Global stress response in Pseudomonas aeruginosa upon malonate utilization.

Versatility in carbon source utilization assists Pseudomonas aeruginosa in its adaptation to various niches. Recently, we characterized the role of malonate, an understudied carbon source, in quorum sensing regulation, antibiotic resistance, and virulence factor production in P. aeruginosa . These results indicate that global responses to malonate metabolism remain to be uncovered. We leveraged a publicly available metabolomic dataset on human airway and found malonate to be as abundant as glycerol, a common airway metabolite and carbon source for P. aeruginosa . Here, we explored and compared adaptations of P. aeruginosa UCBPP-PA14 (PA14) in response to malonate or glycerol as a sole carbon source using transcriptomics and phenotypic assays. Malonate utilization activated glyoxylate and methylcitrate cycles and induced several stress responses, including oxidative, anaerobic, and metal stress responses associated with increases in intracellular aluminum and strontium. Some induced genes were required for optimal growth of P. aeruginosa in malonate. To assess the conservation of malonate-associated responses among P. aeruginosa strains, we compared our findings in strain PA14 with other lab strains and cystic fibrosis isolates of P. aeruginosa . Most strains grew on malonate as a sole carbon source as efficiently as or better than glycerol. While not all responses to malonate were conserved among strains, formation of biomineralized biofilm-like aggregates, increased tolerance to kanamycin, and increased susceptibility to norfloxacin were the most frequently observed phenotypes. Our findings reveal global remodeling of P. aeruginosa gene expression during its growth on malonate as a sole carbon source that is accompanied by several important phenotypic changes. These findings add to accumulating literature highlighting the role of different carbon sources in the physiology of P. aeruginosa and its niche adaptation. Importance: Pseudomonas aeruginosa is a notorious pathogen that causes local and systemic infections in immunocompromised individuals. Different carbon sources can uniquely modulate metabolic and virulence pathways in P. aeruginosa , highlighting the importance of the environment that the pathogen occupies. In this work, we used a combination of transcriptomic analysis and phenotypic assays to determine how malonate utilization impacts P. aeruginosa, as recent evidence indicates this carbon source may be relevant to certain niches associated within the human host. We found that malonate utilization can induce global stress responses, alter metabolic circuits, and influence various phenotypes of P. aeruginosa that could influence host colonization. Investigating the metabolism of malonate provides insight into P. aeruginosa adaptations to specific niches where this substrate is abundant, and how it can be leveraged in the development of much-needed antimicrobial agents or identification of new therapeutic targets of this difficult-to-eradicate pathogen.

Under conditions closely mimicking vaginal fluid, Lactobacillus jensenii strain 62B produces a bacteriocin-like inhibitory substance that targets and eliminates Gardnerella species.

Within the vaginal ecosystem, lactobacilli and Gardnerella spp. likely interact and influence each other's growth, yet the details of this interaction are not clearly defined. Using medium simulating vaginal fluid and a two-chamber co-culturing system to prevent cell-to-cell contact between the bacteria, we examined the possibility that Lactobacillus jensenii 62B (Lj 62B) and/or G. piotii (Gp) JCP8151B produce extracellular factors through which they influence each other's viability. By 24 h post-inoculation (hpi) in the co-culture system and under conditions similar to the vaginal environment - pH 5.0, 37 °C, and 5% CO2, Lj 62B viability was not affected but Gp JCP8151B had been eliminated. Cell-free supernatant harvested from Lj 62B cultures (Lj-CFS) at 20 hpi, but not 16 hpi, also eliminated Gp JCP8151B growth. Neither lactic acid nor H2O2 production by Lj 62B was responsible for this effect. The Lj-CFS did not affect viability of three species of lactobacilli or eight species of Gram-positive and Gram-negative uropathogens but eliminated viability of eight different strains of Gardnerella spp. Activity of the inhibitory factor within Lj-CFS was abolished by protease treatment and reduced by heat treatment suggesting it is most likely a bacteriocin-like protein; fractionation revealed that the factor has a molecular weight within the 10-30 kDa range. These results suggest that, in medium mimicking vaginal fluid and growth conditions similar to the vaginal environment, Lj 62B produces a potential bacteriocin-like inhibitory substance (Lj-BLIS) that clearly targets Gardnerella spp. strains. Once fully characterized, Lj-BLIS may be a potential treatment for Gardnerella-related BV that does not alter the vaginal microflora.

Glycogen availability and pH variation in a medium simulating vaginal fluid influence the growth of vaginal Lactobacillus species and Gardnerella vaginalis.

