Oriane Soetens

Differentiation of cfiA-Negative and cfiA-Positive Bacteroides fragilis Isolates by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT Carbapenem resistance in Bacteroides fragilis is associated with cfiA-encoded class B metallo-beta-lactamase. cfiA-negative and cfiA-positive isolates belong to genotypically distinct groups. Of a total of 248 B. fragilis isolates included in this study, 214 were susceptible, 10 were intermediate, and 24 were resistant to meropenem. We show that matrix-assisted laser desorption ionization–time of flight mass spectrometry is able to differentiate between cfiA-negative and cfiA-positive isolates and predict carbapenem resistance in a routine laboratory setting.

Evaluation of Different Cytomegalovirus (CMV) DNA PCR Protocols for Analysis of Dried Blood Spots from Consecutive Cases of Neonates with Congenital CMV Infections

ABSTRACT Two protocols for the extraction of cytomegalovirus (CMV) DNA and two methods for the amplification of CMV DNA in dried blood spots were evaluated for the retrospective diagnosis of congenital CMV infection. During the period from 1996 to 2006, a urine screening program detected 76 congenitally infected neonates. Stored Guthrie cards with blood from 55 cases and 12 controls were tested. Two spots of dried blood were cut from each card and evaluated in two centers. CMV DNA was extracted from a whole single spot. Center 1 used phenol-chloroform extraction and ethanol precipitation followed by a conventional PCR. Center 2 used the NucliSens easyMAG automated DNA/RNA extraction platform (bioMérieux) followed by a real-time PCR. For evaluation of the extraction method, DNA extracted from each blood spot was evaluated by the amplification method used by the collaborating center. The sensitivities were 66% for center 1 and 73% for center 2. None of the controls were positive. A sensitivity as high as 82% could be obtained by combining the most sensitive extraction method (the phenol-chloroform procedure) with the most sensitive PCR method (real-time PCR). The detection rate was not influenced by the duration of storage of the spots. The sensitivity was higher with blood from congenitally infected cases due to a primary maternal CMV infection, regardless of the protocol used. However, the difference reached significance only for the least-sensitive protocol (P = 0.036).

Microbiology of Bronchoalveolar Lavage Fluid in Children With Acute Nonresponding or Recurrent Community-Acquired Pneumonia: Identification of Nontypeable Haemophilus influenzae as a Major Pathogen

Childhood community-acquired pneumonia (CAP) is a heterogeneous and polymicrobial entity. Using flexible fiberoptic bronchoscopy with bronchoalveolar lavage, we identified nontypeable Haemophilus influenzae as one of the major pathogens involved in recurrent or non-responsive CAP in children.

Outbreak of Infection by Carbapenem-Resistant Acinetobacter baumannii Producing the Carbapenemase OXA-58 in Belgium

ABSTRACT Carbapenem-resistant Acinetobacter baumannii isolates were obtained from 17 patients between September 2004 and August 2005 at the Academisch Ziekenhuis Vrije Universiteit Brussel, Brussels, Belgium. These multidrug-resistant isolates, which belonged to a single clone, remained susceptible to colistin and tigecycline only and produced the carbapenem-hydrolyzing oxacillinase OXA-58. This study highlights the importance of the intercountry spread of this β-lactamase-mediated resistance mechanism and its epidemic evolution.

The ARG11 Gene of Saccharomyces cerevisiae Encodes a Mitochondrial Integral Membrane Protein Required for Arginine Biosynthesis

Prototype strain MG409 (arg11-1) is a severe arginine bradytroph with greatly reduced ornithine and arginine pools, although all known enzymes required for arginine biosynthesis are functional. To identify the function required for normal arginine production impaired in MG409, we have cloned, sequenced, and performed a first molecular characterization of ARG11. We show that the ARG11 open reading frame encodes a putative 292-residue protein with a predicted molecular mass of 31.5 kDa. Sequence similarities, a tripartite organization, and six potential hydrophobic transmembrane spans suggest that Arg11p belongs to the mitochondrial integral inner membrane carrier family. We have used immuno-Western blotting and hemagglutinin epitope-tagged derivatives of Arg11p, Arg8p (a mitochondrial matrix marker), and Arg3p (a cytosolic marker) to demonstrate that Arg11p is confined to the mitochondria and behaves like an integral membrane protein. A deletion created in ARG11 causes the same arginine-leaky behavior as the original arg11-1 mutation, which yields a premature stop codon at residue 266. Arg11p thus appears to fulfill a partially redundant function requiring its 27 carboxyl-terminal amino acids. As a working hypothesis, we propose that Arg11p participates in the export of matrix-made ornithine into the cytosol.

Species Identification of Clinical Prevotella Isolates by Matrix-Assisted Laser Desorption Ionization–Time of Flight Mass Spectrometry

ABSTRACT The performance of matrix-assisted laser desorption–ionization time of flight mass spectrometry (MALDI-TOF MS) for species identification of Prevotella was evaluated and compared with 16S rRNA gene sequencing. Using a Bruker database, 62.7% of the 102 clinical isolates were identified to the species level and 73.5% to the genus level. Extension of the commercial database improved these figures to, respectively, 83.3% and 89.2%. MALDI-TOF MS identification of Prevotella is reliable but needs a more extensive database.

