Effect of colistin-based antibiotic combinations on the eradication of persister cells in Pseudomonas aeruginosa.

OBJECTIVES: Persister cells are responsible for antibiotic treatment failure and the emergence of antibiotic resistance. The synergistic lethal effects of antibiotic combinations on persister cells were investigated using Pseudomonas aeruginosa isolates. METHODS: Persister assays were performed on P. aeruginosa clinical isolates using colistin, amikacin, ciprofloxacin and cefepime, individually and in combination. ATP concentrations were measured and morphological changes in persister cells were observed using transmission electron microscopy (TEM). The expression of relA, spoT and obg genes was evaluated and persister-cell formation was investigated in a relA and spoT double mutant (ΔrelAΔspoT). RESULTS: The P. aeruginosa persister cells were eradicated upon exposure to the colistin-based antibiotic combination colistin + ciprofloxacin. Simultaneous treatment with both antibiotics, rather than sequential treatment, caused more effective eradication. The intracellular ATP concentration was most reduced in colistin persisters. While the spoT gene was only overexpressed in colistin-persister cells, the relA gene was overexpressed in all persister cells compared with untreated parent cells. TEM analysis suggested the possibility that persister cells might be formed by different mechanisms depending on the antibiotic. Cell elongation and cell wall or membrane damage in colistin persisters, DNA condensation in amikacin persisters and outer membrane vesicles in ciprofloxacin persisters were identified. CONCLUSIONS: In P. aeruginosa, the colistin-based antibiotic combination (colistin + ciprofloxacin) was effective for the eradication of persister cells, probably due to the different persister cell-formation mechanisms between the two antibiotics. Simultaneous, rather than sequential, treatment with two antibiotics could be more effective for eradicating persister P. aeruginosa cells.

Eradication of persister cells of Acinetobacter baumannii through combination of colistin and amikacin antibiotics.

OBJECTIVES: Persister cells following antibiotic exposure may cause failure of antibiotic treatment. The synergistic effects of antibiotic combinations with respect to eliminating persister cells were investigated based on their characteristics. METHODS: For Acinetobacter baumannii clinical isolates, persister assays were performed using colistin, amikacin, imipenem and ciprofloxacin in various ways, including exposure to antibiotics in combination and sequentially. Persister phenotypes were observed through analysis of ATP concentration, membrane potential and transmission electron microscopy. RESULTS: Each A. baumannii isolate showed a specific survival rate of persister cells against each antibiotic. The persister cells were eradicated effectively by exposure to the combination of colistin and amikacin, especially in the sequential order of colistin then amikacin. While the persister cells were not identified after 6 h when exposed to the antibiotics in the order colistin then amikacin, they remained at 0.016% when antibiotic exposure was done in the order amikacin then colistin. Although membrane potential was low in both colistin and amikacin persisters, depletion of the intracellular ATP concentration was only observed in colistin persisters. In addition, transmission electron microscopy analysis showed that colistin persisters have a unique morphology with a rough and rippled membrane and many outer membrane vesicles. Empty pore-like structures surrounded by cracks were also observed. CONCLUSIONS: In A. baumannii, the combination of colistin and amikacin was most effective for eradication of persister cells, probably due to different mechanisms of persister cell formation between antibiotics. It was also identified that the sequential order of colistin followed by amikacin was important to eradicate the persister cells.

Development of colistin resistance in pmrA-, phoP-, parR- and cprR-inactivated mutants of Pseudomonas aeruginosa.

OBJECTIVES: Colistin susceptibility in Pseudomonas aeruginosa is associated with a lipopolysaccharide (LPS) structure that is controlled by the modulation of several two-component regulatory systems. In this study, we attempted to elucidate the role of these two-component systems in the development of colistin resistance in P. aeruginosa. METHODS: pmrA-, phoP-, parR- or cprR-inactivated mutants were constructed from a colistin-susceptible P5 strain. Colistin-resistant mutants (P5R, P5ΔpmrA-R, P5ΔphoP-R, P5ΔparR-R and P5ΔcprR-R) were developed in vitro from a wild-type strain (P5) and pmrA-, phoP-, parR- or cprR-inactivated mutants by serial passage in colistin-containing media. Expression levels of the pmrA, phoP, parR, cprR and arnB genes were determined and amino acid alterations of two-component regulatory systems during development of colistin resistance were also investigated. RESULTS: While P5ΔpmrA-R, P5ΔparR-R and P5ΔcprR-R showed elevated expression of the phoP gene, the expression levels of the pmrA, parR and cprR genes were not different between gene-inactivated mutants and the adapted colistin-resistant mutants. P5ΔphoP-R showed no significant elevation in expression of any of the pmrA, parR or cprR genes. The arnB gene was overexpressed in all in vitro-selected colistin-resistant mutants compared with colistin-susceptible wild-type and gene-inactivated mutants. Three amino acid alterations in PhoQ and three in ParS were identified in induced colistin-resistant mutants. CONCLUSIONS: Our data suggest that individual two-component systems may not be essential for the acquisition of colistin resistance in P. aeruginosa. The PhoPQ two-component system may play a major role in the development of colistin resistance in our strains, but alternative or compensatory pathways may exist.