BACKGROUND: Glycogen metabolism by Lactobacillus spp. that dominate the healthy vaginal microbiome contributes to a low vaginal pH (3.5-4.5). During bacterial vaginosis (BV), strict and facultative anaerobes including Gardnerella vaginalis become predominant, leading to an increase in the vaginal pH (> 4.5). BV enhances the risk of obstetrical complications, acquisition of sexually transmitted infections, and cervical cancer. Factors critical for the maintenance of the healthy vaginal microbiome or the transition to the BV microbiome are not well defined. Vaginal pH may affect glycogen metabolism by the vaginal microflora, thus influencing the shift in the vaginal microbiome. RESULTS: The medium simulating vaginal fluid (MSVF) supported growth of L. jensenii 62G, L. gasseri 63 AM, and L. crispatus JV-V01, and G. vaginalis JCP8151A at specific initial pH conditions for 30 d. L. jensenii at all three starting pH levels (pH 4.0, 4.5, and 5.0), G. vaginalis at pH 4.5 and 5.0, and L. gasseri at pH 5.0 exhibited the long-term stationary phase when grown in MSVF. L. gasseri at pH 4.5 and L. crispatus at pH 5.0 displayed an extended lag phase over 30 d suggesting inefficient glycogen metabolism. Glycogen was essential for the growth of L. jensenii, L. crispatus, and G. vaginalis; only L. gasseri was able to survive in MSVF without glycogen, and only at pH 5.0, where it used glucose. All four species were able to survive for 15 d in MSVF with half the glycogen content but only at specific starting pH levels - pH 4.5 and 5.0 for L. jensenii, L. gasseri, and G. vaginalis and pH 5.0 for L. crispatus. CONCLUSIONS: These results suggest that variations in the vaginal pH critically influence the colonization of the vaginal tract by lactobacilli and G. vaginalis JCP8151A by affecting their ability to metabolize glycogen. Further, we found that L. jensenii 62G is capable of glycogen metabolism over a broader pH range (4.0-5.0) while L. crispatus JV-V01 glycogen utilization is pH sensitive (only functional at pH 5.0). Finally, our results showed that G. vaginalis JCP8151A can colonize the vaginal tract for an extended period as long as the pH remains at 4.5 or above.

Anti-Pseudomonas aeruginosa Vaccines and Therapies: An Assessment of Clinical Trials.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that causes high morbidity and mortality in cystic fibrosis (CF) and immunocompromised patients, including patients with ventilator-associated pneumonia (VAP), severely burned patients, and patients with surgical wounds. Due to the intrinsic and extrinsic antibiotic resistance mechanisms, the ability to produce several cell-associated and extracellular virulence factors, and the capacity to adapt to several environmental conditions, eradicating P. aeruginosa within infected patients is difficult. Pseudomonas aeruginosa is one of the six multi-drug-resistant pathogens (ESKAPE) considered by the World Health Organization (WHO) as an entire group for which the development of novel antibiotics is urgently needed. In the United States (US) and within the last several years, P. aeruginosa caused 27% of deaths and approximately USD 767 million annually in health-care costs. Several P. aeruginosa therapies, including new antimicrobial agents, derivatives of existing antibiotics, novel antimicrobial agents such as bacteriophages and their chelators, potential vaccines targeting specific virulence factors, and immunotherapies have been developed. Within the last 2-3 decades, the efficacy of these different treatments was tested in clinical and preclinical trials. Despite these trials, no P. aeruginosa treatment is currently approved or available. In this review, we examined several of these clinicals, specifically those designed to combat P. aeruginosa infections in CF patients, patients with P. aeruginosa VAP, and P. aeruginosa-infected burn patients.

Urinary Catheters Coated with a Novel Biofilm Preventative Agent Inhibit Biofilm Development by Diverse Bacterial Uropathogens.

