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Isolation and characterisation of the biological repeating unit of cepacian, the exopolysaccharide produced by Burkholderia cepacia complex bacteria
No full textThe repeating unit of cepacian, the exopolysaccharide produced by the majority of the Burkholderia cepacia complex microorganisms, was isolated from inner bacterial membranes and investigated by mass spectrometry. The data obtained led to the determination of its structure, thus disclosing the oligosaccharidic sequence synthesised by bacteria, and gave also information on the biosynthetic pathway of cepacian repeating unit, thanks to the presence of saccharidic intermediates shorter than the repeating unit. In addition, from zero to four acetyl substituents were detected on the repeating unit before the polymerisation process which takes place in the periplasmic space. The procedure used can be conveniently exploited as an alternative route to the structural determination of bacteria exopolysaccharides, particularly useful for complex repeating units
Pseudomonas aeruginosa class I integron AAC (6')-Ib variant (aacA4) and CATB10-Ib variant (cat B10) genes, complete cds.
No full textNew chloramphenicol-acetyltransferases (CATB10) encoded by a class
1 integron from a multidrug resistant Pseudomonas aeruginosa
clinical isolat
Investigation of bacterial resistance to the immune system response: Cepacian depolymerisation by reactive oxygenspecies.
No full textReactive oxygen species (ROS) are part of the weapons used by the immune system to kill and degrade infecting
microorganisms. Bacteria can produce macromolecules, such as polysaccharides, that are able to scavenge ROS.
Species belonging to the Burkholderia cepacia complex are involved in serious lung infection in cystic fibrosis patients
and produce a characteristic polysaccharide, cepacian. The interaction between ROS and bacterial polysaccharides was
first investigated by killing experiments, where bacteria cells were incubated with sodium hypochlorite (NaClO) with and
without prior incubation with cepacian. The results showed that the polysaccharide had a protective effect towards
bacterial cells. Cepacian was then treated with different concentrations of NaClO and the course of reactions was
followed by means of capillary viscometry. The degradation products were characterised by size-exclusion chromatography,
NMR and mass spectrometry. The results showed that hypochlorite depolymerised cepacian, removed side chains
and O-acetyl groups, but did not cleave the glycosidic bond between glucuronic acid and rhamnose. The structure of
some oligomers produced by NaClO oxidation is reported
Influence of the culture medium in the evaluation of biofilm inhibitory concentrations for isolates of Burkholderia cenocepacia
No full textBacteria belonging to the Burkholderia cepacia complex (Bcc) principally cause chronic pulmonary infections in cystic fibrosis (CF) patients. They are intrinsically resistant to many antibiotics, so therapeutic options are often limited. Moreover, their ability to form biofilms (BF) can retard contact with some antimicrobials, making these infections difficult to eradicate even by drugs that display a Minimal Inhibitory Concentration (MIC) lower than the breakpoint. But the MIC is evaluated on the planktonic forms whereas the higher resistance of the sessile forms (BIC: Biofilm Inhibitory Concentration) towards some antibiotics commonly used in the clinical management of lung infections has been widely described (REF).
One variable experimental condition in the evaluation of the BIC is the choice of the medium. Data available till now have been obtained using different media, as no guidelines have been established yet. However, preliminary results suggest that the composition of the BF matrix (protein/EPS ratio) varies depending on the medium used to culture bacteria (Cescutti, personal communication).
We evaluated the antibiotic susceptibility of a B. cenocepacia clinical isolate, named BTS2, which has stably colonized a patient for ten years, is a good biofilm producer in the sample taken in 2000 (BTS2-00) while shows a significant reduction in this capacity 9 years later (BTS2-09).
We compared the susceptibility of its sessile forms grown inside the biofilm synthesized in three different media: Muller Hinton Broth (MHB), commonly used for antimicrobial susceptibility tests; Yeast Extract Medium (YEM), where Bcc produce large amount of the EPS cepacian (REF); and Synthetic Cystic Fibrosis Medium (SCFM), a synthetic medium that mimics the nutritional composition of CF sputum (REF). A modification of the Calgary Device method was used (REF). Bacteria were initially grown as biofilm using one of the media listed above. Then, the incubation with the antibiotic was always carried on in MHB.
