Abstract:To assess the contribution of ppGpp in antibiotic tolerance to quinolone in Pseudomonas aeruginosa, knockout mutants of the genes involved or linked with the stringent response, such as relA, spoT and dksA, were constructed and investigated for their antibiotic susceptibility to quinolones. The survival of the dksA and spoT mutants in the presence of 8 g/ml of ofloxacin and 1 g/ml of ciprofloxacin were shown to be approximately 20-180 and 10-40 times respectively, higher than the same for the wild type strain. The intracellular levels of ppGpp determined with high performance liquid chromatography (HPLC) demonstrated that spoT and dksA mutants possess higher basal levels of ppGpp. The data suggest that elevated basal levels of ppGpp may be responsible for rendering these mutants tolerant to quinolones and expand the importance of ppGpp as an antimicrobial target in P. aeruginosa.

The aim of this study was to determine whether Actinobacillus actinomycetemcomitans lipopolysaccharide (LPS-A. actinomycetemcomitans) could stimulate a murine macrophage cell line (RAW264.7 cells) to produce nitric oxide (NO). The cells were treated with LPS-A. actinomycetemcomitans or Escherichia coli LPS (LPS-Ec) for 24 h. The effects of N(G)-monomethyl-L-arginine (NMMA), polymyxin B and cytokines (IFN-gamma, TNF-alpha, IL-4 and IL-12) on the production of NO were also determined. The role of protein tyrosine kinase, protein kinase C and microtubulin organization on NO production were assessed by incubating RAW264.7 cells with genistein, bisindolylmaleide and colchicine prior to LPS-A. actinomycetemcomitans stimulation, respectively. NO levels from the culture supernatants were determined by the Griess reaction. The results showed that LPS-A. actinomycetemcomitans stimulated NO production by RAW264.7 cells in a dose-dependent manner, but was slightly less potent than LPS-Ec. NMMA and polymyxin B blocked the production of NO. IFN-gamma and IL-12 potentiated but IL-4 depressed NO production by LPS-A. actinomycetemcomitans-stimulated RAW264.7 cells. TNF-alpha had no effects on NO production. Genistein and bisindolylmalemaide, but not colchicine, reduced the production of NO in a dose-dependent mechanism. The results of the present study suggest that A. actinomycetemcomitans LPS, via the activation of protein tyrosine kinase and protein kinase C and the regulatory control of cytokines, stimulates NO production by murine macrophages.

The alternative sigma factor 54 has been implicated in diverse functions within the cells. In this study, we have constructed an rpoN mutant of Pseudomonas aeruginosa and investigated its importance as a target for antimicrobial agents, such as quinolones and carbapenems. The stationary-phase cells of the rpoN mutant displayed a survival rate approximately 15 times higher than that of the wild-type cells in the presence of quinolones and carbapenems. The stationary phase led to substantial production of pyoverdine by the P. aeruginosa rpoN mutant. Pyoverdine synthesis correlated with decreased susceptibility to antimicrobial agents. Quantitative real-time PCR revealed that stationary-phase cells of the rpoN mutant grown without an antimicrobial agent had approximately 4-to 140-and 2-to 14-fold-higher levels of transcripts of the pvdS and vqsR genes, respectively, than the wild-type strain. In the presence of an antimicrobial agent, levels of pvdS and vqsR transcripts were elevated 400-and 5-fold, respectively, in comparison to the wild-type levels. Flow cytometry assays using a green fluorescent protein reporter demonstrated increased expression of the vqsR gene in the rpoN mutant throughout growth. A pvdS mutant of P. aeruginosa, deficient in pyoverdine production, was shown to be susceptible to biapenem. These findings suggest that rpoN is involved in tolerance to antimicrobial agents in P. aeruginosa and that its tolerant effect is partly dependent on increased pyoverdine production and vqsR gene expression.Pseudomonas aeruginosa is an opportunistic pathogen that infects immunocompromised hosts, causing infections that are especially difficult to eradicate. P. aeruginosa has evolved a mechanism to partly escape from the effects of antimicrobial agents without necessarily expressing a resistance mechanism. This mechanism has been introduced in the literature as antimicrobial tolerance. Antimicrobial tolerance can be defined as the intrinsic ability of bacteria to survive the killing effects of antimicrobial agents (23). The molecular basis of the tolerance is virtually unexplored. Under certain environmental conditions, such as an alteration in the nutritional supply, entry into the stationary phase, or high cell density, temperature, pH, or osmolarity, planktonic cells can turn on stress response genes and switch to a more tolerant phenotype (12). Stress response genes are regulated by different linked signals, such as quorum sensing, ppGpp, and poly(P) kinase. We have recently reported that increased basal levels of ppGpp under nongrowing conditions might be a signal leading to tolerance to quinolones in P. aeruginosa (25). Transcriptional regulators such as sigma factors are key elements in the bacterial adaptive responses needed for pathogenesis. For example, it has been shown that RpoS, a central regulator of the stress response, also plays a role in tolerance to quinolones and carbapenems in P. aeruginosa (11). RpoN is another important sigma factor that also appears to regulate virulence in P. aeruginos...