Despite the implementation of stringent guidelines for the prevention of catheter-associated (CA) urinary tract infection (UTI), CAUTI remains one of the most common health care-related infections. We previously showed that an antimicrobial/antibiofilm agent inhibited biofilm development by Gram-positive and Gram-negative bacterial pathogens isolated from human infections. In this study, we examined the ability of a novel biofilm preventative agent (BPA) coating on silicone urinary catheters to inhibit biofilm formation on the catheters by six different bacterial pathogens isolated from UTIs: three Escherichia coli strains, representative of the most common bacterium isolated from UTI; one Enterobacter cloacae, a multidrug-resistant isolate; one Pseudomonas aeruginosa, common among patients with long-term catheterization; and one isolate of methicillin-resistant Staphylococcus aureus, as both a Gram-positive and a resistant organism. First, we tested the ability of these strains to form biofilms on urinary catheters made of red rubber, polyvinyl chloride (PVC), and silicone using the microtiter plate biofilm assay. When grown in artificial urine medium, which closely mimics human urine, all tested isolates formed considerable biofilms on all three catheter materials. As the biofilm biomass formed on silicone catheters was 0.5 to 1.6 logs less than that formed on rubber or PVC, respectively, we then coated the silicone catheters with BPA (benzalkonium chloride, polyacrylic acid, and glutaraldehyde), and tested the ability of the coated catheters to further inhibit biofilm development by these uropathogens. Compared with the uncoated silicone catheters, BPA-coated catheters completely prevented biofilm development by all the uropathogens, except P. aeruginosa, which showed no reduction in biofilm biomass. To explore the reason for P. aeruginosa resistance to the BPA coating, we utilized two specific lipopolysaccharide (LPS) mutants. In contrast to their parent strain, the two mutants failed to form biofilms on the BPA-coated catheters, which suggests that the composition of P. aeruginosa LPS plays a role in the resistance of wild-type P. aeruginosa to the BPA coating. Together, our results suggest that, except for P. aeruginosa, BPA-coated silicone catheters may prevent biofilm formation by both Gram-negative and Gram-positive uropathogens.

The influence of a biofilm-dispersing wound gel on the wound healing process.

Topical antimicrobials that reduce the bacterial bioburden within a chronically-infected wound may have helpful or harmful effects on the healing process. We used murine models of full-thickness skin wounds to determine the effects of the novel biofilm-dispersing wound gel (BDWG) and its gel base on the healing of uninfected wounds. The rate of wound closure over 19 days was comparable among the BDWG-treated (BT) wounds and the controls. Compared with the controls, histology of the BT wounds showed formation of a stable blood clot at day 1, more neovascularisation and reepithelialisation at day 3, and more organised healing at day 7. Fluorescence-activated cell sorting analysis showed a lower percentage of neutrophils in wounded tissues of the BT group at days 1 and 3, and significantly more M2 macrophages at day 3. Levels of proinflammatory cytokines and chemokines were increased over the uninjured baseline within the wounds of all treatment groups but the levels were significantly lower in the BT group at day 1, modulating the inflammatory response. Our results suggest that BDWG does not interfere with the wound healing process and may enhance it by lowering inflammation and allowing transition to the proliferative stage of wound healing by day 3.

Dietary supplementation of gingerols- and shogaols-enriched ginger root extract attenuate pain-associated behaviors while modulating gut microbiota and metabolites in rats with spinal nerve ligation.

Neuroinflammation is a central factor in neuropathic pain (NP). Ginger is a promising bioactive compound in NP management due to its anti-inflammatory property. Emerging evidence suggests that gut microbiome and gut-derived metabolites play a key role in NP. We evaluated the effects of two ginger root extracts rich in gingerols (GEG) and shogaols (SEG) on pain sensitivity, anxiety-like behaviors, circulating cell-free mitochondrial DNA (ccf-mtDNA), gut microbiome composition, and fecal metabolites in rats with NP. Sixteen male rats were divided into four groups: sham, spinal nerve ligation (SNL), SNL+0.75%GEG in diet, and SNL+0.75%SEG in diet groups for 30 days. Compared to SNL group, both SNL+GEG and SNL+SEG groups showed a significant reduction in pain- and anxiety-like behaviors, and ccf-mtDNA level. Relative to the SNL group, both SNL+GEG and SNL+SEG groups increased the relative abundance of Lactococcus, Sellimonas, Blautia, Erysipelatoclostridiaceae, and Anaerovoracaceae, but decreased that of Prevotellaceae UCG-001, Rikenellaceae RC9 gut group, Mucispirillum and Desulfovibrio, Desulfovibrio, Anaerofilum, Eubacterium siraeum group, RF39, UCG-005, Lachnospiraceae NK4A136 group, Acetatifactor, Eubacterium ruminantium group, Clostridia UCG-014, and an uncultured Anaerovoracaceae. GEG and SEG had differential effects on gut-derived metabolites. Compared to SNL group, SNL+GEG group had higher level of 1'-acetoxychavicol acetate, (4E)-1,7-Bis(4-hydroxyphenyl)-4-hepten-3-one, NP-000629, 7,8-Dimethoxy-3-(2-methyl-3-buten-2-yl)-2H-chromen-2-one, 3-{[4-(2-Pyrimidinyl)piperazino]carbonyl}-2-pyrazinecarboxylic acid, 920863, and (1R,3R,7R,13S)-13-Methyl-6-methylene-4,14,16-trioxatetracyclo[11.2.1.0∼1,10∼.0∼3,7∼]hexadec-9-en-5-one, while SNL+SEG group had higher level for (±)-5-[(tert-Butylamino)-2'-hydroxypropoxy]-1_2_3_4-tetrahydro-1-naphthol and dehydroepiandrosteronesulfate. In conclusion, ginger is a promising functional food in the management of NP, and further investigations are necessary to assess the role of ginger on gut-brain axis in pain management.

