Evaluation of anti-quorum sensing potential of Averrhoa bilimbi (Kamias) against Pseudomonas aeruginosa ATCC 27853

BACKGROUND AND OBJECTIVE@#Many opportunistic and nosocomial pathogens like Pseudomonas aeruginosa are very reliant on a bacterium-to-bacterium communication system called quorum sensing (QS). Without the aforementioned process, gene expressions associated with virulence factors will not be produced. In this study, the sub-inhibitory concentrations (sub-MICs) of methanolic leaf extract and obtained fractions from Averrhoa bilimbi (kamias) were screened for ability to inhibit quorum sensing-controlled phenotypes of P. aeruginosa ATCC 27853.@*METHODOLOGY@#A. bilimbi crude extract was fractionated through liquid-liquid extraction, producing four (4) fractions: hexane fraction, dichloromethane (DCM) fraction, ethyl acetate (EtOAc) fraction, and water (H2O) fraction. Among the sub-MICs obtained from resazurin-based fluorimetric microtiter assay, only 50 μg/mL was utilized in evaluating the anti-QS properties of crude extract and fr@*RESULTS@# In the swarming motility assay, hexane fraction (9.39 mm ± 0.67) and DCM fraction (10.82 mm ± 0.95) displayed restriction in the treated P. aeruginosa ATCC 27853 swarms against the control (16.20 mm ± 2.55). In the anti-pyocyanin production assay, hexane fraction exhibited an inhibition of 42.66 % ± 12.94. TLC analysis and phytochemical screening revealed that hexane fraction contains steroids, terpenes, triterpenes, and glycolipids; and DCM fraction contains cardiac glycosides, triterpenoids, terpenes, triterpenes, steroids, alkaloids, and glycolipids.@*CONCLUSION@#Hexane and DCM fractions obtained from A. bilimbi significantly inhibited swarming of P. aeruginosa ATCC 27853 while none of the extracts were able to significantly inhibit pyocyanin formation of P. aeruginosa ATCC 27853.

Thymol Rich Thymbra capitata Essential Oil Inhibits Quorum Sensing, Virulence and Biofilm Formation of Beta Lactamase Producing Pseudomonas aeruginosa

Infections with Pseudomonas aeruginosa are difficult to treat not only because it is often associated with multidrug-resistant infections but also it is able to form biofilm. The aim of this study was to evaluate the antibiofilm and anti-Quorum Sensing (QS) activities of Thymbra capitata essential oils (EOs) against Beta Lactamase (BL) producing P. aeruginosa and the reference strain P. aeruginosa 10145. GC/MS analysis showed that thymol (23.25%) is the most dominant compound in T. capitata EOs. The MICs of T. capitata EOs against P. aeruginosa (BL) and P. aeruginosa 10145 were 1.11%. At sub MIC (0.041, 0.014 and 0.0046%), the EOs of T. capitata remarkably inhibited the biofilm formation of both strains tested and complete inhibition of the biofilm formation was reported at 0.041%. The EOs of T. capitata were found to inhibit the swarming motility, aggregation ability and hydrophobic ability of P. aeruginosa (BL) and P. aeruginosa 10145. Interestingly, the EOs of T. capitata reduce the production of three secreted virulence factors that regulated by QS system including pyocyanin, rhamnolipids and LasA protease. The potent antibiofilm and anti-QS activities of T. capitata EOs can propose it as a new antibacterial agent to control pseudomonas infections.

Um novo gene de Pseudomonas aeruginosa envolvido em percepção de quórum / A novel gene involved in Pseudomonas aeruginosa quorum sensing

