Uso del sistema E-test per lo studio di combinazioni antibiotiche verso batteri Gram-negativi multiresistenti in Fibrosi Cistica

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Caratterizzazione di isolati di Pseudomonas aeruginosa provenienti da pazienti affetti da fibrosi cistica

Pseudomonas aeruginosa is the major pulmonary pathogen that causes morbidity and mortality in burned, immunocompromised and cystic fibrosis patients. Among the various virulence factors, type IV-pili, play a major role in mediating bacteria-host cells interactions, in formation of biofilm and for twiching motility.These pili are composed of pilin, 15000-16000 molecular weight monomeric subunit, synthesized from pilA gene. The N-terminal region of pilin protein is strong conserved and is important for the oligomerization.The C-terminal region is less conserved and contains a disulfide-bonded loop (DSL) structure that is thought to interact with the eukaryotic glycolipid receptor “asialo GM1”. Analysis of pilin allele distribution among isolates from various sources revealed the presence of six groups of pilin allels characterized by different DSL sequence and different associated accessory genes in pilA chromosomal locus. 81 P. aeruginosa isolates were recovered from cystic fibrosis patients during a 3 years period. 30 of these strains were grown and their genomic DNA was prepared using a rapid method for gram-negative bacteria. PCR primers were used for amplification of pilA and adjacent sequences revealing the presence of three different amplification products. One of these is highly homologous with pilA gene of PA14 strain, the others are identical to PA103 and PAK pilA genes. Our study revealed in the prevalence of isolates with group II pilin genes from Cystic Fibrosis compared with other groups that are predominant in previous studies

In Vitro Resistance to Macrolides and Clindamycin by Group B Streptococcus Isolated from Pregnant and Nonpregnant Women

Background. Despite the introduction of screening bases intrapartum prophylaxis, Streptococcus agalactiae is still an important etiological agent of perinatal infections. The increasing rate of resistance and the differences in resistance pattern among countries suggest that a program of surveillance at the institutional level is important in determining optimal prophylaxis. In contrast, knowledge on GBS epidemiology in Italy is limited, and no data are available in the Southern region of the country. We sought to determine the occurrence of resistance to macrolides and clindamycin of GBS isolates in pregnant and nonpregnant women. Methods. Between 2005 and 2008, 1346 vaginal and 810 rectovaginal swabs were obtained from pregnant and not-pregnant women. Results. The occurrence of macrolides and clindamycin resistance was 16.5% in 2005 increasing up to 69.9% in 2008. A high percentage of isolates was resistant to tetracycline through all the study period with no statistically significant annual. Conclusions. In our cohort, an increase of in vitro resistance of GBS to macrolides and clindamycin is clearly evident. The discordance with reports from different countries emphasize the crucial role of microbiological methods in setting possible therapeutic strategies

kluyvera ascorbata infection in cystic fibrosis airway disease

Bacilli belonged to genus Kluyvera are Gram negative non-fermentative microorganisms described in the Enterobacteriaceae family, generally considered as saprophytes of the digestive tract.Their role in pathogenesis of infectious disease is unclear, but recently, they are emerged as a cause of clinically significant disease in no-CFpopulation and several anatomic sites are involved as urinary, respiratory and gastrointestinal tracts. In the present note, we describe the identification of Kluyvera ascorbata isolates, obtained from sputum samples of one CF patient (male, 10 years old). The note concerns a patient suffering from chronic obstructive bronchopulmonary disease and chronic Pseudomonas aeruginosa infection. Isolates showed a good growth on MacConkey agar and BCSA, after incubation at 37°C for 24-48 h and they were sensitive to several antibiotics, as cephalosporins, carbapenems, quinolones and trimethoprim-sulfamethoxazole. Clinical observation and the spirometric parameters early before and after isolation of microrganism did not show any worsening status in our patient. The selective pressure given by intensive use of antibiotics in CF patients increases susceptibility to infections by opportunistic microrganisms. Our report underlines the importance of careful bacterial identification and antimicrobial susceptibility study in defining the potential role of new pathogen bacteria in CF lung and to address possible therapeutic strategies that may help to guide antibiotic therapy regimes in CF patients