Recombinant R2-pyocin cream is effective in treating Pseudomonas aeruginosa-infected wounds.

Pseudomonas aeruginosa, a gram-negative opportunistic pathogen, is one of the major species isolated from infected chronic wounds. The multidrug resistance exhibited by P. aeruginosa and its ability to form biofilms that are difficult to eradicate, along with the rising cost of producing new antibiotics, has necessitated the search for alternatives to standard antibiotics. Pyocins are antimicrobial compounds produced by P. aeruginosa that protect themselves from their competitors. We synthesized and purified recombinant P. aeruginosa R2 pyocin and used it in an aqueous solution (rR2P) or formulated in polyethylene glycol (rR2PC) to treat P. aeruginosa-infected wounds. Clinical strains of P. aeruginosa were found to be sensitive (completely), partially sensitive, or resistant to rR2P. In the in vitro biofilm model, rR2P inhibited biofilm development by rR2P-sensitive isolates, while rR2PC eliminated partial biofilms formed by these strains in an in vitro wound biofilm model. In the murine model of excision wounds, and at 24 h post-infection, rR2PC application significantly reduced the bioburden of the clinical isolate BPI86. Application of rR2PC containing two glycoside hydrolase antibiofilm agents eliminated BPI86 from infected wounds. These results suggest that the topical application of rR2PC is an effective therapy for treating wounds infected with R2P-senstive P. aeruginosa strains.

Beneficial effect of dietary geranylgeraniol on glucose homeostasis and bone microstructure in obese mice is associated with suppression of proinflammation and modification of gut microbiome.

Geranylgeraniol (GGOH) is found in edible oils such as olive, linseed, and sunflower oils, which have favorable metabolic effects. However, it is unknown whether these physiological benefits are mediated through the gut microbiome. Thus, the purpose of this study was to test the hypothesis that GGOH supplementation would improve glucose homeostasis and benefit the bone microstructure in obese mice through suppression of inflammation and modification of gut microbiota composition. Thirty-six male C57BL/6J mice were divided into 3 groups: a low-fat diet, a high-fat diet (HFD), and an HFD supplemented with 800 mg GGOH/kg diet (GG) for 14 weeks. Glucose and insulin tolerance tests were measured at baseline and end of study. The concentrations of adipokine cytokines (resistin, leptin, monocyte chemoattractant protein-1, interleukin-6) were measured via ELISA. Bone microarchitecture and quality were measured by micro-CT. Microbiome analysis was performed using 16S rRNA amplicon sequencing on cecal content. Relative to the HFD group, the GG group: (1) improved glucose tolerance and insulin sensitivity; (2) reduced production of pro-inflammatory adipokines, (3) increased serum procollagen I intact N-terminal propeptide (bone formation marker) concentrations, while decreasing serum collagen type 1 cross-linked C-telopeptide (bone resorption marker) levels, and (4) increased stiffness at both femur and LV-4 and cortical thickness at femoral midshaft. Compared to the HFD group, the GG group had an increased abundance of Butyricicoccus pullicaecorum and decreased Dorea longicatena in the cecal microbiome. Collectively, GGOH improves glucose homeostasis and bone microstructure in obese mice, probably via suppression of pro-inflammation and modification of microbiome composition.

Malonate utilization by Pseudomonas aeruginosa affects quorum-sensing and virulence and leads to formation of mineralized biofilm-like structures.

