Antibacterial, anti-swarming and anti-biofilm formation activities of Chamaemelum nobile against Pseudomonas aeruginosa

AbstractINTRODUCTION:Chamomile ( Chamaemelum nobile ) is widely used throughout the world, and has anti-inflammatory, deodorant, bacteriostatic, antimicrobial, carminative, sedative, antiseptic, anti-catarrhal, and spasmolytic properties. Because of the increasing incidence of drug-resistant bacteria, the development of natural antibacterial sources such as medical herbs for the treatment of infectious diseases is necessary. Extracts from different plant parts such as the leaves, flowers, fruit, and bark of Combretum albiflorum, Laurus nobilis , and Sonchus oleraceus were found to possess anti-quorum sensing (QS) activities. In this study, we evaluated the effect of C. nobile against Pseudomonas aeruginosa biofilm formationMETHODS:The P. aeruginosa samples were isolated from patients with different types of infection, including wound infection, septicemia, and urinary tract infection. The flowers of C. nobile were dried and the extract was removed using a rotary device and then dissolved in dimethyl sulfoxide at pH 7.4. The microdilution method was used to evaluate the minimum inhibitory concentration (MIC) of this extract on P. aeruginosa , and biofilm inhibition was assayed.RESULTS:Eighty percent of the isolated samples (16/20) could form a biofilm, and most of these were isolated from wound infections. The biofilm inhibitory concentration of the C. nobile extract was 6.25-25mg/ml, whereas the MIC was 12.5-50mg/ml.CONCLUSIONS:The anti-QS property of C. nobile may play an important role in its antibacterial activity, thus offering an additional strategy in the fight against bacterial infections. However, molecular investigation is required to explore the exact mechanisms of the antibacterial action and functions of this phytocompound.

Monoterpene isolated from the essential oil of Trachyspermum ammi is cytotoxic to multidrug-resistant Pseudomonas aeruginosa and Staphylococcus aureus strains

Abstract INTRODUCTION: The aim of this study was to determine whether an herbal extract containing monoterpene exhibited activity against multidrug-resistant Staphylococcus aureus and Pseudomonas aeruginosa isolated from clinical infection samples. METHODS: The essential oil of Trachyspermum ammi (L.) Sprague ex Turrill (Apiaceae) fruit was extracted by hydrodistillation. Fruit residues were treated with hydrochloric acid and re-hydrodistilled to obtain volatile compounds. Compounds in the distilled oil were identified using gas-chromatography (GC) and GC-mass spectrometry (MS). The antibiotic susceptibility of all bacterial isolates was analyzed using both the disc diffusion method and determination of the minimum inhibitory concentration (MIC). The sensitivity of antibiotic-resistant isolates to essential oil was also determined by using the disc diffusion method and MIC determination. RESULTS: Of 26 clinical isolates, 92% were multidrug-resistant (MDR). Aromatic monoterpenes (thymol, paracymene, and gamma-terpinene) were the major (90%) components of the oil. Growth of S. aureus strains was successfully inhibited by the oil, with an inhibitory zone diameter (IZD) between 30-60mm and MIC <0.02μL/mL. The oil had no antimicrobial activity against clinical isolates of P. aeruginosa; rather, it prevented pigment production in these isolates. CONCLUSIONS: This study revealed that the essential oil of Trachyspermum ammi, which contains monoterpene, has good antibacterial potency. Monoterpenes could thus be incorporated into antimicrobial ointment formulas in order to treat highly drug-resistant S. aureus infections. Our findings also underscore the utility of research on natural products in order to combat bacterial multidrug resistance.

Algumas exigências metabólicas de Pseudomonas denitrificans

Para estudos da bioquímica da desnitrificação os desnitrificadores são cultivados em meio parcialmente sintético, com extrato de levedura ou de carne ou ainda peptona. Procurou-se então um meio de cultura completamente sintético no qual P. denitrificans pudesse desenvolver-se e fazer desnitrificação. Para tanto, células de um "strain" dessa bactéria foram crescidas durantes, 24 e 48 horas em tubos de ensaio contendo 10 ml de meio de cultura consistindo de succinato de sódio, nitrato de potássio, extrato de levedura o tampão de fosfato 1M, valôr pH 6,8. Dêsse meio (controle) 0,1 ml foi inoculado em 10 ml do meio de cultura em estudo, mantido então em condições parcialmente anaeróbicas. Após 24 horas uma alíquota foi retirada e suspensa em água destilada e a turbidês lida em espectrofotômetro Beckman, a 420 my. Os dados obtidos após longa série de ensaios permitiram concluir que a fim de se obter em 48 horas um crescimento da mesma ordem que o obtido com o meio controle, o meio de cultura sintético deve conter micronutrientes (Zn, Fe, Mn, Cu, Co, B e Mo, em EDTA), sulfato de sódio, sulfato de magnésio e ácido glutâmico, além de KNO3, succinato e tampão de fosfato.