Microbiology of airway disease in a cohort of patients with Cystic Fibrosis

BACKGROUND: Recent reports document an increasing incidence of new Gram-negative pathogens such as Stenotrophomonas maltophilia and Alcaligenes xylosoxidans isolated from patients with Cystic Fibrosis, along with an increase in common Gram-negative pathogens such as Pseudomonas aeruginosa and Burkholderia cepacia complex. Furthermore, the increase in multidrug-resistance of such organisms makes the therapeutic management of these patients more problematic. Therefore, careful isolation and identification, and accurate studies of susceptibility to antibiotics are critical for predicting the spread of strains, improving therapeutic measures and facilitating our understanding of the epidemiology of emerging pathogens. The first aim of this study was to determine the incidence and the prevalence of colonization by Gram-negative organisms isolated from respiratory samples of Cystic Fibrosis patients in the Regional Referral Cystic Fibrosis Centre of Naples; the second was to evaluate the spectrum of multidrug-resistance of these organisms. METHODS: Patients (n = 300) attending the Regional Cystic Fibrosis Unit were enrolled in this study over 3 years. Sputum was processed for microscopic tests and culture. An automated system, Phoenix (Becton Dickinson, Sparks, Maryland, USA), was used for phenotypic identification of all strains; the API 20 NE identification system (bioMérieux, Marcy l'Etoile, France) was used when the identification with the Phoenix system was inaccurate. A PCR-RFLP method was used to characterize the organisms in the Burkholderia cepacia complex. A chemosusceptibility test on microbroth dilutions (Phoenix) was used. Primary outcomes such as FEV1 were correlate with different pathogens. RESULTS: During the period of study, 40% of patients was infected by Pseudomonas aeruginosa, 7% by Burkholderia cepacia complex, 11% by Stenotrophomonas maltophilia and 7% by Alcaligenes xylosoxidans. Of the strains isolated, 460 were multidrug-resistant. Multiresistant were Pseudomonas aeruginosa and Burkholderia cepacia complex. CONCLUSION: The results confirm previously reported data; in particular, they show an increase the isolation of non-fermentative Gram-negative bacteria in Cystic Fibrosis patients. They also demonstrate increased resistance to antibiotics. Beta-lactams are rarely effective, with exception of ceftazidime, which is the most efficacious agent against multiresistant strains. Aminoglycosides and quinolones are poorly efficacious

Uso del sistema E-test per lo studio di combinazioni antibiotiche verso batteri Gram-negativi multiresistenti in Fibrosi Cistica

Objectives: Cystic Fibrosis patients are prone to infection by Gram-negative bacteria, such as Pseudomonas aeruginosa and Burkholderia cepacia, which become very resistant with recurrent antibiotic treatments.The purpose of this study was to evaluate the susceptibility patterns of 12 isolates of Burkholderia cepacia and 8 isolates of Pseudomonas aeruginosa, isolated from Cystic Fibrosis patients to five individual antibiotics (ceftazidime, ciprofloxacin, piperacillin/tazobactam, levofloxacin and trimethoprim-sulfamethoxazole) and to four antibiotic combinations (ceftazidime associated with one of the other antibiotics). Methods: Susceptibility tests were carried out using an agar diffusion method, the E-test (AB Biodisk, Solna, Sweden). Results: Strains were selected because of their resistance to individual antimicrobial agents, tested with automated system (Phoenix, BD), which ranged from 41.6% for ceftazidime to 83.3% for ciprofloxacin for Burkholderia cepacia and from 25% for ceftazidime to 100% for trimethoprim-sulfamethoxazole for Pseudomonas aeruginosa. By using E-test,we were able to demonstrate synergy against 2 strains of Pseudomonas aeruginosa (25%) with ceftazidime- piperacillin/tazobactam. No synergy was detected against all strains of Burkholderia cepacia. Conclusions:These results suggest that the E-test offers a simple, labour-efficient and accurate method for MIC determination on agar medium and the susceptibility to antibiotic combinations greatly improves the guide to antibiotic therapy for infections to Gram-negative bacteria in Cystic Fibrosis patients

Respiratory infections by Achromobacter xylosoxidans in a cohort of Cystic Fibrosis patients: identification, antimicrobial susceptibility and molecular epidemiology

Pulmonary infections by Gram-negative bacteria such as Achromobacter xylosoxidans are recovered frequently in patients with Cystic Fibrosis. Aims of this study were to value the isolation frequency of A.xylosoxidans strains in a cohort of Cystic Fibrosis patients, to investigate their antimicrobial sensitivity and to establish possible clonal likeness among strains.A retrospective study was undertaken between January 2004 and December 2008 on 300 patients receiving care at the Regional Cystic Fibrosis Centre of “Federico II” University, Naples. Sputum samples were collected and selective media as well as commercial systems for bacterial identification were used. The activity of antimicrobial agents was determined using diffusion and micro-dilution methods. For DNA-fingerprinting, a genomic DNA macrorestriction followed by pulsed-field gel electrophoresis was carried out. A total of 238 strains from 51 patients were isolated. Strains were resistant to aztreonam, about half of these were resistant to gentamicin and trimethoprim-sulphamethoxazole. They were sensitive to piperacillin, piperacillin/tazobactam, and also to carbapenems, quinolones, cephalosporines. Macrorestriction analysis applied on some isolates showed substantial heterogeneity among strains.Actually, the prognostic role of A. xylosoxidans in Cystic Fibrosis is unclear, but this finding must imply difficulties on therapeutic approach. So, it is need to be on the look out regard such microorganisms. Preliminary results of DNA-fingerprinting indicate no evidence of clonal likeness and then of patient-to-patient spread

