Phenotypic and genotypic characterization of Pseudomonas aeruginosa from cystic fibrosis patients.

Pseudomonas aeruginosa accounts for about one half of all pulmonary infections of cystic fibrosis (CF) patients. In this study, we analyzed 135 P. aeruginosa strains isolated from the expectorations of 55 CF adult patients attending a CF referral center over a period of five years. We assessed the genotype of the strains by pulsed-field gel electrophoresis (PFGE) and analyzed some phenotypic characteristics, such as O serotype, enzyme and mucous production, antibiotics susceptibility, and motility. PFGE allowed the typification of 97.1% of strains, revealing the presence of nine different genomic patterns. The pattern indicated as B was the most frequent, whereas patterns H and I were the most uncommon. Serotyping failed to identify 37.8% of strains and 29 out of 55 patients harbored almost one non-typable (NT) strain. During the five years of the study, we observed a progressive reduction of O6 and O10 types, but an increase of the O1 type and of NT strains. Most strains produced protease, hemolysin, and gelatinase, and were mobile. Several patients harbored the same serotype or genotype in sequential isolates, though characterized by a different susceptibility to antimicrobials. We did not observe a relationship between bacterial genotype and phenotype. This could be due to the fact that PFGE is not sensitive enough to detect subtle genotypic differences. The epidemiological importance of the genotypic characterization of bacteria-colonizing CF subjects and the surveillance measures to be adopted in CF centers are briefly discussed.

Burkholderia respiratory tract infections in Italian patients with cystic fibrosis: molecular characterization.

OBJECTIVES: To characterize by molecular techniques Burkholderia strains responsible for respiratory tract infections in cystic fibrosis (CF) patients (children and adults), to assign the Burkholderia cepacia complex (Bcc) isolates to a genomovar and to assess the presence of cblA and esmR genes in bacteria. Unique or sequential Burkholderia isolates (n=48) that had been collected from eight CF children and 17 adults over several (4-6) years were investigated; moreover 11 reference strains were analyzed. METHODS: The microorganisms were identified by using biochemical methods, genotyped by pulse field gel electrophoresis (PFGE) and random-amplified polymorphic DNA fingerprinting-PCR (RAPD-PCR), and assessed by PCR assays for the genomovar and cblA and esmR genes of Bcc. RESULTS: Among isolates 70.8% were identified as Bcc genomovar III-A; one child was infected by Burkholderia ambifaria and four adults were colonized with Burkholderia gladioli. The cblA gene was not detected in any of the isolates, while the esmR gene was detected in the 52.1% of the strains, all belonging to genomovar III-A. CONCLUSION: Molecular analysis of strains revealed in CF patients a colonization with a persistent Burkholderia flora with strains of one genotype. The prevalence of Bcc of genomovar III-A in the two categories of patients and of B. gladioli in four adults demonstrated that transmission may have occurred between subjects. Moreover the B. ambifaria infection demonstrated in a child may be environmentally derived.

Multicenter comparison of ESP Culture System II with BACTEC 460TB and with Lowenstein-Jensen medium for recovery of mycobacteria from different clinical specimens, including blood.

The recently developed ESP Culture System II (AccuMed, Chicago, Ill.) was compared with radiometric BACTEC 460TB (Becton Dickinson, Towson, Md.) and with Lowenstein-Jensen medium for recovery of mycobacteria from over 2,500 clinical specimens both of respiratory and nonrespiratory origin, including blood. The majority of the 219 mycobacterial isolates (129) belonged to the Mycobacterium tuberculosis complex, followed by 37 isolates of the Mycobacterium avium complex (MAC) and 53 isolates of eight other mycobacterial species. Rates of recovery obtained with BACTEC, ESP, and Lowenstein-Jensen medium were 89, 79, and 64%, respectively, with such differences being statistically significant. Different media and systems appeared to behave differently when the more frequently detected organisms were considered: M. tuberculosis complex isolates grew better with BACTEC, and MAC isolates grew better with ESP. An analysis of the combinations of Lowenstein-Jensen medium with BACTEC and with ESP did not reveal significant differences in recovery rates. With regard to the times needed for the detection of positive cultures, they were significantly longer on Lowenstein-Jensen medium (average, 28 days) than with the remaining two systems, between which there was no difference (average, 18 days). We conclude, therefore, that the ESP system, when used in combination with a solid medium, performs as well as the thoroughly validated radiometric BACTEC system and offers the advantages of full automation and absence of radioisotopes.

