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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
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
ITS2 metabarcoding analysis complements lichen mycobiome diversity data
Full text linkLichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The
complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and
high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its
sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi.
Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a wellcharacterized,
alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as
asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes.
We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence
in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity
widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary
analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes
pKBuS13, a KPC-2-Encoding Plasmid from Klebsiella pneumoniae Sequence Type 833, Carrying Tn4401b Inserted into an Xer Site-Specific Recombination Locus
Full text linkHere, we report the first detection of a Klebsiella pneumoniae carbapenemase 2 (KPC-2)-producing Klebsiella pneumoniae strain belonging to sequence type 833 (ST833), collected in an Italian hospital from a patient coming from South America. Its bla KPC determinant was carried by a ColE1 plasmid, pKBuS13, that showed the Tn4401b::bla KPC-2 transposon inserted into the regulatory region of an Xer site-specific recombination locus. This interfered with the correct resolution of plasmid multimers into monomers, lowering plasmid stability and leading to overestimation of the number of plasmids harbored by a single host cell. Sequencing of the fragments adjacent to Tn4401b detected a region that did not have significant matches in databases other than the genome of a carbapenem-resistant Escherichia coli strain collected during the same year at a hospital in Boston. This is interesting in an epidemiologic context, as it suggests that despite the absence of tra genes and the instability under nonselective conditions, the circulation of pKBuS13 or of analogous plasmids might be wider than reported
Integroni di classe 1 ed antibiotico-resistenze in batteri gram-negativi isolati da Larus cachinnans michahellis
No full textThe vaginal microbiota of healthy female cats
Full text linkThe vaginal microbiota of the queen (i.e., female cat) has never been described using culture independent methods. The objectives of the present research were to describe the vaginal microbiota of healthy domestic shorthair queens using both 16S rRNA sequencing and culture, and to assess the effects of age, living environment, and reproductive season on its composition. Thirty queens undergoing elective ovariectomy were included in the study. The vaginal samples were collected just before surgery, from animals under general anaesthesia. Two consecutive mini-swabs were introduced in the queens' vaginal tract. A preliminary study with 10 healthy queens aimed to negate sampling order's effect. Two consecutive samples for sequencing (5 queens, 10 swabs) and culture (5 queens, 10 swabs) were collected, confirming a match (100 % in culture, Bray-Curtis P = 0.96 in sequencing). The experiment included 20 queens that were prospectively grouped based on age (prepubertal N = 10, adult N = 10), living environment (indoor N = 10, outdoor N = 10), and time of the year, whether during the reproductive season (N = 10) or during seasonal anoestrous (N = 10). Bacteria were identified through metataxonomic analysis, amplifying the V1-V2 regions of 16S rRNA gene, and through standard culture followed by MALDI-TOF MS. The feline vaginal microbiota is dominated by Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteria. Escherichia-Shigella, Streptococcus, and Pasteurella were the most abundant genera. Although culture underestimated bacterial richness and diversity compared to sequencing, Escherichia and Streptococcus were the most isolated bacteria. No bacterial growth was observed in 15 % of samples (N = 3/20), whereas growth of one or two bacterial species was observed in 64.7 % (N = 11/17) and 35.3 % (N = 6/17) of cases, respectively. No differences in terms of alpha (Kruskal-Wallis rank sum test P = 0.65) and beta diversity (Bray-Curtis, Unweighted and Weighted UniFrac analyses P > 0.5) were observed. Although a difference in alpha diversity based on phylogenetic tree (P = 0.02) was detected between indoor and outdoor queens. In conclusion, mixed and monoculture of Escherichia coli, Streptococcus canis, Staphylococcus felis, and Enterococcus spp. are normal findings within the cat vagina. Age and reproductive season do not influence the feline vaginal microbiota, whereas further research is needed to elucidate the role of the living environment