Pseudomonas aeruginosa é uma gamaproteobactéria com capacidade de colonizar diversos tipos de ambiente e infectar hospedeiros filogeneticamente distintos. Em humanos, comporta-se como um patógeno oportunista,estando frequentemente relacionada à infecções em indivíduos imunocomprometidos e indivíduos portadores de fibrose cística. Um mecanismo importante para a versatilidade de P. aeruginosa é o sistema de percepção de quórum (QS), onde a bactéria pode vincular expressão gênica à densidade populacional e às características do ambiente. Atualmente, sabe-se que muitos outros reguladores estão interligados com QS, entre eles, a proteína reguladora RsmA e os pequenos RNAs RsmZ e RsmY. Além disso, diversos fatores importantes para a patogenicidade da bactéria são reguladas por QS. Em P. aeruginosa PA14, um fator importante para a patogenicidade em diversos hospedeiros é a proteína KerV, cujo envolvimento com QS foi descrito pela primeira vez neste trabalho. A linhagem D12, que possui uma deleção no gene kerV, mostrou alterações em fenótipos regulados por QS, como a maior produção de piocianina, composto que contribui para virulência e persistência das infecções causada por P. aeruginosa. Por ser facilmente detectável e pela regulação de sua síntese não ter sido completamente explorada em PA14, a expressão dos genes responsáveis pela produção de piocianina é um interessante repórter na investigação do possível envolvimento de KerV com QS. Além de piocianina, D12 apresenta níveis reduzidos de ramnolipídeos. Esses fenótipos somados se assemelham aos fenótipos da mutação de rsmA, sugerindo o envolvimento de KerV com os sistemas QS e Gac-Rsm direta ou indiretamente. Neste trabalho, mostramos que KerV exerce um efeito negativo na regulação dos operons phz1 e phz2, responsáveis pela síntese de piocianina, alterando a expressão desses genes. KerV exerce também um efeito positivo na expressão da proteína RsmA, responsável pela repressão de diversos genes alvos, onde RsmA se liga ao sítio de ligação ao ribossomo no mRNA, impedindo a tradução. Ensaios de gel shift mostraram que a ligação direta de RsmA na sequência líder de phzA1 e phzA2 ocorre, elucidando a maneira pela qual KerV está envolvido na regulação da expressão dos operons phz em P. aeruginosa PA14. Mostramos também que phz2 é ativo e contribui para a síntese de piocianina, pois na ausência de phz1, os níveis do pigmento são maiores do que aqueles detectados em PA14. Isso sugere uma maior expressão de phz2 e uma regulação diferencial dos operons de acordo com as condições ambientais como possível estratégia para manter os níveis desse composto. Uma evidência dessa regulação diferencial é vista no mutante lasR. Na fase inicial de crescimento, esse mutante não produz piocianina, porém quando exposto a tempos mais longos de cultivo, a produção de piocianina é maior quando comparada a PA14. Isso é reflexo da ativação da expressão de phz1 no mutante lasR em fase estacionária tardia, enquanto phz2 permanece não expresso. Isso indica que phz2 é dependente de LasR, ainda que indiretamente. Já phz1, embora tenha sua expressão influenciada por LasR no estágio inicial de crescimento, na fase estacionária é regulado por outros fatores independentes de las

Pseudomonas aeruginosa is a gammaproteobacterium that colonizes several environments and infects phylogenetically distinct hosts. It behaves as an opportunistic pathogen in humans, often related to infection in immunocompromised individuals and cystic fibrosis patients. An important mechanism for P. aeruginosa versatility is the quorum sensing (QS) network, that allows bacteria to link gene expression to population density and environmental traits. Several additional regulators are interconnected with QS, as the regulatory mRNA binding protein RsmA and the non-coding small RNAs RsmZ and RsmY. Futhermore, key factors for pathogenicity are QS-regulated. In P. aeruginosa PA14, an important pathogenicity-related factor is the KerV protein, described for the first time here as involved in QS. D12 strain, that harbor a deletion in the kerV gene, shows alterations in QS-regulated phenotypes, such as high production of pyocyanin, a compound that contributes to virulence and persistence of P. aeruginosa infections. As the production of pyocyanin is easily detected and all mechanisms involved in its synthesis regulation are not fully described, the expression of genes responsible for production of this pigment is a good reporter to investigate KerV involvement in the QS network. Additionally, D12 also shows lower levels of rhamnolipids, another QS-regulated trait. Taken together, these phenotypes resemble the effects of a rsmA mutation, suggesting KerV involvement with QS and Gac-Rsm systems. In this work, we propose that KerV exerts a negative effect in the regulation of phz1 and phz2 operons, responsible for pyocyanin synthesis, by alterating the expression of these genes. KerV also has a positive effect on rsmA expression, responsible for the repression of several genes by blocking the ribosome binding site preventing the translation. Gel shift assays showed that RsmA binds directly in the leader sequence of phzA1 and phzA2, elucidating the manner in which KerV is involved in the regulation of phz operons expression in P. aeruginosa PA14. We also demonstrate that phz2 is actively expressed and contributes to pyocyanin production in PA14, since in the phz1 mutant the levels of pyocyanin are even higher than in the wild type strain. This suggests a phz2 higher expression and a differential regulation of phz operons according to environmental changes as a mechanism to maintain the levels of pyocyanin synthesis. An evidence for this regulation is the synthesis of pyocyanin by the lasR mutant, which does not make pyocyanin at early growth stages. However, at late stationary phase, pyocyanin production is even higher than in the wild-type strain, reflecting the LasR-independent regulation of phz1 expression, while phz2 operon remains silent