Isolamento e caracterização de Pseudomonas maltophilia (Hugh & Ryschenkow, 1960) de material clínico humano, na cidade do Rio de Janeiro

Os autores estudaram as propriedades morfo-bioquímicas e a sensibilidade às substâncias antimicrobianas, de uma nova e rara espécie de Pseudomonas, a Pseudomonas maltophilia (Hugh & Ryschenkow, 1960), isolada de secração vaginal. Como características marcantes, dentre mais de 65 testadas, as amostras estudadas mostraram ser: oxidase negativa e lisina descarboxilase positiva; produziram desoxiribonuclease e um pigmento escuro que se difunde no meio; atacaram oxidativamente a maltose tanto em meio complexo nitrogenado como em meio de Hugh & Leifson e hidrolisaram a esculina. As amostras foram sensíveis ao cloranfenicol, gentamicina, kanamicina, colistin e gabromicina.

Pseudomonas aeruginosa adhesion to normal and injured respiratory mucosa

Human nasal polyps outgrowth culture were used to study the adhesion of Pseudomonas aeruginosa to respiratory cells. By transmission electron microscopy, bacteria associated with ciliated cells were identified trapped at the extremities of cilia, usually as aggregates of several bacterial cells. They were never seen at the interciliary spaces or attached along cilia. Bacteria were also seen to adhere to migrating cells of the periphery of the outgrowth culture. Using a model of repair of wounded respiratory epithelial cells in culture, we observed that the adhesion of P. aeruginosa to migrating cells of the edges of the repairing wounds was significantly higher than the adhesion to non-migrating cells and that adherent bacteria were surrounded by a fibrocnectin-containing fibrillar material The secretion of extracellular matrix components is involved in the process of epithelium repair following injury. To investigate the molecular basis of P. aeruginosa adhesion to migrating cells, bacteria were treated with a fibronectin solution before their incubation with the respiratory cells. P. aeruginosa treatment by fibronectin significantly increased their adhesion to migrating cells. Accordingly, we hypothesize that during cell migration, fibronectin secreted by epithelial cells may favour P. aeruginosa adhesion by establishing a bridge between the bacteria and the epithelial cell receptors. Such a mechanism may represent a critical step for P. aeruginosa infection of healing injured epithelium.

Isolation of Pseudomonas cepacia in cystic fibrosis patient

Pulmonary infection on cystic fibrosis (CF) patients are associated with a limited qualitative number of microorganisms. During the colonization process, Staphylococcus aureus usually preceedes Pseudomonas aeruginosa. This latter is at first non-mucoid, being replaced or associated to a mucoid morphotype which is rare in other diseases. In 1980, Pseudomonas cepacia appeared as an important agent in CF pulmonary infections with a mean frequency of about 6.1% isolations in different parts of the world. The primus colonization mainly occurs in the presence of pre-existent tissue lesions and the clinical progress of the disease is variable. In some patients it can be fulminant; in others it can cause a gradual and slow decrease in their pulmonary functions. The concern with this germ isolation is justified by its antibiotic multiple resistence and the possibility of direct transmission from a colonized patient to a non-colonized one. We reported the first case of P. cepacia infection in a CF patient in our area. The microbiological attendance to this patient had been made from 1986 to 1991 and the first positive culture appeared in 1988. The sensitivity profile showed that the primus colonization strain was sensitive to 9 of 17 tested antibiotics, however in the last culture the strain was resistent to all antibiotics. These data corroborate the need for monitoring the bacterial flora on CF patients respiratory system.