Mycobacterium tuberculosis grows linearly at the single-cell level with larger variability than model organisms.

The ability of bacterial pathogens to regulate growth is crucial to control homeostasis, virulence, and drug response. Yet, we do not understand the growth and cell cycle behaviors of Mycobacterium tuberculosis (Mtb), a slow-growing pathogen, at the single-cell level. Here, we use time-lapse imaging and mathematical modeling to characterize these fundamental properties of Mtb. Whereas most organisms grow exponentially at the single-cell level, we find that Mtb exhibits a unique linear growth mode. Mtb growth characteristics are highly variable from cell-to-cell, notably in their growth speeds, cell cycle timing, and cell sizes. Together, our study demonstrates that growth behavior of Mtb diverges from what we have learned from model bacteria. Instead, Mtb generates a heterogeneous population while growing slowly and linearly. Our study provides a new level of detail into how Mtb grows and creates heterogeneity, and motivates more studies of growth behaviors in bacterial pathogens.

Types and functions of heterogeneity in mycobacteria.

The remarkable ability of Mycobacterium tuberculosis to survive attacks from the host immune response and drug treatment is due to the resilience of a few bacilli rather than a result of survival of the entire population. Maintenance of mycobacterial subpopulations with distinct phenotypic characteristics is key for survival in the face of dynamic and variable stressors encountered during infection. Mycobacterial populations develop a wide range of phenotypes through an innate asymmetric growth pattern and adaptation to fluctuating microenvironments during infection that point to heterogeneity being a vital survival strategy. In this Review, we describe different types of mycobacterial heterogeneity and discuss how heterogeneity is generated and regulated in response to environmental cues. We discuss how this heterogeneity may have a key role in recording memory of their environment at both the single-cell level and the population level to give mycobacterial populations plasticity to withstand complex stressors.

Tuberculosis treatment failure associated with evolution of antibiotic resilience.

The widespread use of antibiotics has placed bacterial pathogens under intense pressure to evolve new survival mechanisms. Genomic analysis of 51,229 Mycobacterium tuberculosis (Mtb)clinical isolates has identified an essential transcriptional regulator, Rv1830, herein called resR for resilience regulator, as a frequent target of positive (adaptive) selection. resR mutants do not show canonical drug resistance or drug tolerance but instead shorten the post-antibiotic effect, meaning that they enable Mtb to resume growth after drug exposure substantially faster than wild-type strains. We refer to this phenotype as antibiotic resilience. ResR acts in a regulatory cascade with other transcription factors controlling cell growth and division, which are also under positive selection in clinical isolates of Mtb. Mutations of these genes are associated with treatment failure and the acquisition of canonical drug resistance.

O-GlcNAcylation ameliorates the pathological manifestations of Alzheimer's disease by inhibiting necroptosis.

O-GlcNAcylation (O-linked ß-N-acetylglucosaminylation) is notably decreased in Alzheimer's disease (AD) brain. Necroptosis is activated in AD brain and is positively correlated with neuroinflammation and tau pathology. However, the links among altered O-GlcNAcylation, ß-amyloid (Aß) accumulation, and necroptosis are unclear. Here, we found that O-GlcNAcylation plays a protective role in AD by inhibiting necroptosis. Necroptosis was increased in AD patients and AD mouse model compared with controls; however, decreased necroptosis due to O-GlcNAcylation of RIPK3 (receptor-interacting serine/threonine protein kinase 3) was observed in 5xFAD mice with insufficient O-linked ß-N-acetylglucosaminase. O-GlcNAcylation of RIPK3 suppresses phosphorylation of RIPK3 and its interaction with RIPK1. Moreover, increased O-GlcNAcylation ameliorated AD pathology, including Aß burden, neuronal loss, neuroinflammation, and damaged mitochondria and recovered the M2 phenotype and phagocytic activity of microglia. Thus, our data establish the influence of O-GlcNAcylation on Aß accumulation and neurodegeneration, suggesting O-GlcNAcylation-based treatments as potential interventions for AD.