Comparison of antibiotic resistance and virulence factors in pigmented and Non-pigmented Pseudomonas aeruginosa / Comparación de la resistencia antibiótica y los factores de virulencia en las Pseudomonas aeruginosa pigmentadas y no pigmentadas

OBJECTIVES: Pseudomonas aeruginosa produces multiple virulence factors that have been implicated in pathogenesis and quorum sensing. The aim of this study was to determine differences in the virulence factors of pigmented and non-pigmented P aeruginosa isolates. METHODS: Associations were assessed between pigment production (pyocyanin and pyoverdin) and production of DNase, elastase, lipase, protease, siderophore, twitching motility, antibiotic resistance patterns and virulence-associated genes in 57 non-duplicate P aeruginosa isolates from wounds, sputum, urine, high vaginal swab (HVS), ear, eye and respiratory tract swabs and aspirates of peritoneum and ulcers. RESULTS: Most (82.5%) of the isolates produced either pigment. Pigmented isolates produced more frequently and significant more (p < 0.05) DNase, elastase, lipase protease, and siderophore. Imipenem was the only antibiotic to which all isolates were susceptible (p < 0.05), while 93% and 32% were resistant to tetracycline and norfloxacin, respectively. There was however no significant difference between pigmented and non-pigmented isolates when antibiotic resistance was compared. While isolates had multiple virulence-associated genes, exoS (51%), rhlA (37%) and rhlB (46%) were the predominant genes detected. Except for exoY, genes were present in pigmented isolates more frequently than in non-pigmented isolates. CONCLUSION: The results of this study suggest that antibiotic resistance per se might not be associated with the pigment production in P aeruginosa. However, pigment production appeared to be more significantly associated with multi-drug resistance, presence ofvirulence-associated genes, and expression ofcertain virulence factors, most notably elastase, protease, siderophore and DNase activity. Since pigment production is easy to determine, this might to be a good starting point to identify the virulence status ofan isolate.

OBJETIVO: Pseudomonas aeruginosa produce múltiples factores de virulencia que han estado implicados en patogénesis y detección de quórum (quorum sensing). El objetivo de este estudio fue determinar las diferencias en los factores de virulencia de aislados de P aeruginosa pigmentada y no pigmentada. MÉTODO: Se evaluaron las asociaciones entre la producción de pigmentos (piocianina y pioverdina) y la producción de Dnasa, elastasa, lipasa, proteasa, sideróforos, motilidad asociada a superficies (twitching), patrones de resistencia antibiótica, y genes asociados con virulencia en 57 aislados de P aeruginosa no duplicados, de heridas, esputo, orina, exudado vaginal, exudados de oídos, ojos, y vías respiratorias, y aspirados de peritoneo y úlceras. RESULTADOS: La mayor parte (82.5%) de los aislados produjeron uno de los pigmentos. Los aislados pigmentados produjeron con mayor frecuencia y más significativamente (p < 0.05). Dnasa, elastasa, lipasa, proteasa, y siderósforos. Imipenem fue el único antibiótico al que todos los aislados eran susceptibles (p < 0.05), mientras que el 93% y el 32% fueron resistentes a la tetraciclina y a la norfloxacina, respectivamente. Sin embargo, no hubo diferencia significativa entre los aislados pigmentados y los no pigmentados cuando se comparaba la resistencia antibiótica. Si bien los aislados tenían múltiples genes asociados con la virulencia, exoS (51%), rhlA (37%) y rhlB (46%) fueron los genes predominantes detectados. Con excepción de exoY, los genes estuvieron presentes en aislados pigmentados con mayor frecuencia que en los aislados no pigmentados. CONCLUSIÓN: Los resultados de este estudio sugieren que la resistencia antibiótica per se podría no estar asociada con la producción de pigmentos en P aeruginosa. Sin embargo, la producción de pigmentos parecía estar asociada más significativamente con la resistencia a las multidrogas, la presencia de genes asociados con la virulencia, y la expresión de ciertos factores de virulencia, en particular la actividad de la elastasa, la proteasa, los sideróforos, y la Dnasa. Puesto que la producción de pigmentos es fácil de determinar, esto podría ser un buen punto de partida para identificar el estado de virulencia de un aislado.