In young children, persistent wheezing is associated with bronchial bacterial infection: a retrospective analysis

BackgroundYoung children with persistent wheezing pose a diagnostic and therapeutical challenge to the pediatrician.We aimed to evaluate bacterial bronchial infection as a possible reason for non response to conventional asthma therapy, and to identify and characterise the predominant pathogens involved.MethodsWe retrospectively analysed microbiological and cytological findings in a selected population of young wheezers with symptoms unresponsive to inhaled corticosteroid (ICS) therapy, who underwent flexible bronchoscopy with bronchoalveolar lavage (BAL). Procedural measures were taken to limit contamination risk and quantitative bacterial culture of BAL fluid (significance cut-off ≥ 104 colony-forming units/ml) was used. Modern microbiological methods were used for detection of a wide panel of pathogens and for characterisation of the bacterial isolates.Results33 children aged between 4 and 38 months, without structural anomalies of the conductive airways were evaluated. Significant bacterial BAL cultures were found in 48,5 % of patients. Haemophilus influenzae was isolated in 30,3 %, Streptococcus pneumoniae in 12,1 % and Moraxella catarrhalis in 12,1 %. All H. influenzae isolates were non-encapsulated strains and definitely distinguished from non-haemolytic H. haemolyticus. Respiratory viruses were detected in 21,9 % of cases with mixed bacterial-viral infection in 12,1 %. Cytology revealed a marked neutrophilic inflammation.ConclusionsBacterial infection of the bronchial tree is common in persistent preschool wheezers and provides a possible explanation for non response to ICS therapy. Non-typeable H. influenzae seems to be the predominant pathogen involved, followed by S. pneumoniae and M. catarrhalis.

Modified Real-Time PCR for Detecting, Differentiating, and Quantifying Ureaplasma urealyticum and Ureaplasma parvum

We evaluated a previously described quantitative real-time PCR (qPCR) for quantifying and differentiating Ureaplasma parvum and U. urealyticum. Because of nonspecific reactions with Staphylococcus aureus DNA in the U. parvum PCR, we developed a modified qPCR and designed new primers. These oligonucleotides eradicated cross-reactions, indicating higher specificity. The detection limits of the qPCR were determined at 1 and 3 colony-forming units/ml for U. parvum and U. urealyticum, respectively. The quantification limits of the assay for both Ureaplasma species ranged from 2.10(6) to 2.10(1) copy numbers per PCR. A total of 300 patient samples obtained from the lower genital tract were tested with this newly designed qPCR assay and compared with culture results. Of the samples, 132 (44.0%) were culture positive, whereas 151 (50.3%) tested positive using qPCR. The U. parvum and U. urealyticum species were present in 79.5% and 12.6% of the qPCR-positive samples, respectively. Both species were found in 7.9% of those samples. Quantification of U. parvum and U. urealyticum in the samples ranged from less than 2.5 × 10(3) to 7.4 × 10(7) copies per specimen. In conclusion, the modified qPCR is a suitable method for rapid detection, differentiation, and quantification of U. parvum and U. urealyticum.

Prosthetic valve endocarditis caused by Bordetella holmesii, an Acinetobacter lookalike.

We report a case of fulminant endocarditis on a prosthetic homograft aortic valve caused by Bordetella holmesii, which was successfully managed by surgical valve replacement and antibiotic treatment. B. holmesii, a strictly aerobic, small, Gram-negative coccobacillus, has been implicated as an infrequent cause of a pertussis-like syndrome and other respiratory illnesses. However, B. holmesii is also a rare cause of septicaemia and infective endocarditis, mostly in immunocompromised patients. To our knowledge, this is the first report of B. holmesii endocarditis on a prosthetic aortic valve. Routine laboratory testing initially misidentified the strain as Acinetobacter sp. Correct identification was achieved by 16S rRNA gene and outer-membrane protein A (ompA) gene sequencing. Interestingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry also produced an accurate species-level identification. Subsequent susceptibility testing and review of the literature revealed ceftazidime, cefepime, carbapenems, aminoglycosides, fluoroquinolones, piperacillin/tazobactam, tigecycline and colistin as possible candidates to treat infections caused by B. holmesii.

Simple Algorithm for Identification of Bordetella pertussis Pertactin Gene Variants

ABSTRACT Studies performed in several countries have demonstrated the recent emergence and subsequent dominance of circulating Bordetella pertussis strains harboring pertactin and pertussis toxin variants not included in pertussis vaccines. Determination of the pertactin gene variants is commonly performed using a time-consuming and expensive sequence analysis. We developed a simple and reliable pertactin typing algorithm suitable for large-scale screening. The assay correctly identified all pertactin alleles in representative strains. The typing of 231 clinical strains of B. pertussis routinely isolated in Belgium showed that this algorithm was adequate to identify less-frequent prn types like prn9 and prn11.