Pseudomonas aeruginosa is an opportunistic pathogen that uses malonate among its many carbon sources. We recently reported that, when grown in blood from trauma patients, P. aeruginosa expression of malonate utilization genes was upregulated. In this study, we explored the role of malonate utilization and its contribution to P. aeruginosa virulence. We grew P. aeruginosa strain PA14 in M9 minimal medium containing malonate (MM9) or glycerol (GM9) as a sole carbon source and assessed the effect of the growth on quorum sensing, virulence factors, and antibiotic resistance. Growth of PA14 in MM9, compared to GM9, reduced the production of elastases, rhamnolipids, and pyoverdine; enhanced the production of pyocyanin and catalase; and increased its sensitivity to norfloxacin. Growth in MM9 decreased extracellular levels of N-acylhomoserine lactone autoinducers, an effect likely associated with increased pH of the culture medium; but had little effect on extracellular levels of PQS. At 18 hr of growth in MM9, PA14 formed biofilm-like structures or aggregates that were associated with biomineralization, which was related to increased pH of the culture medium. These results suggest that malonate significantly impacts P. aeruginosa pathogenesis by influencing the quorum sensing systems, the production of virulence factors, biofilm formation, and antibiotic resistance.

During bacteremia, Pseudomonas aeruginosa PAO1 adapts by altering the expression of numerous virulence genes including those involved in quorum sensing.

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that produces numerous virulence factors and causes serious infections in trauma patients and patients with severe burns. We previously showed that the growth of P. aeruginosa in blood from severely burned or trauma patients altered the expression of numerous genes. However, the specific influence of whole blood from healthy volunteers on P. aeruginosa gene expression is not known. Transcriptome analysis of P. aeruginosa grown for 4 h in blood from healthy volunteers compared to that when grown in laboratory medium revealed that the expression of 1085 genes was significantly altered. Quorum sensing (QS), QS-related, and pyochelin synthesis genes were downregulated, while genes of the type III secretion system and those for pyoverdine synthesis were upregulated. The observed effect on the QS and QS-related genes was shown to reside within serum fraction: growth of PAO1 in the presence of 10% human serum from healthy volunteers significantly reduced the expression of QS and QS-regulated genes at 2 and 4 h of growth but significantly enhanced their expression at 8 h. Additionally, the production of QS-regulated virulence factors, including LasA and pyocyanin, was also influenced by the presence of human serum. Serum fractionation experiments revealed that part of the observed effect resides within the serum fraction containing <10-kDa proteins. Growth in serum reduced the production of many PAO1 outer membrane proteins but enhanced the production of others including OprF, a protein previously shown to play a role in the regulation of QS gene expression. These results suggest that factor(s) within human serum: 1) impact P. aeruginosa pathogenesis by influencing the expression of different genes; 2) differentially regulate the expression of QS and QS-related genes in a growth phase- or time-dependent mechanism; and 3) manipulate the production of P. aeruginosa outer membrane proteins.

Osteoprotective effect of green tea polyphenols and annatto-extracted tocotrienol in obese mice is associated with enhanced microbiome vitamin K2 biosynthetic pathways.

The role of the gut microbiome in bone health has received significant attention in the past decade. We investigated the effects of green tea polyphenols (GTP) and annatto-extracted tocotrienols (AT) on bone properties and gut microbiome in obese mice. Male mice were assigned to a two (no AT vs. 400 mg/kg diet AT) × two (no GTP vs. 0.5% w/v GTP) factorial design, namely control, G, T, and G+T group respectively, for 14 weeks. The 4th lumbar vertebra (LV-4) and femur were harvested for bone microstructural analysis using µ-CT. Microbiome analysis using 16S rRNA gene sequencing of cecal feces was performed. AT increased bone volume at distal femur. GTP increased serum procollagen type 1 N-terminal propeptide concentration, bone volume at the distal femur and the LV-4, and trabecular number at distal femur; whereas GTP decreased trabecular separation at distal femur. Interactions between GTP and AT were observed in serum C-terminal telopeptide of type I collagen level (control>G=T=G+T) as well as the cortical bone area (control

Hereditary hemochromatosis promotes colitis and colon cancer and causes bacterial dysbiosis in mice.

Hereditary hemochromatosis (HH), an iron-overload disease, is a prevalent genetic disorder. As excess iron causes a multitude of metabolic disturbances, we postulated that iron overload in HH disrupts colonic homeostasis and colon-microbiome interaction and exacerbates the development and progression of colonic inflammation and colon cancer. To test this hypothesis, we examined the progression and severity of colitis and colon cancer in a mouse model of HH (Hfe-/-), and evaluated the potential contributing factors. We found that experimentally induced colitis and colon cancer progressed more robustly in Hfe-/- mice than in wild-type mice. The underlying causes were multifactorial. Hfe-/- colons were leakier with lower proliferation capacity of crypt cells, which impaired wound healing and amplified inflammation-driven tissue injury. The host/microflora axis was also disrupted. Sequencing of fecal 16S RNA revealed profound changes in the colonic microbiome in Hfe-/- mice in favor of the pathogenic bacteria belonging to phyla Proteobacteria and TM7. There was an increased number of bacteria adhered onto the mucosal surface of the colonic epithelium in Hfe-/- mice than in wild-type mice. Furthermore, the expression of innate antimicrobial peptides, the first-line of defense against bacteria, was lower in Hfe-/- mouse colon than in wild-type mouse colon; the release of pro-inflammatory cytokines upon inflammatory stimuli was also greater in Hfe-/- mouse colon than in wild-type mouse colon. These data provide evidence that excess iron accumulation in colonic tissue as happens in HH promotes colitis and colon cancer, accompanied with bacterial dysbiosis and loss of function of the intestinal/colonic barrier.