L' ANTIBIOTICO RESISTENZA NELL' AMBIENTE MONITORAGGIO IN BATTERI ISOLATI DA AVIFAUNA
No full text2001/2002Development of antibiotic resistance in bacteria is mainly due to the presence of resistance genes and to the selective pressure exerted by antibiotic use. Resistance can result from spontaneous mutations or acquisition of genes coding for a resistance mechanism. Antibacterial agents are used in agricultural techniques, in human and animai therapy and prevention of bacteria infections, and are added continuously to animai feeds to promote growth. As a result of exposure to antibiotics, the level of antibiotic resistance of bacteria belonging to the normal intestina! flora of human and animals increases. These bacteria not only constitute an enormous reservoir of resistance genes for potentially pathogenic bacteria, but also their level of resistance is considered to be a good indicator for the selection pressure exerted by antibiotic use in that population and for the resistance problems to be expected in pathogens. Indeed, reservoirs of antibiotic resistance in humans and in animals can interact in different ways: food and water are the most probable vectors of trasmission to the intestina! flora. Population of gulls (Larus cachinnans michahellis) has increased in North-East of Italy during the past decade. This increase has been attribuited to the ability of gulls to adapt to urban areas, in fact they are able to nest on roofs and feed of urban waste. Gulls ability to over fly large areas suggests that their feces may have a potential role in bacterial dissemination and more underhand antibiotic resistance in urban and rural environment. Transfer of antibiotic resistance genes between different species of bacteria c an be facilitated by mobile DNA elements, such as transposons and plasmids. Integrons have been identified on these mobile elements, and they often contain one or more genes that encode antibiotic resistance. Nine classes of integrases have been described, but class l is prevalent in Enterobacteriaceae. In this work l 02 cloacal swabs of Larus cachinnans michahellis were collected in natural reserve during spring in 2000 and 200 l. 214 strains of Enterobacteriaceae were isolated and identified as Proteus sp.(n = 89), E.coli (n = 84), Klebsiella sp.(n. = 18), Sa/monella sp. (n = 17) and Citrobacter sp. (n = 6). Also 162 Gram-positive bacteria strains were identified as Enterococcusfaecalis (n= 101) and Staphylococcus sp. (n= 61). Isolated avian strains were tested for susceptibility to a battery of antibiotics representing various classes of them. Gram-positive isolates showed low levels of resistance, but Enterobacteriaceae were resistant to a lot of antibiotics, especially to ampicillin (Sa/monella sp., E. coli and Proteus sp.), to tetracycline (Sa/monella sp., andE. coli), to streptomycin (Proteus sp., E. coli, Klebsiella sp. and Sa/monella sp.), to nalidixic acid (Proteus sp. andE. coli). The high resistance levels found in Gram-negative strains are very important if we consider the natural habitat of monitorated avifauna, and we could explain this fact as a result of the spread of environmental antibiotic contaminants with their consequent selection pressure and the dissemination of antibiotic resistance genes by horizontal transfer. Gram-negative avian strains were also screened for class l integrase using a specific probes which hybridizes to conserved regions of integron encoded gene intll. 25 of the 214 isolates were positive and showed to have class l integrons. Their sizes were detected by PCR with specific primers 5'CS-3'CS, flanking variable region of integron. Integrons ranged from 500 hp to 4800 bp. The incidence of class l integrons was correlated with multiple-drug resistances exhibited by avian Enterobacteriaceae to streptomycin, trimethoprimsulfamethoxazolo, tetracycline and chloramphenicol. The prevalent size ofintegrons was 1000 hp, 1600 hp and 1700 hp. Also 80 human clinical Enterobacteriaceae strains were screened for the integron presence, 16 strains showed to carry integrons ranged from 1000 pb to 2000 pb.The molecular characterization of integrons from avian and human strains, using different restriction endonucleases like Alul, Cfol, Mspl and Rsal, revealed the same types of integrons: 1000 pb integron, with the same restriction pattem, was found in 2 E. coli from Larus, in l E. coli and 5 Proteus sp. from human. An identica! 