Comparison of thymidine phosphorylase expression and prognostic factors in gallbladder and bile duct cancer.

BACKGROUND: Biliary tract cancers have limitations in information about different location-related pathogenesis and clinico-pathological characteristics. The goal of this study was to investigate anatomical site-related similarities and differences in biliary tract cancers and to assess the expression and clinical significance of functional proteins such as p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1. METHODS: One hundred and sixty-one patients with biliary tract adenocarcinomas, who underwent curative or palliative surgery in a single institution between October 1994 and December 2003 were evaluated, retrospectively. The level of protein expression of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 was assessed by immunohistochemistry. RESULTS: With respect to clinico-pathological characteristics, gallbladder cancer was more frequent in women, and bile duct cancer was more common in men. Perineural invasion was more common in bile duct cancer. Recurrence as a distant metastasis was more common in gallbladder cancer. Immunohistochemical analysis revealed that thymidine phosphorylase expression was significantly higher in gallbladder cancer than in bile duct cancer. Positive thymidine phosphorylase and p53 staining were associated with an advanced stage. Differentiation, vascular invasion, perineural invasion, lymphatic invasion, lymph node metastasis, and TNM stage independently predicted poor prognosis in biliary tract cancer. These correlations were seen more clearly in gallbladder cancer. The immunohistochemical staining patterns of p53, cyclin D1, survivin, thymidine phosphorylase, and ERCC1 showed no prognostic significance in biliary tract cancers. CONCLUSIONS: We concluded that gallbladder and bile duct cancers are considered to be separate diseases with different clinico-pathological characteristics and prognostic factors. In addition, we hypothesize that high expression of thymidine phosphorylase by gallbladder cancer results in a higher response rate to capecitabine by gallbladder cancer than bile duct cancer.

Variation in the formation of persister cells against meropenem in Klebsiella pneumoniae bacteremia and analysis of its clinical features.

We investigated variations in the rate of persister cell formation against meropenem in 68 Klebsiella pneumoniae isolates from blood. The persister cell formation rates varied markedly but were not significantly different between the patient survival group and death group at 30 days. In addition, they were not associated with the patients' underlying diseases. However, the isolates of CC15 and CC23 showed higher survival rates against 10× MIC of meropenem than CC11. The survival rate of persister cells was less for amikacin and colistin than that for ciprofloxacin. When combinations of meropenem and other antibiotics were administered, persister formation rates decreased compared with those against only meropenem. However, no synergistic effect to remove persister cells was observed. Further investigation is needed to understand persister cell formation in K. pneumoniae with respect to the mechanism involved and clinical implications and that diverse strategies should be explored to remove persister cells.

Transition of colistin dependence into colistin resistance in Acinetobacter baumannii.

We recently demonstrated a high rate of colistin dependence in Acinetobacter baumannii isolates exposed to colistin in vitro. In the present study, we obtained a colistin-resistant (H08-391R) and colistin-dependent mutant (H08-391D) from a colistin-susceptible parental strain (H08-391). We found that the colistin-dependent mutant converted into a stable colistin-resistant mutant (H08-391D-R) in vitro after four serial passages without colistin. H08-391D and H08-391D-R were both found to harbor defective lipid A, as indicated by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry analysis. Additionally, both contained an ISAba1 insertion in lpxC, which encodes a lipid A biosynthetic enzyme. Further, membrane potential measurements using the fluorescent dye 3,3'-diethyloxacarbocyanine iodide (DiOC2[3]) showed that the membrane potential of H08-391D and H08-391D-R was significantly decreased as compared to that of the parental strain, H08-391. Moreover, these mutant strains exhibited increased susceptibilities to antibiotics other than colistin, which may be attributed to their outer membrane fragility. Such phenomena were identified in other A. baumannii strains (H06-855 and its derivatives). Taken together, our study reveals that the colistin-dependent phenotype is a transient phenotype that allows A. baumannii to survive under colistin pressure, and can transition to the extremely resistant phenotype, even in an antibiotic-free environment.