Some of the tested drugs showed different activity levels on the BF depending on the culture medium used, but the most important difference was observed after incubation with aztreonam, whose BICs in different media diverged more than 50x even in absence of significant differences of the BF production. So, as it seems difficult to ascribe only to the thickness of the BF matrix the protection against the drug, we are planning to analyse its ability to spread into the different BF matrices following the course of a labelled molecule by confocal microscopy analysis
The biofilm produced by Burkholderia cepacia complex: molecular aspects and relationship with exopolysaccharides
No full textIntroduction. In cystic fibrosis patients, Burkholderia cepacia complex (Bcc) can cause serious pulmonary chronic infections thanks in part
to the ability to form biofilm, matrix rich in exopolysaccharides. In Bcc grown in the planktonic state, the main exopolysaccharide is cepacian
while in biofilm its presence is controversial.
Methods and Results. Two clinical isolates, named BTS7 and BTS2, were studied.
BTS7 produces abundant cepacian but not much biofilm (quantified by colorimetric method).At least two of the genes involved in cepacian
biosynthesis are not necessary for biofilm production as two BTS7 derivatives, bceB and bceQ knocked out by transposon mutagenesis,
produce biofilm levels comparable to the wild-type.
BTS2 sinthesyzes cepacian only if cultured on a specific medium. It has been colonizing a patient for almost ten years, showing a significant
reduction of biofilm production during this period.This reduction did not appear together with the lack of factors required for the initial
adhesion to the surface, or to differences in some of the Bcc genes involved in biofilm formation.
Moreover, sequencing of its bce locus revealed a bceX gene, absent in BTS7, coding for a trascriptional regulator. Its product may negatively
regulate the production of cepacian but not the one of other polysaccharides, promoting the formation of biofilm.
Conclusions. Cepacian seems to be marginal in the production of biofilm.The reduced ability to produce biofilm of BTS2 suggests possible
gene mutations occurred over time. Using custom arrays we will compare the gene expression of the BTS2 isolates, to identify the genes
responsible for the observed phenotypic changes
Study of class I integron in a Burkholderia cepacia complex strain isolated from blood colture
No full textThe Burkholderia cepacia complex (Bcc) consists of several species that cause lung infections in patients with cystic fibrosis but are also capable to colonize immunocompromised patients. Once established, the infection is usually difficult to eradicate, as Bcc is intrinsically resistant to many antibiotics. Besides, the acquisition of additional resistance determinants by horizontal gene transfer makes very difficult the therapeutic approach to these infections. Among horizontally acquired DNAs, integrons have been frequently reported in many Gramnegative bacteria that affect human health, but they have not been found frequently in Burkholderia isolates until now. In the present work we report on a Bcc isolate, recovered from the blood of an immunocompromised patient, that carries a 2.3 kb class I integron already described in a Salmonella enterica isolate eight years ago, coding for aacA4, aadA1 and catB2 in its cassette array
Analisi genetica dei livelli di acetilazione dell'esopolisaccaride prodotto da Burkholderia cepacia
No full textIntroduzione: Le infezioni causate da Burkholderia cepacia complex (Bcc) in pazienti affetti da fibrosi cistica (CF)
sono spesso correlate ad un aumento di mortalità e l’innata resistenza di questi microrganismi ad un ampio range di
antibiotici complica il trattamento di tali pazienti. Uno dei meccanismi che rendono i batteri più resistenti all’azione
degli antibiotici è la capacità di produrre biofilm.
Metodi: Mediante microdiluizione in brodo e l’utilizzo della tecnologia “Calgary Biofilm Device” sono state valutate le
minime concentrazioni di antibiotico in grado di inibire la crescita di un ceppo Bcc cresciuto in forma planctonica
(MIC) e all’interno di biofilm (BIC). Sono stati paragonati due isolati indipendenti dello stesso ceppo (BTS-2) che, a
distanza di 6 anni (BTS2-00 e BTS2-06), ha notevolmente ridotto la propria capacità di produrre biofilm.
