Qualidade microbiológica e perfil de sensibilidade antimicrobiana dos isolados de tilápias (Oreochromis spp.) de pesque-pague da microrregião do Estado de São Paulo

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Epidemiologia molecular e estudo dos fatores de virulência de Staphylococcus aureus resistentes à oxacilina isolados de feridas em pacientes atendidos em unidades básicas de saúde da cidade de Botucatu

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Efeito da combinação de antibióticos e sinvastatina sobre microrganismos de interesse endodôntico e na expressão de marcadores odontoblásticos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Phenotypical characterization and adhesin identification in escherichia coli strains isolated from dogs with urinary tract infections

Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.

Targeting the Transposase Domain of the DNA Repair Component Metnase to Enhance Chemotherapy

Previous studies have shown that the DNA repair component Metnase (SETMAR) mediates resistance to DNA damaging cancer chemotherapy. Metnase has a nuclease domain that shares homology with the Transposase family. We therefore virtually screened the tertiary Metnase structure against the 550,000 compound ChemDiv library to identify small molecules that might dock in the active site of the transposase nuclease domain of Metnase. We identified eight compounds as possible Metnase inhibitors. Interestingly, among these candidate inhibitors were quinolone antibiotics and HIV integrase inhibitors, which share common structural features. Previous reports have described possible activity of quinolones as antineoplastic agents. Therefore, we chose the quinolone ciprofloxacin for further study, based on its wide clinical availability and low toxicity. We found that ciprofloxacin inhibits the ability of Metnase to cleave DNA and inhibits Metnase-dependent DNA repair. Ciprofloxacin on its own did not induce DNA damage, but it did reduce repair of chemotherapy-induced DNA damage. Ciprofloxacin increased the sensitivity of cancer cell lines and a xenograft tumor model to clinically relevant chemotherapy. These studies provide a mechanism for the previously postulated antineoplastic activity of quinolones, and suggest that ciprofloxacin might be a simple yet effective adjunct to cancer chemotherapy. Cancer Res; 72(23); 6200-8. (C) 2012 AACR.

Occurrence and sorption of fluoroquinolones in poultry litters and soils from sao Paulo State, Brazil

Animal production is one of the most expressive sectors of Brazilian agro-economy. Although antibiotics are routinely used in this activity, their occurrence, fate, and potential impacts to the local environment are largely unknown. This research evaluated sorption-desorption and occurrence of four commonly used fluoroquinolones (norfloxacin, ciprofloxacin, danofloxacin, and enrofloxacin) in poultry litter and soil samples from Sao Paulo State, Brazil. The sorption-desorption studies involved batch equilibration technique and followed the OECD guideline for pesticides. All compounds were analyzed by HPLC, using fluorescence detector. Fluoroquinolones' sorption potential to the poultry litters (K-d <= 65 L kg(-1)) was lower than to the soil (K-d similar to 40,000 L kg(-1)), but was always high (>= 69% of applied amount) indicating a higher specificity of fluoroquinolones interaction with soils. The addition of poultry litter (5%) to the soil had not affected sorption or desorption of these compounds. Desorption was negligible in the soil (<= 0.5% of sorbed amount), but not in the poultry litters (up to 42% of sorbed amount). Fluoroquinolones' mean concentrations found in the poultry litters (1.37 to 6.68 mg kg(-1)) and soils (22.93 mu g kg(-1)) were compatible to those found elsewhere (Austria, China, and Turkey). Enrofloxacin was the most often detected compound (30% of poultry litters and 27% of soils) at the highest mean concentrations (6.68 mg kg(-1) for poultry litters and 22.93 mu g kg(-1) for soils). These results show that antibiotics are routinely used in poultry production and might represent one potential source of pollution to the environment that has been largely ignored and should be further investigated in Brazil. (C) 2012 Elsevier B.V. All rights reserved.

Nosocomial outbreak of Pantoea agglomerans bacteraemia associated with contaminated anticoagulant citrate dextrose solution: new name, old bug?