Metabolic benefits of annatto-extracted tocotrienol on glucose homeostasis, inflammation, and gut microbiome.

Emerging evidence suggests that the gut microbiome plays an important role in the pathophysiology of both obesity and type 2 diabetes mellitus. We previously reported that dietary annatto-extracted tocotrienol exerts beneficial effects by modulating inflammatory responses in mice fed a high-fat diet (HFD). The purpose of this study was to test the hypothesis that tocotrienol supplementation when combined with an HFD would result in an altered gut microbiota composition. For 14 weeks, forty-eight male C57BL/6J mice were assigned to 4 groups-low-fat diet, HFD, HFD supplemented with annatto-extracted tocotrienol at 800 mg/kg diet (AT), and HFD supplemented with metformin at 200 mg/kg diet. Glucose homeostasis was assessed by glucose and insulin tolerance tests, serum and pancreas insulin levels, and histological assessments of insulin and glucagon in pancreatic tissue. The concentrations of adipokines were measured in white adipose tissues. For the gut microbiome analysis, cecal content was collected, DNA was extracted, and 16S rRNA gene sequencing was performed. AT supplementation improved glucose homeostasis and lowered resistin, leptin, and interleukin-6 levels in white adipose tissue. Relative to the HFD group, AT-supplemented mice showed a decrease in the Firmicutes to Bacteroidetes ratio and had a lower abundance of Ruminococcus lactaris, Dorea longicatena, and Lachnospiraceae family. The relative abundance of Akkermansia muciniphila was increased in the AT group compared to the low-fat diet group. The association between the metabolic improvements and the identified bacterial taxa suggests a potential metabolic modulation caused by AT supplementation through the gut microbiota composition in mice fed an HFD.

New markers for sepsis caused by Pseudomonas aeruginosa during burn infection.

INTRODUCTION: Sepsis is a leading cause of mortality in burn patients. One of the major causes of sepsis in burn patients is Pseudomonas aeruginosa. We hypothesized that during dissemination from infected burn wounds and subsequent sepsis, P. aeruginosa affects the metabolome of the blood resulting in changes to specific metabolites that would serve as biomarkers for early diagnosis of sepsis caused by P. aeruginosa. OBJECTIVES: To identify specific biomarkers in the blood after sepsis caused by P. aeruginosa infection of burns. METHODS: Gas chromatography with time-of-flight mass spectrometry was used to compare the serum metabolome of mice that were thermally injured and infected with P. aeruginosa (B-I) to that of mice that were neither injured nor infected, mice that were injured but not infected, and mice that were infected but not injured. RESULTS: Serum levels of 19 metabolites were significantly increased in the B-I group compared to controls while levels of eight metabolites were significantly decreased. Thymidine, thymine, uridine, and uracil (related to pyrimidine metabolism), malate and succinate (a possible sign of imbalance in the tricarboxylic acid cycle), 5-oxoproline (related to glutamine and glutathione metabolism), and trans-4-hydroxyproline (a major component of the protein collagen) were increased. Products of amino acid metabolism were significantly decreased in the B-I group, including methionine, tyrosine, indole-3-acetate, and indole-3-propionate. CONCLUSION: In all, 26 metabolites were identified, including a unique combination of five metabolites (trans-4-hydroxyproline, 5-oxoproline, glycerol-3-galactoside, indole-3-acetate, and indole-3-propionate) that could serve as a set of biomarkers for early diagnosis of sepsis caused by P. aeruginosa in burn patients.

Application of Lactobacillus gasseri 63 AM supernatant to Pseudomonas aeruginosa-infected wounds prevents sepsis in murine models of thermal injury and dorsal excision.

