginosa PAO1 persister cells. However, not all QS inhibiting BFs can revert persistence at growth non-inhibitory concentrations, suggesting that QS inhibition itself is not sufficient for persister control.     

Activities of a broad-spectrum antimicrobial peptide analogue SAMP-A4-C8 and its combat against pneumonia in Staphylococcus aureus-infected mice

Antimicrobial peptides and their analogues have become substitutes for antibiotics in recent years. The antimicrobial peptide analogue SAMP-A4-C8 (n-octanoic-VRLLRRRI) with high antimicrobial activity was found in our lab. We speculate that it may kill pathogens by some lethal mechanism of action. In the present investigation, the microbicidal activities of SAMP-A4-C8 and its mechanism of action were investigated. The results demonstrated that SAMP-A4-C8 had lethal activities against Staphylococcus aureus and Candida albicans by cell disruption. Based on its microbicidal activities, we believe that it is worth further research for its potential as drug candidate. The results showed that SAMP-A4-C8, with low propensity to induce the resistance of S. aureus and C. albicans, could kill the persister cells of S. aureus and C. albicans, exhibited biofilm forming inhibition activity and preformed biofilm eradication ability against S. aureus and C. albicans, and displayed therapeutic potential on pneumonia in S. aureus-infected mice by reducing lung inflammation. The present study provided a promising drug candidate in the war against multidrug resistance.     

Heterogeneous Persister Cells Formation in Acinetobacter baumannii

Bacterial persistence is a feature that allows susceptible bacteria to survive extreme concentrations of antibiotics and it has been verified in a number of species, such as Escherichia coli, Pseudomonas aeruginosa, Staphylococcus spp., Mycobacterium spp. However, even though Acinetobacter baumannii is an important nosocomial pathogen, data regarding its persistence phenotype are still lacking. Therefore, the aim of this study was to evaluate the persistence phenotype in A. baumannii strains, as well as its variation among strains after treatment with polymyxin B and tobramycin. Stationary cultures of 37 polymyxin B-susceptible clinical strains of A. baumannii were analyzed for surviving cells after exposure to 15 µg/mL of polymyxin B for 6 h, by serial dilutions and colony counting. Among these, the 30 tobramycin-susceptible isolates also underwent tobramycin treatment at a concentration of 160 µg/mL and persister cells occurrence was evaluated equally. A high heterogeneity of persister cells formation patterns among isolates was observed. Polymyxin B-treated cultures presented persister cells corresponding from 0.0007% to 10.1% of the initial population and two isolates failed to produce detectable persister cells under this condition. A high variability could also be observed when cells were treated with tobramycin: the persister fraction corresponded to 0.0003%–11.84% of the pre-treatment population. Moreover, no correlation was found between persister subpopulations comparing both antibiotics among isolates, indicating that different mechanisms underlie the internal control of this phenotype. This is the first report of persister cells occurrence in A. baumannii. Our data suggest that distinct factors regulate the tolerance for unrelated antibiotics in this species, contrasting the multi-drug tolerance observed in other species (eg. dormancy-mediated tolerance). Supporting this observation, polymyxin B – an antibiotic that is believed to act on non-dividing cells as well – failed to eradicate persister cells in the majority of the isolates, possibly reflecting a disconnection between persistence and dormancy.     

Antibacterial activity of hypocrellin A against Staphylococcus aureus

The antibacterial activity and acting mechanism of hypocrellin A (HA) were conducted regarding in vitro activity of HA on Staphylococcus aureus GZ86 by analyzing the growth, permeability, and morphology of the bacterial cells following treatment with HA. The experimental results indicated 1.5?mg/l HA could completely inhibit the growth of 10^7?CFU/ml S. aureus cells in liquid beef extract-peptone medium under a halogen?Ctungsten lamp for 120?min. Meanwhile, HA resulted in the leakage of reducing sugars and proteins and induced the respiratory chain dehydrogenases into inactive state, suggesting that HA were able to destroy the permeability of the bacterial membranes. When the cells of S. aureus were exposed to 2.5?mg/l HA under a halogen?Ctungsten lamp for 120?min, many pits and gaps were observed in bacterial cells by scanning electron microscopy, and the cell wall was fragmentary, indicating the bacterial cells were damaged severely. The experiments strongly confirmed the contribution of multiform reactive oxygen species (ROS) to bactericidal effect. In conclusion, the combined results suggested that ROS may damage the structure of bacterial cell wall and depress the activity of some membranous enzymes, which cause S. aureus bacteria to die eventually.     