Risultati: I dati preliminari finora ottenuti indicano che l’isolato BTS2-00, produttore di abbondanti quantità di biofilm,
presenta valori di BIC significativamente superiori alle MIC per alcuni degli antibiotici saggiati. Tale incremento è però
molto meno marcato nel caso dell’isolato BTS2-06, che produce biofilm in quantità circa 5 volte inferiore. Le MIC dei
due isolati sono comparabili nella maggior parte dei casi (con l’eccezione della ciprofloxacina per la quale si può
ipotizzare l’acquisizione di un determinante di resistenza avvenuto nel corso del tempo) mentre le BIC di BTS2-06 sono
sempre inferiori rispetto a quelle dell’isolato BTS2-00.
Conclusioni: L’osservazione che BTS2 presenti un incremento della resistenza delle forme sessili rispetto a quelle
planctoniche nei confronti di alcuni degli antibiotici saggiati, non più evidente quando il ceppo riduce la quantità di
biofilm prodotto, avvalora l’ipotesi che la matrice del biofilm costituisca una barriera efficace contro il passaggio di
alcuni antibiotici e dimostra l’importanza di utilizzare test adeguati per valutare l’efficacia della terapia nei pazienti CF
Fitness cost associated with the chromosomal integron In70.2 in Pseudomonas aeruginosa clinical isolates
No full textAn epidemiologic survey performed at the Trieste University Hospital (northeastern Italy) between 1999 and 2002 revealed a remarkable spread of an MDR Pseudomonas aeruginosa strain, named TS-832035, which carried the chromosomal integron In70.2 containing four gene cassettes (blaVIM-1, aacA4, aphA15 and aadA1) in its variable region and conferring resistance to ß-lactams, including carbapenems, and to several aminoglycosides. Moreover, some other P. aeruginosa isolates, strictly related to TS-832035 but lacking in the integron In70.2, were detected, but they remained a minor component within the cluster during the three years of surveillance.They showed an MDR phenotype like TS- 832035, differing only for the susceptibility level to carbapenems. The genomic relatedness between TS-832035 and TS-103 was investigated by random amplification of polymorphic DNA (RAPD) typing, pulsed-field gel electrophoresis (PFGE) analysis of SpeI-digested genomic DNA, and multilocus sequence typing (MLST). The cost of the integron In70.2 on the fitness of TS-832035 was determined by performing growth kinetics and direct competition assays against the clonal isolate TS-103 in three media differing for nutrient availability: a rich medium (Luria Bertani (LB) Broth) and a minimal medium (28 g/l K2HPO4, 12 g/l KH2PO4, 0.4 g/l MgSO4, 7H2O, 4 g/l (NH4)2SO4) added with a rich carbon source (0.4% w/v glucose) or with a poorer carbon source (0.4% w/v sodium acetate). Growth kinetic data were obtained by measuring optical density at 600 nm (OD600). For competition assays, the number of CFU/ml of each isolate was estimated by colony-hybridization. We proved the clonality of the two isolates by molecular investigations.The results of the growth kinetics showed the existence of a significant in vitro fitness cost associated with the integron In70.2, more evident in a poorer medium.The sensitivity of the two isolates to the antimicrobial agents tested was the same, except for the different levels of resistance to carbapenems (MIC 16 μg/ml versus 64-128 μg/ml). Although we can not exclude that other factors may have favoured the in vivo spread of TS-832035, our results suggest that the increased level of resistance to carbapenems has conferred on this isolate a selective advantage able to compensate metabolic cost associated to the integron
Characterization of integrons in Burkholderia cepacia clinical isolates