Pseudomonas aeruginosa: study of Antibiotic Resistance and Molecular Typing in Hospital Infection Cases in a Neonatal Intensive Care Unit from Rio de Janeiro City, Brazil

This study had the objective of to analyze the demographic and bacteriologic data of 32 hospitalized newborns in an neonatal intensive care unit of a public maternity hospital in Rio de Janeiro city, Brazil, seized by Pseudomonas aeruginosa sepsis during a period ranged from July 1997 to July 1999, and to determine the antimicrobial resistance percentage, serotypes and pulsed field gel electrophoresis (PFGE) patterns of 32 strains isolated during this period. The study group presented mean age of 12.5 days, with higher prevalence of hospital infection in males (59.4%) and vaginal delivery (81.2%), than females (40.6%) and cesarean delivery (18.8%), respectively. In this group, 20 (62.5%) patients received antimicrobials before positive blood cultures presentation. A total of 87.5% of the patients were premature, 62.5% presented very low birth weight and 40.6% had asphyxia. We detected high antimicrobial resistance percentage to b-lactams, chloramphenicol, trimethoprim/sulfamethoxazole and tetracycline among the isolated strains. All isolated strains were classified as multi-drug resistant. Most strains presented serotype O11 while PFGE analysis revealed seven distinct clones with isolation predominance of a single clone (75%) isolated from July 1997 to June 1998.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.

Emergence of dhfrXVb and blaCARB-4 gene cassettes in class 1 integrons from clinical Pseudomonas aeruginosa isolated in Amazon region

Integrons play a role in horizontal acquisition and expression of genes, as well as gene reservoir, contributing for the resistance phenotype, particularly relevant to bacteria of clinical importance. We aimed to determine the composition and the organization of the class 1 integron variable region present in Pseudomonas aeruginosa clinical isolates from Brazil. Strains carrying class 1 integrons were resistant to the majority of antibiotics tested, except to imipenem and ceftazidime. Sequence analysis of the integron variable region revealed the presence of the blaCARB-4 gene into two distinct cassette arrays: aacA4-dhfrXVb-blaCARB-4 and aadB-aacA4-blaCARB-4 . dhfrXVb gene cassette, which is rare in Brazil and in P. aeruginosa species, was found in one isolate. PFGE analysis showed the spread of blaCARB-4 among P. aeruginosa clones. The occurrence of blaCARB-4 and dhfrXVb in Brazil may contribute for developing resistance to clinically important antibiotics, and shows a diversified scenarium of these elements occurring in Amazon clinical settings, where no study about integron dinamycs was performed to date.

Changing the epidemiology of carbapenem-resistant Pseudomonas aeruginosa in a Brazilian teaching hospital: the replacement of São Paulo metallo-β-lactamase-producing isolates

In Brazil, carbapenem-resistant Pseudomonas aeruginosa isolates are closely related to the São Paulo metallo-β-lactamase (SPM) Brazilian clone. In this study, imipenem-resistant isolates were divided in two sets, 2002/2003 and 2008/2009, analysed by pulsed field gel electrophoresis and tested for the Ambler class B metallo-β-lactamase (MBL) genes blaSPM-1, blaIMP and blaVIM. The results show a prevalence of one clone related to the SPM Brazilian clone in 2002/2003. In 2008/2009, P. aeruginosa isolates were mostly MBL negative, genetically diverse and unrelated to those that had been detected earlier. These findings suggest that the resistance to carbapenems by these recent P. aeruginosa isolates was not due to the spread of MBL-positive SPM-related clones, as often observed in Brazilian hospitals.

The infection of microvascular endothelial cells with ExoU-producing Pseudomonas aeruginosa triggers the release of von Willebrand factor and platelet adhesion

An increased plasma concentration of von Willebrand factor (vWF) is detected in individuals with many infectious diseases and is accepted as a marker of endothelium activation and prothrombotic condition. To determine whether ExoU, a Pseudomonas aeruginosa cytotoxin with proinflammatory activity, enhances the release of vWF, microvascular endothelial cells were infected with the ExoU-producing PA103 P. aeruginosa strain or an exoU-deficient mutant. Significantly increased vWF concentrations were detected in conditioned medium and subendothelial extracellular matrix from cultures infected with the wild-type bacteria, as determined by enzyme-linked immunoassays. PA103-infected cells also released higher concentrations of procoagulant microparticles containing increased amounts of membrane-associated vWF, as determined by flow cytometric analyses of cell culture supernatants. Both flow cytometry and confocal microscopy showed that increased amounts of vWF were associated with cytoplasmic membranes from cells infected with the ExoU-producing bacteria. PA103-infected cultures exposed to platelet suspensions exhibited increased percentages of cells with platelet adhesion. Because no modulation of the vWF mRNA levels was detected by reverse transcription-polymerase chain reaction assays in PA103-infected cells, ExoU is likely to have induced the release of vWF from cytoplasmic stores rather than vWF gene transcription. Such release is likely to modify the thromboresistance of microvascular endothelial cells.