We describe an outbreak investigation of Pantoea agglomerans bacteraemia associated with anticoagulant citrate-dextrose 46% (ACD) solution prepared in-house. A healthy man presented with septic shock during plasmapheresis for granulocyte donation. The solution used for priming and blood samples were sent for culture. Identification of the isolate to species level was performed by gyrB sequencing. Typing was performed by pulsed-field gel electrophoresis (PFGE). In total, eight cases were identified during a three-week period. P. agglomerans was also cultured from six ACD solution bags. Isolates from patients and ACD bags were identical by PFGE. All isolates were susceptible to ampicillin, cephazolin, gentamicin, ciprofloxacin, cefepime and imipenem. (C) 2011 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

Antimicrobial activity of triantibiotic paste, 2% chlorhexidine gel, and calcium hydroxide on an intraoral-infected dentin biofilm model

Introduction: The purpose of this study was to evaluate the antimicrobial activity of calcium hydroxide, 2% chlorhexidine gel, and triantibiotic paste (ie, metronidazole, minocycline, and ciprofloxacin) by using an intraorally infected dentin biofilm model. Methods: Forty bovine dentin specimens were infected intraorally using a removable orthodontic device in order to induce the biofilm colonization of the dentin. Then, the samples were treated with the medications for 7 days. Saline solution was used as the control. Two evaluations were performed: immediately after the elimination of the medication and after incubation in brain-heart infusion medium for 24 hours. The Live/Dead technique (Invitrogen, Eugene, OR) and a confocal microscope were used to obtain the percentage of live cells. Nonparametric statistical tests were performed to show differences in the percentage of live cells among the groups (P < .05). Results: Calcium hydroxide and 2% chlorhexidine gel did not show statistical differences in the immediate evaluation. However, after application of the brain-heart infusion medium for 24 hours, 2% gel chlorhexidine showed a statistically lesser percentage of live cells in comparison with calcium hydroxide. The triantibiotic paste significantly showed a lower percentage of live cells in comparison with the 2% chlorhexidine gel and calcium hydroxide groups in the immediate and secondary (after 24 hours) evaluations. Conclusions: The triantibiotic paste was most effective at killing the bacteria in the biofilms on the intraorally infected dentin model in comparison with 2% chlorhexidine gel and calcium hydroxide

Phenotypical characterization and adhesin identification in Escherichia coli strains isolated from dogs with urinary tract infections

Pathogenic strains of Escherichia coli are the most common bacteria associated with urinary tract infections in both humans and companion animals. Standard biochemical tests may be useful in demonstrating detailed phenotypical characteristics of these strains. Thirteen strains of E. coli isolated from dogs with UTIs were submitted to biochemical tests, serotyping for O and H antigens and antimicrobial resistance testing. Furthermore, the presence of papC, sfa, and afa genes was evaluated by PCR, and genetic relationships were established using enterobacterial repetitive intergenic consensus PCR (ERIC-PCR). The antimicrobial that showed the highest resistance rate among the isolates was nalidixic acid (76.9%), followed by cephalotin (69.2%), sulfamethoxazole + trimethoprim (61.5%), tetracycline (61.5%), streptomycin (53.8%), ciprofloxacin (53.8%), ampicillin (46.2%), gentamicin (30.8%) and chloramphenicol (23.1%). No isolate was resistant either to meropenem or nitrofurantoin. Among the five clusters that were identified using ERIC-PCR, one cluster (A) had only one strain, which belonged to a serotype with zoonotic potential (O6:H31) and showed the genes papC+, sfa+, afa-. Strains with the genes papC-, sfa+, afa- were found in two other clusters (C and D), whereas all strains in clusters B and E possessed papC-, sfa-, afa- genes. Sucrose and raffinose phenotypic tests showed some ability in discriminating clusters A, B and C from clusters D and E.