No full textBurkholderia cepacia is an opportunistic pathogen able to colonize the airways of Cystic Fibrosis (CF) patients, frequently developing chronic infections. In 20% of cases these infections cause severe and poorly controlled pathological situations because of the intrinsic antibiotic resistance expressed by the microorganism. CF patients are often subjected to antibiotic therapy: this facilitates the acquisition of antibiotic resistance determinants by the infecting bacteria. Integrons are mobile genetic elements that are widespread in bacterial populations and favor the acquisition of gene cassettes coding for these determinants.The presence of class 1 integrons was investigated by PCR with primers specific for the 5’ and 3’ ends in Burkholderia isolates recovered from patients in treatment at the CF center of Friuli Venezia Giulia. The same integron, carrying an uncommon allelic form (Ib) of the aacA4 gene in its cassette array and conferring resistance to some aminoglycosides, was found in two independent isolates (different RAPD profiles) infecting two different patients. In both isolates the integron was carried by plasmids and was still present 3 and 6 years later the first finding. Despite the exchange of integrons between bacterial pathogens is fully described, these items were not frequently found in Burkholderia isolates. Although the clinical relevance of the integron we identified is low (a single gene cassette encoding a widespread resistance),we feel concerned that these genetic elements begin to circulate in this bacterial species, as this could make more and more troublesome the treatment of infections notoriously difficult to eradicate
Variazioni di suscettibilità ad antibiotici e al peptide BMAP-27 in isolati multipli di P. aeruginosa ottenuti da pazienti con fibrosi cistica
No full textE’ noto che, nel tessuto polmonare dei pazienti con fibrosi cistica (FC), P. aeruginosa evolve lentamente, accumulando piccole mutazioni, che accentuano le caratteristiche favorevoli al persistere dell’infezione. Questo fatto, in aggiunta all’evenienza d’infezioni plurime, fa sì che la popolazione di P. aeruginosa, che colonizza un singolo paziente, possa mostrare fenotipi multipli. E’ ipotizzabile che la diversificazione stessa favorisca la persistenza, garantendo un ampio spettro di caratteristiche potenzialmente utili a fronteggiare l’attività dei sistemi di difesa e degli antibiotici.
In questo lavoro ci siamo proposti di valutare la diversificazione della popolazione di P. aeruginosa nei confronti della suscettibilità a 5 molecole antibiotiche appartenenti a classi differenti (aztreonam, cefepime, ceftazidime, ciprofloxacina, tobramicina) ed alla catelicidina di origine bovina BMAP-27.
I ceppi di P. aeruginosa sono stati isolati ed identificati presso l’Ospedale Bambino Gesù di Roma da 19 pazienti FC con infezione cronica. Ciascun paziente ha fornito da 2 a 4 isolati, provenienti da un unico campione di espettorato e scelti per la diversa morfologia delle colonie. La tipizzazione è stata eseguita mediante macrorestrizione del genoma con SpeI, seguita dalla risoluzione dei frammenti con PFGE. Le MIC sono state valutate con il metodo della microdiluizione in brodo, seguendo le linee guida CLSI. BMAP-27 è stato sintetizzato in fase solida, utilizzando la chimica Fmoc.
La tipizzazione degli isolati ha dimostrato che 8/19 pazienti erano colonizzati da un solo ceppo, del quale era possibile distinguere, in base alla morfologia delle colonie, 2 o 3 varianti fenotipiche; 10 pazienti erano infettati da 2 ceppi diversi ed 1 paziente era infettato da tre ceppi. Alcuni ceppi infettavano più pazienti (fino ad un massimo di 7).
Lo studio della suscettibilità agli antibiotici ha evidenziato l’ampia diffusione di resistenze e la presenza di differenze significative nelle MIC di ceppi con profilo PFGE diverso coinfettanti lo stesso paziente. In 5 pazienti sono state riscontrate anche variazioni significative di MIC (8-32x) tra varianti fenotipiche dello stesso ceppo, presenti simultaneamente nell’espettorato.
Le MIC di BMAP-27 erano comprese tra 2 e 16 μM (MIC90= 8 μM; MIC50= 4 μM) e, rispetto agli antibiotici convenzionali, mostravano una variabilità inferiore, sia tra ceppi diversi che tra varianti fenotipiche dello stesso ceppo (al massimo 4x).
Lo studio conferma che, nel polmone del singolo paziente FC, possono essere presenti contemporaneamente più ceppi di P. aeruginosa, e varianti degli stessi, con suscettibilità diverse agli antibiotici. Inoltre, dimostra una minore variabilità delle MIC del peptide BMAP-27
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