Introduction. Severely burned patients are susceptible to bacterial infection within their burn wounds, which frequently leads to sepsis, multiple organ failure and death. The opportunistic pathogen Pseudomonas aeruginosa, an organism inherently resistant to multiple antibiotics, is a common cause of sepsis in these patients.Aim. Development of a topical treatment unrelated to conventional antibiotics is essential for prevention of P. aeruginosa infection and sepsis, leading to a role for the direct application of probiotics or their by-products.Methodology. We examined the effectiveness of 20× concentrated supernatant from Lactobacillus gasseri strain 63 AM (LgCS) grown in de Man, Rogosa and Sharpe broth in inhibiting P. aeruginosa biofilms in vitro, as well as in reducing wound bioburden and P. aeruginosa sepsis in vivo.Results. LgCS inhibited the growth of P. aeruginosa strain PAO1, prevented its biofilm development and eliminated partially developed PAO1 biofilms. In the murine model of thermal injury, a single injection of LgCS following injury and PAO1 infection reduced mortality to 0 % and prevented systemic spread (sepsis). Furthermore, a second injection of LgCS 24 h after the first eliminated PAO1 from the wound. In the murine dorsal excision infection model, either LgCS or ceftazidime treatment of the PAO1-infected wound significantly reduced the mortality rate among infected mice, while combining LgCS with ceftazidime eliminated mortality.Conclusion. These results suggest the potential of LgCS in preventing sepsis from P. aeruginosa infection in severely burned and other immunocompromised patients.

Pseudomonas aeruginosa Alters Its Transcriptome Related to Carbon Metabolism and Virulence as a Possible Survival Strategy in Blood from Trauma Patients.

Trauma patients (TPs) are highly susceptible to infections, which often lead to sepsis. Among the numerous causative agents, Pseudomonas aeruginosa is especially important, as P. aeruginosa sepsis is often fatal. Understanding the mechanism of its pathogenesis in bloodstream infections is imperative; however, this mechanism has not been previously described. To examine the effect of trauma-induced changes in blood on the expression of P. aeruginosa genes, we grew strain UCBPP-PA14 (PA14) in blood samples from eight TPs and seven healthy volunteers (HVs). Compared with its growth in blood from HVs, the growth of PA14 in blood from TPs significantly altered the expression of 285 genes. Genes whose expression was significantly increased were related to carbon metabolism, especially malonate utilization and mannitol uptake, and efflux of heavy metals. Genes whose expression was significantly reduced included genes of the type VI secretion system, genes related to uptake and metabolism of amino acids, and genes related to biosynthesis and transport of the siderophores pyoverdine and pyochelin. These results suggest that during systemic infection in trauma patients, and to adapt to the trauma-induced changes in blood, P. aeruginosa adjusts positively and negatively the expression of numerous genes related to carbon metabolism and virulence, respectively. IMPORTANCE While a considerable body of knowledge regarding sepsis in trauma patients is available, the potential influence of trauma-induced changes in the blood of these patients on the pathogenesis of Pseudomonas aeruginosa is basically an unexplored area. Rather than using standard laboratory media, we grew P. aeruginosa in whole blood from either healthy volunteers or trauma patients. The specific changes in the P. aeruginosa transcriptome in response to growth in blood from trauma patients reflect the adaptation of this organism to the bloodstream environment. This knowledge is vital for understanding the strategies this pathogen uses to adapt and survive within the host during systemic infection. Such information will help researchers and clinicians to develop new approaches for treatment of sepsis caused by P. aeruginosa in trauma patients, especially in terms of recognizing the effects of specific therapies (e.g., iron, zinc, or mannitol) on the organism. Further, this information can most likely be extrapolated to all patients with P. aeruginosa septicemia.

Prospective observational case series evaluating middle ear fluid and tympanostomy tubes through pyrosequencing.

OBJECTIVE: One of the most common sequelae of tympanostomy tube (TT) placement is post-tympanostomy tube otorrhea (PTTO). Granulation tissue formation has been reported in 5-13.8% of patients with TT placement. The purpose of this study is to determine the biodistribution of microorganisms on TTs and middle ear fluid obtained from patients with PTTO. METHODS: This was a prospective observational study of subjects (6-months-16-years) who underwent standard of care treatment for chronic PTTO. Data was collected on diagnosis, existence of antibiotic resistance, duration of tube placement, and evidence of recurrent infection and/or PTTO. TTs and middle ear fluid were subjected to pyrosequencing; additionally, ear fluid samples were sent for culture-based laboratory diagnostics. RESULTS: DNA-pyrosequencing analysis of bacteria from fluid and TTs of pediatric subjects with PTTO revealed a mixture of both aerobic and anaerobic populations. Retained tubes with minimal otorrhea revealed a predominance of Staphylococcus species, normal external auditory canal (EAC) microbiome, within middle ear fluid as well as on TTs. However, TTs with active mucopurulent otorrhea and granulation tissue unveiled prominence of Gram-negative facultative anaerobes such as Pseudomonas and Eikenella. Discrepancies in prominent bacteria were seen between standard culture-based techniques versus pyrosequencing. CONCLUSION: Retained tympanostomy tubes are colonized primarily with normal flora of the EAC. However, mucopurulent otorrhea associated with granulation tissue formation revealed a prominence of Gram-negative facultative anaerobes. Standard culture-based diagnostics may identify bacteria, which are not predominant species of infection. Future studies are necessary to assess the association of Gram-negative facultative anaerobes with granulation tissue formation.