Colistin resistance in Pseudomonas aeruginosa that is not linked to arnB.

PURPOSE: It is known that the arnB (or pmrH) gene encoding uridine 5'-(beta-1-threo-pentapyranosyl-4-ulose diphosphate) aminotransferase plays a critical role in colistin resistance in Pseudomonas aeruginosa through the addition of 4-amino-4-deoxy-l-arabinose (l-Ara4N) to lipid A. In this study, we attempted to obtain a colistin-resistant mutant from an arnB-deleted mutant through exposure to colistin. METHODOLOGY: We constructed an arnB deletion mutant (P5ΔarnB :: nptIII) from a colistin-susceptible strain (P5) by allelic replacement mutagenesis, and colistin-resistant mutants were selected in vitro using P5 and P5ΔarnB :: nptIII. The growth rate, lipid A structure, biofilm-forming activity and cell viability in diverse stressful conditions (osmotic, oxidative, acidic and heat stress) were investigated. Expression of phoP, pmrA, parR, and cprR was evaluated by qRT-PCR. RESULTS: An arnB deletion mutant was shown to develop colistin resistance through the addition of l-Ara4N to lipid A, despite a low survival rate (over 1000-fold lower than that of the wild-type strain) in the media with colistin. Two colistin-resistant mutants showed higher survival rates than colistin-susceptible strains against 5 % NaCl. In the presence of acidic and heat stress, P5ΔarnB :: nptIII-CstR exhibited higher survival rates during conditions of 1 % HCl and 42 °C than the other strains. Both phoP and pmrA genes were overexpressed significantly in both colistin-resistant mutants, but parR and cprR genes were not. CONCLUSION: We revealed that colistin resistance could be developed despite arnB deletion in P. aeruginosa through the addition of l-Ara4N to lipid A, which was accompanied by diverse physiological changes.

Evolved resistance to colistin and its loss due to genetic reversion in Pseudomonas aeruginosa.

The increased reliance on colistin for treating multidrug-resistant Gram-negative bacterial infections has resulted in the emergence of colistin-resistant Pseudomonas aeruginosa. We attempted to identify genetic contributors to colistin resistance in vitro evolved isogenic colistin-resistant and -susceptible strains of two P. aeruginosa lineages (P5 and P155). Their evolutionary paths to acquisition and loss of colistin resistance were also tracked. Comparative genomic analysis revealed 13 and five colistin resistance determinants in the P5 and P155 lineages, respectively. Lipid A in colistin-resistant mutants was modified through the addition of 4-amino-L-arabinose; this modification was absent in colistin-susceptible revertant strains. Many amino acid substitutions that emerged during the acquisition of colistin resistance were reversed in colistin-susceptible revertants. We demonstrated that evolved colistin resistance in P. aeruginosa was mediated by a complicated regulatory network that likely emerges through diverse genetic alterations. Colistin-resistant P. aeruginosa became susceptible to the colistin upon its withdrawal because of genetic reversion. The mechanisms through which P. aeruginosa acquires and loses colistin resistance have implications on the treatment options that can be applied against P. aeruginosa infections, with respect to improving bactericidal efficacy and preventing further resistance to antibiotics.

Comparison of the Virulence-Associated Phenotypes of Five Species of Acinetobacter baumannii Complex.

In this study, we compared the virulence-associated factors of Acinetobacter baumannii complex species. Sixty-three isolates of five A. baumannii complex species, including 19 A. baumannii, 15 A. nosocomialis, 13 A. seifertii, 13 A. pittii, and 3 A. calcoaceticus isolates, were included in this study. For all isolates, biofilm formation, A549 cell adherence, resistance to normal human serum, and motility were evaluated. A. baumannii complex isolates showed diversity in biofilm formation, A549 cell adherence, and serum resistance, and no strong positive relationships among these virulence characteristics. However, A. seifertii showed relatively consistent virulence-associated phenotypes. In addition, A. baumannii clone ST110 exhibited consistently high virulence-associated phenotypes. Motility was observed in seven isolates, and all four A. baumannii ST110 isolates showed twitching motility. Although some inconsistencies in virulence-associated phenotypes were seen, high virulence characteristics were observed in A. seifertii, which has been mainly reported in Korea and shows high rates of colistin resistance.