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Pseudomonas aeruginosa is a Gram-negative bacterium and causes respiratory infection especially in elderly patients. Royal jelly has been used worldwide as a traditional remedy and as a nutrient; however, the effect against P. aeruginosa is unclear. The aim of this study was to analyze antibacterial, antiadherent, and anti-inflammatory effects of royal jelly against P. aeruginosa. Wild-type strain PAO1 and clinical isolates of P. aeruginosa were used for antibacterial assay and antiadherent assay to abiotic surface and epithelial cells, which are pharynx (Detroit 562) and lung (NCI-H292) epithelial cells. In anti-inflammatory assay, epithelial cells were pretreated with royal jelly before bacterial exposure to investigate its inhibitory effect on interleukin (IL-8) and macrophage inflammatory protein-3α/CCL20 overproduction. Although royal jelly did not have antibacterial activity at concentration of 50% w/v, antiadherent activity was confirmed on the abiotic surface and epithelial cells under concentration of 25%. Pretreatment with royal jelly significantly inhibited overproduction of IL-8 and CCL20 from both cells. These results demonstrated that royal jelly inhibits P. aeruginosa adherence and protects epithelial cells from excessive inflammatory responses against P. aeruginosa infection. Our findings suggested that royal jelly may be a useful supplement as complementary and alternative medicine for preventing respiratory infection caused by P. aeruginosa.

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Pseudomonas aeruginosa (P. aeruginosa) is an opportunistic bacterium, which could aggressively infect immunocompromised patients and thus, cause high mortality rate. In addition, P. aeruginosa in oropharynx could be aspirated and cause ventilator associated pneumonia. Royal jelly is one of bee's products that has been used for therapeutic needs including antibacteria. Adherence factor of P. aeruginosa were flagelum, pili and lectin. The aim of the study was to determine the effect of royal jelly to P. aeruginosa adhesion. Suspension of P. aeruginosa (ATCC® 27853 TM ) was incubated at 37 °C for 18 h. Treatment groups were exposed to royal jelly with several concentrations, 2%, 4%, 6%; while distilled water was being used as negative control. Bacterial adhesion test was determined using spectrophotometer λ = 600 nm to measure optical density values of adhered bacterial suspension in tubes. The result of one-way ANOVA showed significant differences (p<0.05) of optical density values among groups indicating that royal jelly affected the bacterial adhesion. LSD results showed significant difference of optical density values between 2%, 4%, and 6% royal jelly compared to distilled water. Six percent of royal jelly had the least optical density value compared to the other groups. In conclusion, royal jelly has the ability to decrease adhesion of P. aeruginosa. Six percent of royal jelly has better ability to decrease adhesion of P. aeruginosa than other concentrations.

ABSTRAKInflamasi merupakan respon alami tubuh terhadap adanya kerusakan jaringan. Salah satu medikamen untuk mengatasi inflamasi adalah antiinflamasi non steroid (AINS). Penggunaan AINS mempunyai beberapa efek samping dan dalam beberapa hal penggunaan tanaman obat dinilai lebih aman. Rosela merupakan salah satu tanaman obat yang mempunyai potensi sebagai antiinflamasi. Penelitian ini bertujuan untuk mengetahui efek pemberian sistemik ekstrak etanolik rosela terhadap ekspresi COX-2 dan jumlah neutrofil fase inflamasi pada proses penyembuhan luka. Bunga rosela didapatkan dari perkebunan di Dusun Bulusari Desa Pojok Kecamatan Tarokan Kabupaten Kediri Jawa Timur. Pembuatan ekstrak rosela dilakukan di LPPT unit I UGM Yogjakarta dengan cara perkolasi. Tikus putih galur Wistar sebanyak 36 ekor diberi perlukaan dengan punch biopsi ɵ 3 mm pada mukosa bukal. Subjek dibagi menjadi 3 kelompok, masing-masing kelompok 12 ekor tikus. Pembagian kelompok terdiri dari kontrol negatif (saline), kontrol positif (ibuprofen 20 mg/kg BB) dan perlakuan (ekstrak rosela 500 mg/kg BB). Pemberian minum sesuai kelompoknya sehari sekali selama 4 hari. Pada hari ke-1, ke-2, ke-3 dan ke-4 tikus dikorbankan lalu jaringan mukosa yang mengalami perlukaan dibuat preparat histologis. Pewarnaan Hematoksilin Eosin (HE) dilakukan untuk mengamati jumlah neutrofil. Ekspresi COX-2 diamati pada preparat dengan pewarnaan imunohistokimia menggunakan rabbit polyclonal antibody COX-2 (Lab Vision, USA). Jumlah neutrofil dan ekspresi COX-2 dihitung di bawah mikroskop cahaya lalu data dianalisi menggunakan ANAVA dan LSD. Hasil penelitian menunjukkan bahwa ekspresi COX-2 dan jumlah neutrofil lebih rendah pada kelompok perlakuan dibanding kontrol. Pengamatan klinis pada hari ke-4 juga tampak luka seluruh subjek telah menutup sempurna setelah pemberian minum rosela. Disimpulkan bahwa ekstrak etanolik rosela mempunyai kemampuan menghambat ekspresi COX-2 dan menurunkan jumlah neutrofil sehingga dapat digunakan sebagai bahan anti-inflamasi. Maj Ked Gi. Juni 2014; 21(1): 13 -19.Kata kunci: ekstrak etanolik rosela (Hibiscus sabdariffa), ekspresi COX-2, jumlah neutrofil, penyembuhan luka ABSTRACT: Expression of COX-2 and The Number of Neutrophil in Inflammation stage of Wound Healing Process after Systemic Administration of Ethanolic Extract Rosela. Inflammation is an initial stage of body's natural response to tissue damage.The use empirically plants often used for traditional medicine because it is easily found in the community and fewer side effects. Flavanoid presence of roselle (Hibiscus sabdariffa) is thought to have antiinflammatory effects. This study aimed to know the effect of systemic administration of Roselle ethanolic extract toward COX-2 expression and neutrophils number in the inflammatory phase of wound healing processes. Roselle was obtained from plantations in Bulusari hamlet