Mutations in the quinolone resistance-determining regions of gyrA and parC in Enterobacteriaceae isolates from Brazil

Mutations in the quinolone resistance-determining regions (QRDR) in chromosomal gyrA and parC genes and fluoroquinolone susceptibility profiles were investigated in quinolone-resistant Enterobacteriaceae isolated from community and hospitalized patientsin the Brazilian Southeast region. A total of 112 nalidixic acid-resistant enterobacterial isolates collected from 2000 to 2005 were investigated for mutations in the topoisomerases genes gyrA and parC by amplifying and sequencing the QRDR regions. Susceptibility to fluoroquinolones was tested by the agar dilution method. Amongst the 112 enterobacterial isolates, 81 (72.3%) were resistant to ciprofloxacin and 5 (4.5%) showed reduced susceptibility. Twenty-six (23.2%) were susceptible to ciprofloxacin. Several alterations were detected in gyrA and parC genes. Escherichia coli isolates (47.7%) showed double mutations in the gyrA gene and a single one in the parC gene. Two unusual aminoacid substitutions are reported, an Asp87-Asn in a Citrobacter freundii isolate with reduced susceptibility to fluoroquinolones and a Glu84-Ala in one E. coli isolate.Only a parC gene mutation was found in fluoroquinolone-susceptible Enterobacter aerogenes. None of the isolates susceptible to ciprofloxacin presented mutations in topoisomerase genes. This comprehensive analysis of QRDRs in gyrA and parC genes, covering commonly isolated Enterobacteriaceae in Brazil is the largest reported up to now.

An assessment of the concentrations of pharmaceutical compounds in wastewater treatment plants on the island of Gran Canaria(Spain)

[EN] An assessment of the concentrations of thirteen different therapeutic pharmaceutical compounds was conducted on water samples obtained from different wastewater treatment plants (WWTPs) using solid phase extraction and high- and ultra-high-performance liquid chromatography with mass spectrometry detection (HPLC-MS/MS and UHPLC-MS/MS), was carried out. The target compounds included ketoprofen and naproxen (anti-inflammatories), bezafibrate (lipid-regulating), carbamazepine (anticonvulsant), metamizole (analgesic), atenolol (?-blocker), paraxanthine (stimulant), fluoxetine (antidepressant), and levofloxacin, norfloxacin, ciprofloxacin, enrofloxacin and sarafloxacin (fluoroquinolone antibiotics). The relative standard deviations obtained in method were below 11%, while the detection and quantification limits were in the range of 0.3 ? 97.4 ng·L-1 and 1.1 ? 324.7 ng·L-1, respectively. The water samples were collected from two different WWTPs located on the island of Gran Canaria in Spain over a period of one year. The first WWTP (denoted as WWTP1) used conventional activated sludge for the treatment of wastewater, while the other plant (WWTP2) employed a membrane bioreactor system for wastewater treatment. Most of the pharmaceutical compounds detected in this study during the sampling periods were found to have concentrations ranging between 0.02 and 34.81 ?g·L-1.

Optimización de un método basado en SPE-LC-MS/MS para la determinación de multiresiduos de compuestos farmacéuticos en aguas depuradas

Universidad de Las Palmas de Gran Canaria, Facultad de Ciencias del Mar, Doctorado en Gestión Costera.

Assessment of the presence of pharmaceutical compounds in seawater samples from coastal area of Gran Canaria Island (Spain)