Chryseobacterium respiratory tract infections in patients with cystic fibrosis

OBJECTIVES: To investigate the prevalence of infections by Chryseobacterium in a cohort of cystic fibrosis (CF) patients, to assess the antimicrobial susceptibility of these strains, to examine their DNA fingerprinting and to evaluate some clinical outcomes of patients infected by these bacteria. METHODS: Patients (300) attending a Regional Cystic Fibrosis Unit were enrolled in this study over 4 years. Natural or induced sputum samples were processed for microscopic and cultural tests. For phenotypic identification, automated and manual systems were used. For chemosusceptibility test, an automatic broth microdilution test and a disk-diffusion test were used and strains underwent DNA fingerprinting by pulsed-field gel electrophoresis (PFGE). RESULTS: Thirty-five strains of Chryseobacterium were isolated from 22 patients. These strains showed a broad-spectrum antimicrobial resistance, with activity only for trimethoprim-sulfamethoxazole and quinolones. PFGE profiles of all isolates were generally heterogeneous, suggesting independent circulation. CONCLUSIONS: This is the first report about clinical isolates of Chryseobacterium spp from CF patients in an Italian Centre. The infection by Chryseobacterium was not associated to a deterioration of pulmonary function and mortality: therefore, all patients infected by Chryseobacterium were co-infected by Pseudomonas aeruginosa and 3 of these were also co-infected by Burkholderia cepacia complex

DNA-fingerprinting di stipiti di Chryseobacterium spp isolati da pazienti con Fibrosi Cistica

Objectives: Pulmonary infections by Gram-negative bacteria, as Pseudomonas aeruginosa, Burkholderia cepacia, Stenotrophomonas maltophilia, are the major cause of morbidity in Cystic Fibrosis patients. In the past decade, several pathogens as Alcaligenes spp and no tuberculosis mycobacteria have been recovered in these patients. Bacteria of genus Chryseobacterium are widespread Gram-negative microrganisms and involved in human infections. Aims of this study were to value the isolation frequency of Chryseobacterium strains in a cohort of Cystic Fibrosis patients, to investigate their antimicrobial sensibility and to establish possible clonal likeness between strains. Methods:A retrospective study was undertaken between January 2003 and December 2005 on 300 patients receiving care at the Regional Cystic Fibrosis Centre of Naples University “Federico II”. Sputum samples were checked: for bacterial identification, selective media and commercial identification systems were used.The activity of antimicrobial agents was determined using diffusion and microdiluthion methods. For DNA-fingerprinting, a genomic DNA macrorestriction followed by pulsed-field electrophoresis was carried out. Results:A total of 26 strains from 17 patients were isolated (7 C. meningosepticum, 14 C. indologenes, 5 C. gleum). Strains were resistant to cephalosporins and carbapenems; some were sensitive to ciprofloxacin, levofloxacin and trimethoprim-sulphamethoxazole. Macrorestriction analysis showed substantial heterogeneity among strains. Conclusions: Actually, the prognostic role of Chryseobacterium in Cystic Fibrosis is unclear and although the small number of isolations, it is need to be on the look out regard such microorganisms. The considerable resistance implies difficulties on therapeutic approach. Results of DNA-fingerprinting indicate no evidence of clonal likeness and then of patient-to-patient spread

Proton pump inhibitors as a risk factor for paediatric Clostridium difficile infection.

BACKGROUND: Proton pump inhibitors (PPIs) and H(2) receptor antagonists (H(2)RAs) may play an important role on the onset of Clostridium difficile-associated disease (CDAD) in adults. The impact of Clostridium difficile on children treated with gastric acid-suppressing agents remains unknown. AIM: To investigate the relationship between CDAD and exposure to acid suppressive therapy in hospitalized paediatric patients. METHODS: We reviewed the medical records of children, with a diagnosis of protracted diarrhoea and abdominal pain, whose stool was analysed for C. difficile toxins. We identified 68 patients with CDAD. For each patient, we randomly selected one control subjects with stool analysis negative for C. difficile. Comorbid illnesses, previous hospitalizations, antibiotics, corticosteroids, immunosuppressants and gastric acid suppressing exposures were recorded. RESULTS: The use of PPI was significantly higher in C. difficile positive group compared with C. difficile negative group [odds ratio (OR): = 4.5; 95% confidence interval (CI) = 1.4-14.4]. We also found a trend for the use of H(2)RAs in patients infected by C. difficile compared with C. difficile negative comparison group (OR: = 3.8; 95% CI = 0.7-18.9). CONCLUSIONS: Children exposed to PPIs therapy seem to be at higher risk for the development of Clostridium difficile-associated disease