1600 pb integron was found in 6 avian strains (3 E. coli, l Klebsiella sp. and 2 Proteus sp.) and 3 Proteus sp. from human; 1700 pb integron was found in 2 E. coli from Larus and 4 E. coli from human. PFGE analysis ofthe strains from animals and humans carring the same types of integrons revelead different pattems. The presence of identica! integrons in different isolates from animals and humans implies horizontal transfer of the complete integron arrangement. 1000, 1600 and 1700 pb integrons were further characterized by sequencing. In integrons of l 000 pb sequence analysis identified a single aadA l gene cassette, integrons of 1600 pb contained dhfrl and aadA genes; integrons of 1700 pb contained d.frl7 and aadA5 genes. aadA, aadA l and aadA5 gene cassettes encoding an Aminoglicoside adenyltransferase and conferring resistance to streptomycin an d spectinomycin, dhfr an d dfr 17 gene cassettes encoding a dihydrofolate reductase and conferring resistance to trimethoprimXV Ciclo1967Versione digitalizzata della tesi di dottorato cartacea
Relationship between antibiotic susceptibility and biofilm production in a clinical strain of Burkholderia cepacia complex
No full textInfections caused by Burkholderia cepacia complex in patients with cystic fibrosis (CF) are often related to increased mortality. One of the mechanisms that makes the bacteria more resistant to the action of antibiotics is the ability to produce biofilm. It has been compared the antibiotic resistance of two clinical isolates of a biofilm-producing strain called BTS-2, responsible for chronic pulmonary infection in a CF patient. Bacteria were incubated in bothYeast Extract Mannitol Medium (YEM) and Mueller Hinton Broth (MHB) at 30°C for 24 hours. Biofilm was quantified by spectrophotometer readings (OD570) of the extracted crystal violet. By microdilution broth, we evaluated the minimum concentration of antibiotic inhibiting the growth of the planktonic form (MIC); by the technology “Calgary Biofilm Device” and by Resazurin, we evaluated the susceptibility of sessile forms (BIC). Data demonstrate that the microorganism in the course of time changed its sensitivity to some of the antibiotics tested.The comparison between sessile and planktonic forms of BTS2 pre-incubated in MHB showed that sessile forms increased their resistance to antibiotics in a few cases only. BICs of BTS2 pre-incubated in YEM, where the strain produced an amount of biofilm significantly lower than in MHB, showed a higher susceptibility to 4 of the 10 antibiotics tested. Our data showed that the susceptibility of sessile forms of a strain is not always lower than the susceptibility of its planktonic forms and that culture medium can affect significantly the biofilm production
Insights into the Oral Bacterial Microbiota of Sows
Full text linkThe investigation of bacterial microbiota represents a developing research field in veterinary medicine intended to look for correlations between animal health and the balance within bacterial populations. The aim of the present work was to define the bacterial microbiota of the oral cavity of healthy sows, which had not been thoroughly described so far. In total, 22 samples of oral fluid were collected and analyzed by 16S-rRNA gene sequencing. CLC Genomics Workbench 20.0 (QIAGEN Digital Insights, Aarhus, Denmark) was then used to examine the results. The predominant orders were Lactobacillales, Clostridiales, and Corynebacteriales. Lactobacillaceae, Corynebacteriaceae, Moraxellaceae, Aerococcaceae, and Staphylococcaceae were the most represented families. As regards the most abundant genera, Lactobacillus, Corynebacterium, Acinetobacter, Staphylococcus, Rothia, Aerococcus, and Clostridium can be pointed out as the bacterial core microbiota. Sows were also divided into “gestating” and “lactating” groups, and mild differences were found between pregnant and lactating sows. The data herein described represent an original contribution to the knowledge of the porcine bacterial microbiota. Moreover, the choice of sows as experimental animals was strategic for identifying the adult microbial community. These data provide a basis for further studies on the oral bacterial microbiota of pigs
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