Rapid identification of Mycobacterium tuberculosis complex on urine samples by Gen-Probe amplification test.

The aim of the study was to evaluate the applicability to urine samples of the Amplified Mycobacterium tuberculosis Direct Detection Test (AMTD), which is currently used to identify this organism in respiratory specimens within a few hours. The study was performed on 95 patients, comprising 35 subjects with a high index of suspicion for active tuberculosis of the urinary tract and 60 subjects with evidence of non-mycobacterial disease. One urine specimen from each subject was examined by microscopy, culture and AMTD. AMTD was positive in 38 specimens and negative in 57. Assuming culture as the reference standard, the sensitivity, specificity, positive predictive value and negative predictive value of AMTD were 100%, 91.93%, 86.84% and 100%, respectively. Reassessing the discrepancies between AMTD and culture by review of patients' charts, the sensitivity, specificity, positive predictive value and negative predictive value of AMTD were 100%, 93.44%, 89.47% and 100%. The results of the study as well as the characteristics of AMTD encourage its use for the rapid recognition of urinary tract tuberculosis, although its findings should be interpreted cautiously when the clinical picture is not consistent with an active tuberculosis.

Pseudomonas aeruginosa clinical isolates: serotypes, resistance phenotypes and plasmid profiles.

78 Pseudomonas aeruginosa strains were isolated from the respiratory tract of 56 patients, 15 of which were affected by cystic fibrosis (CF). The epidemiological typing scheme was based on serotyping, antibiotic resistance pattern and plasmid DNA profile. All strains (except 2 mucoid strains) were typed using a rapid slide O-agglutination technique. Most common serotypes in both group were 0:1, 0:10 and 0:6. Moreover we observed a correlation among 0:12 serotype and CF patients. Plasmid DNA analysis showed that 45.2% (on average) of strains isolated from patients with and w/o CF harboured 1-3 plasmids ranging in size from 1 to 15 Md. Plasmid prevalence was higher in strains isolated from CF patients in specimens collected after antibiotic therapy. A correlation was found between 1 and 1.9 Md plasmids and resistance to aminoglycosides. Our results indicate that the analysis of antibiotic resistance phenotypes combined with plasmid analysis may be useful, in association to serotyping, to characterize the circulation of P. aeruginosa strains and the spread of resistance in these bacteria.

Rapid identification of mycobacteria from culture using acridinium-ester-labelled DNA probes.

OBJECTIVES: Our study used newly developed acridinium-ester-labelled DNA (AE-DNA) probes on 183 mycobacterial isolates, performing the tests on 12B Bactec vials and Loewenstein-Jensen (LJ) slants. METHODS: The probe results were verified using the conventional method as a reference. RESULTS: The probe for M. tuberculosis complex correctly identified 131 of 133 M. tuberculosis isolates, with two false negatives and no false positives, for a sensitivity of 98.5% and a specificity of 100%. The M. gordonae probe correctly identified 27 out of 27 M. gordonae isolates, with no false negatives and one false positive, for a sensitivity of 100% and a specificity of 90.9%. One hundred sixty-eight of the 183 isolates were screened in accordance with an algorithm, designed primarily for the rapid detection and identification of M. tuberculosis. CONCLUSION: The use of the algorithm considerably reduced detection time: M. tuberculosis strains were identified within 16.2 +/- 2.3 days, while identification by conventional method required 35.7 +/- 5.5 days. Probe testing resulted in a cost increase of 67.8%.

[Contribution of the serum anti-mycobacterial IgG measurement to the diagnosis of tuberculosis. Personal cases]. / Contributo della determinazione delle IgG sieriche antimicobatteriche alla diagnosi di tubercolosi. Casistica personale.

Data of a transversal research on 36 suspected tuberculosis (tb) inpatients of St. Luigi Hospital are reported. Serum antimycobacterial antibodies Class IgG to A60 were measured by means of ELISA. Final diagnosis was tb in 19 subjects, while 9 had a tb history over different years. Sensitivity was 78.94% and specificity 75%. According to with other recent reports, our results are poorly discriminating. However, the test, if valued with other clinical data, can contribute to the ascertainment of tb.