Physiologic Stresses Reveal a Salmonella Persister State and TA Family Toxins Modulate Tolerance to These Stresses

Bacterial persister cells are considered a basis for chronic infections and relapse caused by bacterial pathogens. Persisters are phenotypic variants characterized by low metabolic activity and slow or no replication. This low metabolic state increases pathogen tolerance to antibiotics and host immune defenses that target actively growing cells. In this study we demonstrate that within a population of Salmonella enterica serotype Typhimurium, a small percentage of bacteria are reversibly tolerant to specific stressors that mimic the macrophage host environment. Numerous studies show that Toxin-Antitoxin (TA) systems contribute to persister states, based on toxin inhibition of bacterial metabolism or growth. To identify toxins that may promote a persister state in response to host-associated stressors, we analyzed the six TA loci specific to S. enterica serotypes that cause systemic infection in mammals, including five RelBE family members and one VapBC member. Deletion of TA loci increased or decreased tolerance depending on the stress conditions. Similarly, exogenous expression of toxins had mixed effects on bacterial survival in response to stress. In macrophages, S. Typhimurium induced expression of three of the toxins examined. These observations indicate that distinct toxin family members have protective capabilities for specific stressors but also suggest that TA loci have both positive and negative effects on tolerance.     

Emergence of Daptomycin Resistance in Daptomycin-Na?ve Rabbits with Methicillin-Resistant Staphylococcus aureus Prosthetic Joint Infection Is Associated with Resistance to Host Defense Cationic Peptides and mprF Polymorphisms

Background

Previous studies of both clinically-derived and in vitro passage-derived daptomycin–resistant (DAP-R) Staphylococcus aureus strains demonstrated the coincident emergence of increased DAP MICs and resistance to host defense cationic peptides (HDP-R).

Methods

In the present investigation, we studied a parental DAP-susceptible (DAP-S) methicillin-resistant Staphylococcus aureus (MRSA) strain and three isogenic variants with increased DAP MICs which were isolated from both DAP-treated and DAP-untreated rabbits with prosthetic joint infections. These strains were compared for: in vitro susceptibility to distinct HDPs differing in size, structure, and origin; i.e.; thrombin-induced platelet microbicidal proteins [tPMPs] and human neutrophil peptide-1 [hNP-1]; cell membrane (CM) phospholipid and fatty acid content; CM order; envelope surface charge; cell wall thickness; and mprF single nucleotide polymorphisms (SNPs) and expression profiles.

Results

In comparison with the parental strain, both DAP-exposed and DAP-naive strains exhibited: (i) significantly reduced susceptibility to each HDP (P<0.05); (ii) thicker cell walls (P<0.05); (iii) increased synthesis of CM lysyl-phosphatidylglycerol (L-PG); (iv) reduced content of CM phosphatidylglycerol (PG); and (v) SNPs within the mprF locus No significant differences were observed between parental or variant strains in outer CM content of L-PG, CM fluidity, CM fatty acid contents, surface charge, mprF expression profiles or MprF protein content. An isolate which underwent identical in vivo passage, but without evolving increased DAP MICs, retained parental phenotypes and genotype.

Conclusions

These results suggest: i) DAP MIC increases may occur in the absence of DAP exposures in vivo and may be triggered by organism exposure to endogenous HDPs: and ii) gain-in-function SNPs in mprF may contribute to such HDP-DAP cross-resistance phenotypes, although the mechanism of this relationship remains to be defined.     

Vibrio splendidus persister cells induced by host coelomic fluids show a similar phenotype to antibiotic-induced counterparts

Persister cells are dormant variants of regular cells that are multidrug tolerant and have heterogeneous phenotypes; these cells are a potential threat to hosts because they can escape the immune system or antibiotic treatments and reconstitute infectious. Skin ulcer syndrome (SUS) frequently occurs in the sea cucumber (Apostichopus japonicus), and Vibrio splendidus is one of the main bacterial pathogens of SUS. This study found that the active cells of V. splendidus became persister cells more readily in the presence of A. japonicus coelomic fluids. We showed that the A. japonicus coelomic fluids plus antibiotics induce 100-fold more persister cells in V. splendidus compared with antibiotics alone via nine sets of experiments including assays for antibiotic resistance, metabolic activity, and single-cell phenotypes. Furthermore, the coelomic fluids-induced persister cells showed similar phenotypes as the antibiotic-induced persister cells. Further investigation showed that guanosine pentaphosphate/tetraphosphate (henceforth ppGpp) and SOS response pathway involved in the formation of persister cells as determined using real-time RT-PCR. In addition, single-cell observations showed that, similar to the antibiotic-induced V. splendidus persister cells, the coelomic fluids-induced persister cells have five resuscitation phenotypes: no growth, expansion, elongation, elongation and then division, and elongation followed by death/disappearance. In addition, dark foci formed in the majority of persister cells for both the antibiotic-induced and coelomic fluids-induced persister cells. Our results highlight that the pathogen V. splendidus might escape from the host immune system by entering the persister state during the process of infection due to exposure to coelomic fluids.