Heparinase Is Essential for Pseudomonas aeruginosa Virulence during Thermal Injury and Infection.

The opportunistic pathogen Pseudomonas aeruginosa is a major cause of sepsis in severely burned patients. If it is not eradicated from the wound, it translocates to the bloodstream, causing sepsis, multiorgan failure, and death. We recently described the P. aeruginosa heparinase-encoding gene, hepP, whose expression was significantly enhanced when P. aeruginosa strain UCBPP_PA14 (PA14) was grown in whole blood from severely burned patients. Further analysis demonstrated that hepP contributed to the in vivo virulence of PA14 in the Caenorhabditis elegans model. In this study, we utilized the murine model of thermal injury to examine the contribution of hepP to the pathogenesis of P. aeruginosa during burn wound infection. Mutation of hepP reduced the rate of mortality from 100% for mice infected with PA14 to 7% for mice infected with PA14::hepP While comparable numbers of PA14 and PA14::hepP bacteria were recovered from infected skin, only PA14 was recovered from the livers and spleens of infected mice. Despite its inability to spread systemically, PA14::hepP formed perivascular cuffs around the blood vessels within the skin of the thermally injured/infected mice. Intraperitoneal inoculation of the thermally injured mice, bypassing the need for translocation, produced similar results. The rate of mortality for mice infected with PA14::hepP was 0%, whereas it was 66% for mice infected with PA14. As before, only PA14 was recovered from the livers and spleens of infected mice. These results suggest that hepP plays a crucial role in the pathogenesis of PA14 during burn wound infection, most likely by contributing to PA14 survival in the bloodstream of the thermally injured mouse during sepsis.

Isolation and characterization of HepP: a virulence-related Pseudomonas aeruginosa heparinase.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen that causes serious infections in immunocompromised hosts including severely burned patients. In burn patients, P. aeruginosa infection often leads to septic shock and death. Despite numerous studies, the influence of severe thermal injuries on the pathogenesis of P. aeruginosa during systemic infection is not known. Through RNA-seq analysis, we recently showed that the growth of P. aeruginosa strain UCBPP-PA14 (PA14) in whole blood obtained from severely burned patients significantly altered the expression of the PA14 transcriptome when compared with its growth in blood from healthy volunteers. The expression of PA14_23430 and the adjacent gene, PA14_23420, was enhanced by seven- to eightfold under these conditions. RESULTS: Quantitative real-time PCR analysis confirmed the enhancement of expression of both PA14_23420 and PA14_23430 by growth of PA14 in blood from severely burned patients. Computer analysis revealed that PA14_23430 (hepP) encodes a potential heparinase while PA14_23420 (zbdP) codes for a putative zinc-binding dehydrogenase. This analysis further suggested that the two genes form an operon with zbdP first. Presence of the operon was confirmed by RT-PCR experiments. We characterized hepP and its protein product HepP. hepP was cloned from PA14 by PCR and overexpressed in E. coli. The recombinant protein (rHepP) was purified using nickel column chromatography. Heparinase assays using commercially available heparinase as a positive control, revealed that rHepP exhibits heparinase activity. Mutation of hepP resulted in delay of pellicle formation at the air-liquid interface by PA14 under static growth conditions. Biofilm formation by PA14ΔhepP was also significantly reduced. In the Caenorhabditis elegans model of slow killing, mutation of hepP resulted in a significantly lower rate of killing than that of the parent strain PA14. CONCLUSIONS: Changes within the blood of severely burned patients significantly induced expression of hepP in PA14. The heparinase encoded by hepP is a potential virulence factor for PA14 as HepP influences pellicle formation as well as biofilm development by PA14 and the protein is required for full virulence in the C. elegans model of slow killing.