Evidência do antagonismo entre Pseudomonas aeruginosa e bactérias indicadoras de contaminação fecal em água / Evidence of the antagonism between Pseudomonas aeruginosa and indicating bacteria of fecal contamination in water

Sabe-se que a bactéria Pseudomonas aeruginosa (PA) pode interferir em análises colimétricas. Apesar disto, sua enumeração é questionada neste tipo de análise, por ser considerado um microrganismo oportunista, não habitante do trato intestinal de animais de sangue quente. No Brasil, a bactéria é padrão de potabilidade em águas minerais. Por conta da sua grande versatilidade metabólica, a bactéria teria vantagens sobre os demais microrganismos na água. No presente trabalho, 16 linhagens de PA foram investigadas como o objetivo de evidenciar o antagonismo contra dois representantes do grupo coliforme: Escherichia coli (EC) e Enterobacter aerogenes (EA). O teste foi realizado utilizando a técnica dos blocos de gel de agarose modificada. Todas as linhagens de PA apresentaram atividades antimicrobiana contra EA eEC, formando halos de inibição de até 29mm de diâmetro. O fenômeno do antagonismo foi evidenciado quando a piocianina estava presente.

Evidência do antagonismo entre Pseudomonas aeruginosa e bactérias indicadoras de contaminação fecal em água / Evidence of the antagonism between Pseudomonas aeruginosa and indicating bacteria of fecal contamination in water

Sabe-se que a bactéria Pseudomonas aeruginosa (PA) pode interferir em análises colimétricas. Apesar disto, sua enumeração é questionada neste tipo de análise, por ser considerado um microrganismo oportunista, não habitante do trato intestinal de animais de sangue quente. No Brasil, a bactéria é padrão de potabilidade em águas minerais. Por conta da sua grande versatilidade metabólica, a bactéria teria vantagens sobre os demais microrganismos na água. No presente trabalho, 16 linhagens de PA foram investigadas como o objetivo de evidenciar o antagonismo contra dois representantes do grupo coliforme: Escherichia coli (EC) e Enterobacter aerogenes (EA). O teste foi realizado utilizando a técnica dos blocos de gel de agarose modificada. Todas as linhagens de PA apresentaram atividades antimicrobiana contra EA eEC, formando halos de inibição de até 29mm de diâmetro. O fenômeno do antagonismo foi evidenciado quando a piocianina estava presente.

The Pseudomonas aeruginosa (PA) interference towards the coliform group is known in water analysis. Its enumeration has been questioned through the years because it is considered an opportunistic pathogen not inhabitant of intestinal tract of warm-blooded animaIs. In Brazil, PA is indicator only in mineral waters. Due to its metabolic versatility, it would have advantages under other organisms in water. The aim of this work was to evidence the antagonistic phenomenon. It was investigated 16 strains of PA towards Escherichia coli (EC) e Enterobacter aerogenes(EA). Tests were done by using the modified agarose disks technique. All PA strains had antagonistic activity. The event were more evidenced when pyocyanin were present.