Metallo-β-lactamase-production in meropenem-susceptible Pseudomonas aeruginosa isolates: risk for silent spread

The aim of this study was to characterize two metallo-β-lactamases (MBLs)-producing Pseudomonas aeruginosa clinical isolates showing meropenem susceptibility. Antimicrobial susceptibility was assessed by automated testing and Clinical and Laboratory Standards Institute agar dilution method. MBL production was investigated by phenotypic tests. Molecular typing was determined by pulsed field gel electrophoresis (PFGE). MBL-encoding genes, as well as their genetic context, were identified by polymerase chain reaction (PCR) and sequencing. The location of blaIMP-16 was determined by plasmid electrophoresis, Southern blot and hybridization. Transcriptional levels of blaIMP-16, mexB, mexD, mexF, mexY, ampC and oprD were determined by semi-quantitative real time PCR. The P. aeruginosa isolates studied, Pa30 and Pa43, showed imipenem and meropenem susceptibility by automated testing. Agar dilution assays confirmed meropenem susceptibility whereas both isolates showed low level of imipenem resistance. Pa30 and Pa43 were phenotypically detected as MBL producers. PFGE revealed their clonal relatedness. blaIMP-16 was identified in both isolates, carried as a single cassette in a class 1 integron that was embedded in a plasmid of about 60-Kb. Pa30 and Pa43 overexpressed MexAB-OprM, MexCD-OprJ and MexXY-OprM efflux systems and showed basal transcriptional levels of ampC and oprD. MBL-producing P. aeruginosa that are not resistant to meropenem may represent a risk for therapeutic failure and act as silent reservoirs of MBL-encoding genes.

The draft genome sequence of multidrug-resistant Pseudomonas aeruginosa strain CCBH4851, a nosocomial isolate belonging to clone SP (ST277) that is prevalent in Brazil

The high occurrence of nosocomial multidrug-resistant (MDR) microorganisms is considered a global health problem. Here, we report the draft genome sequence of a MDR Pseudomonas aeruginosa strain isolated in Brazil that belongs to the endemic clone ST277. The genome encodes important resistance determinant genes and consists of 6.7 Mb with a G+C content of 66.86% and 6,347 predicted coding regions including 60 RNAs.

Mutational and acquired carbapenem resistance mechanisms in multidrug resistant Pseudomonas aeruginosa clinical isolates from Recife, Brazil

An investigation was carried out into the genetic mechanisms responsible for multidrug resistance in nine carbapenem-resistant Pseudomonas aeruginosaisolates from different hospitals in Recife, Brazil. Susceptibility to antimicrobial agents was determined by broth microdilution. Polymerase chain reaction (PCR) was employed to detect the presence of genes encoding β-lactamases, aminoglycoside-modifying enzymes (AMEs), 16S rRNA methylases, integron-related genes and OprD. Expression of genes coding for efflux pumps and AmpC cephalosporinase were assessed by quantitative PCR. The outer membrane proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The blaSPM-1, blaKPC-2 and blaGES-1 genes were detected in P. aeruginosaisolates in addition to different AME genes. The loss of OprD in nine isolates was mainly due to frameshift mutations, premature stop codons and point mutations. An association of loss of OprD with the overexpression of MexAB-OprM and MexXY-OprM was observed in most isolates. Hyper-production of AmpC was also observed in three isolates. Clonal relationship of the isolates was determined by repetitive element palindromic-PCR and multilocus sequence typing. Our results show that the loss of OprD along with overexpression of efflux pumps and β-lactamase production were responsible for the multidrug resistance in the isolates analysed.

Interação de bactérias fluorescentes do gênero Pseudomonas e de Bacillus spp. com a rizosfera de diferentes plantas

Embora haja muitos trabalhos na literatura com rizobactérias promotoras do crescimento de plantas (RPCPs), existem poucos que expliquem seu mecanismo de ação. É possível que algumas rizosferas favoreçam a colonização radicular por RPCPs, facilitando o estabelecimento da interação planta-bactéria, como se houvesse certa especificidade entre ambas. O objetivo deste trabalho foi verificar se a rizosfera de alface, em comparação com a de outras espécies vegetais, favorece o estabelecimento de bactérias fluorescentes do gênero Pseudomonas, em comparação com as do gênero Bacillus. Coletaram-se amostras do sistema radicular de alface, rúcula, chicória, salsa e tiririca em oito propriedades de produtores comerciais de hortaliças, na região de Campinas, SP. Foi feita a contagem de Pseudomonas spp. fluorescentes e de Bacillus spp. por diluição em série e plaqueamento. De maneira geral, observou-se maior crescimento de Pseudomonas spp. fluorescentes na rizosfera de alface-crespa em relação à de outras plantas, mas isso não ocorreu com Bacillus spp.