[EN]This study presents the evaluation of seven pharmaceutical compounds belonging to different commonly used therapeutic classes in seawater samples from coastal areas of Gran Canaria Island. The target compounds include atenolol (antihypertensive), acetaminophen (analgesic), norfloxacin and ciprofloxacin (antibiotics), carbamazepine (antiepileptic) and ketoprofen and diclofenac (anti-inflammatory). Solid phase extraction (SPE) was used for the extraction and preconcentration of the samples, and liquid chromatography tandem mass spectrometry (LC-MS/MS) was used for the determination of the compounds. Under optimal conditions, the recoveries obtained were in the range of 78.3% to 98.2%, and the relative standard deviations were less than 11.8%. The detection and quantification limits of the method were in the ranges of 0.1–2.8 and 0.3–9.3 ng·L−1, respectively. The developed method was applied to evaluate the presence of these pharmaceutical compounds in seawater from four outfalls in Gran Canaria Island (Spain) during one year. Ciprofloxacin and norfloxacin were found in a large number of samples in a concentration range of 9.0–3551.7 ng·L−1. Low levels of diclofenac, acetaminophen and ketoprofen were found sporadically.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

Indagine sulla presenza di Helicobacter pullorum in allevamenti avicoli italiani

From September 2005 to December 2006, in order to define the prevalence of Helicobacter pullorum in broiler chickens, laying hens and turkey, a total of 365 caecum contents of animals reared in 76 different farms were collected at the slaughterhouse. A caecum content of a ostrich was also sampled. In addition, with the aim of investigating the occurrence of H. pullorum in humans, 151 faeces were collected at the Sant’Orsola-Malpighi University Hospital of Bologna from patients suffering of gastroenteritis. A modified Steele–McDermott membrane filter method was used. Gram-negative curved rod bacteria were preliminary identified as H. pullorum by a PCR assay based on 16S rRNA, then subjected to a RFLP-PCR assay to distinguish between H. pullorum and H. canadensis. One isolate from each farm was randomly selected for phenotypic characterization by biochemical methods and 1D SDSPAGE analysis of whole cell proteins profiles. Minimum Inhibitory Concentration (MIC) for seven different antibiotics were also determined by agar dilution method. Moreover, to examine the intraspecific genomic variability, two strains isolated from 17 different farms were submitted to genotyping by Pulse-Field Gel Electrophoresis (PFGE). In order to assess the molecular basis of fluorquinolone resistance in H. pullorum, gyrA of H. pullorum CIP 104787T was sequenced and nucleotide sequences of the Quinolone Resistance Determining Region (QRDR) of a total of 18 poultry isolates, with different MIC values for ciprofloxacin and nalidixic acid, were compared. According to the PCR and PCR-RFLP results, 306 out of 366 animals examined were positive for H. pullorum (83,6%) and 96,1% of farms resulted infected. All positive samples showed a high number of colonies (>50) phenotipically consistent with H. pullorum on the first isolation media, which suggests that this microrganism, when present, colonizes the poultry caecum at an elevate load. No human sample resulted positive for H. pullorum. The 1D SDS-PAGE whole protein profile analysis showed high similarity among the 74 isolates tested and with the type strain H. pullorum CIP 104787T. Regarding the MIC values, a monomodal distribution was found for ampicillin, chloramphenicol, gentamicin and nalidixic acid, whereas a bimodal trend was noticed for erythromycin, ciprofloxacin and tetracycline (indicating an acquired resistance for these antibiotics). Applying the breakpoints indicated by the CSLI, we may assume that all the H. pullorum tested are sensitive only to gentamicin. The intraspecific genomic variability observed in this study confirm that this species don’t have a clonal population structure, as motioned by other autors. The 2490 bp gyrA gene of H. pullorum CIP104787T with an Open Reading Frame (ORF) encoding a polypeptide of 829 amino acids was for the first time sequenced and characterized. All ciprofloxacin resistant poultry isolates showed ACA®ATA (Thr®Ile) substitution at codon 84 of gyrA corresponding to codons of gyrA 86, 87 and 83 of the Campylobacter jejuni, H. pylori and Escherichia coli, respectively. This substitution was functionally confirmed to be associated with the ciprofloxacin resistant phenotype of poultry isolates. This is the first report of isolation of H. pullorum in turkey and in ostrich, indicating that poultry species are the reservoir of this potential zoonotic microorganisms. In order to understand the potential role as food-borne human pathogen of H. pullorum, further studies must be carried on.