Effect of andrographolide on QS regulating virulence factors production in Pseudomonas aeruginosa / 中国中药杂志

<p><b>OBJECTIVE</b>To investigate the effect of andrographolide on virulence factors production in Pseudomonas aeruginosa.</p><p><b>METHOD</b>Growth rate, pyocyanin, proteolytic activity and elastase activity were measured with or without the presence of andrographolide. The effect of andrographolide on pyocyanin production, proteolytic activity and elastase activity in PAO-JP2 was investigated simultaneously.</p><p><b>RESULT</b>The andrographolide did not affect the growth of PAO1 in planktonic culture. The production of pyocyanin, proteolytic activity and elastase activity were significanthy suppressed in P. aeruginosa cultures grown in the presence of andrographolide. However, these effects were not observed in PAO-JP2.</p><p><b>CONCLUSION</b>The inhibiting effect of andrographolide on virulence factors production in P. aeruginosa may play a role in its anti-infection activity.</p>

Identification System of Nonfermentative Gram Negative Bacilli Using Microplate / 대한임상미생물학회지

BACKGROUND: The accurate and rapid identification (ID) of nonfermentative gram-negative bacilli (NFB) is essential for diagnostic and therapeutic purposes and for epidemiologic studies of hospital infections. Commercial identification systems of NFB are easy to use but too expensive. The aim of the study was to develop a simple system for the identification of NFB species which are frequently isolated from clinical specimens. METHODS: Eighteen biochemical tests used in NFB microplate ID system were pyocyanin in Tech media; pyoverdin in Flo media; glucose fermentation, acid formation from glucose, maltose, lactose, sucrose, and mannitol in oxidation-fermentation media; Nitrate and nitrite reduction in nitrate media; fornithine decarboxylase, lysine decarboxylase, and arginine dihydrolase in Moeller decarboxylase media; acetamide, urease, citrate, 42degrees C growth, and oxidase test. For the establishment of NFB's biochemical data in microplate ID system, 175 consecutive isolates of NFB from clinical specimens isolated during the period of April 2000 were simultaneously tested by microplate method and API 32GN. RESULTS: Ninety-two percent of clinical isolates of NFB were identified to the species level by NFB microplate ID system. CONCLUSIONS: The NFB microplate ID system is simple to use, rapid and economical. Further modification are needed to improve the accuracy and identification rate of NFB isolates.

new phenazinomycin antibiotic with a significant anti pseudomonas effect in chronic suppurative otitis media

A new henazinomycin-related antibiotic [H1] was isolated as a cultural product of Pseudomonas Aeruginosa. This antibiotic possesses antibacterial activity against certain Gram-positive bacteria and gram-negative bacteria including enteric bacteria and Ps. Aeruginosa. This minimum inhibitory concentration [MIC] as determined by the broth microdilution assay ranged between 25-65 micro g/ml. The product yield using a novel fermentation and purification technique was 100-605 micro g/ml, while by classical method a yield of only 10-50 micro g/ml was obtained. The physico-chemical properties of the antibiotic such as melting point, ionization constant and spectrophotometeric analysis were determined. Preliminary drug toxological studies [LD50] were determined in mice but did not show toxic or important undesirable effects even at relatively high concentrations of 400 mg/kg using the intraperitoneal route of administration. Clinically the H1 antibiotic in the form of ear drops [500 micro g/ml] proved effective in the eradication of 82.6% [71/86] gentamicin-resistant Ps. aeruginosa chronic middle ear infections with dramatic reduction in morbidity and negligible side effects

Studies on Pseudomonas Aeruginosa Isolated from Chronic Wounds / 대한정형외과학회잡지

49 strains of Pseudomonas aeruginosa were isolated from chronic wounds associated with orthopedic surgical field at Taegu armed forces general hospital. In the present report, authors have studied the Pyocin type of isolated strains by Gillies and Govans method. Also observed various interrelationship in associaton with Pyocin type, Pyocyanine formation, Oxidase reaction and antibiotic sensitivity. The results were obtained as follows; 1) Among the 49 strains tested in term of pyocin type, 41 strains(81.6%) were found to be typable and 8 strains (16.4%) to be untypable. Among the pyocin type of 41 typables, unclassified type was the most prominent type 17 strains (41.5%) and followed by type I(11 strains), 10 (3 strains), 5 (2 strains) and other type. 2) In the relationship among pyocyanine formation, oxidase reaction and pyocin type, no definite conclusion can be reached due to the paucity of tested strains except that more strains belong to the pyocin type I of the Pyocyanine negative strains. 3) In antibiotic sensitivity of isolated strains tested by plate dilution method, most of all strains were resistant to Kanamycin and Neomycin but few strains were moderate sensitive to Colistin, Polymyxin B, and Terramycin. 4) The Pyocin type of isolated strains were not related with antibiotic sensitivity to Colistin, Polymyxin B and Terramycin substantially.