Clinical and Microbiological Features of Inquilinus sp. Isolates from Five Patients with Cystic Fibrosis

Patients with cystic fibrosis (CF) may be colonized with unusual gram-negative bacilli whose identification is difficult and clinical impact unclear. We describe the clinical and microbiological features of five colonizations with organisms belonging to the recently described genus Inquilinus in CF patients. Isolates were identified from Burkholderia cepacia selective medium by means of 16S rRNA analysis. All of them were resistant to colistin, penicillins, cephalosporins, and monobactams but exhibited a remarkable susceptibility to imipenem. One of the five patients was transiently colonized with a nonmucoid isolate, whereas the four other patients were persistently colonized over the period of follow-up (8 to 21 months) with mucoid isolates. Pulsed-field gel electrophoresis of SpeI-digested genomic DNA was powerful for strain genotyping and demonstrated the clonality of Inquilinus sp. colonization for the two patients tested. Clinical evolution after the onset of Inquilinus was heterogeneous, but for at least one patient the lung function worsened and eradication of Inquilinus sp. was unsuccessful despite several imipenem courses. Finally, Inquilinus spp. may represent a new threat for CF patients due to their mucoid characteristic, their multiresistant pattern to antibiotics, and their ability to persist in the respiratory tract

Evaluation of a commercial immunochromatographic assay for rapid routine identification of PBP2a-positive Staphylococcus aureus and coagulase-negative staphylococci

International audienceWe evaluated the performance of an immunochromatographic assay (PBP2a Culture Colony Test - Alere\texttrademark), detecting protein-binding penicillin 2a on staphylococci primary isolates in only 6minutes. The assay is highly sensitive for the direct detection of MRSA on various culture media whereas it requires cefoxitin induction for methicillin-resistant coagulase-negative staphylococci

Exposure of Staphylococcus aureus to subinhibitory concentrations of β-lactam antibiotics induces heterogeneous vancomycin-intermediate Staphylococcus aureus

International audienceGlycopeptides are known to select for heterogeneous vancomycin-intermediate Staphylococcus aureus (h-VISA) from susceptible strains. In certain clinical situations, h-VISA strains have been isolated from patients without previous exposure to glycopeptides, such as cystic fibrosis patients, who frequently receive repeated treatments with beta-lactam antibiotics. Our objective was to determine whether prolonged exposure to beta-lactam antibiotics can induce h-VISA. We exposed 3 clinical vancomycin-susceptible methicillin-resistant Staphylococcus aureus (MRSA) strains to ceftazidime, ceftriaxone, imipenem, and vancomycin (as a control) at subinhibitory concentrations for 18 days in vitro. Population analyses showed progressive increases in vancomycin resistance; seven of the 12 derived strains obtained after induction were classified as h-VISA according to the following criteria: area under the curve (AUC) on day 18/AUC of Mu3 of >=90% and/or growth on brain heart infusion (BHI) agar with 4 mg/liter vancomycin. The derived isolates had thickened cell walls proportional to the level of glycopeptide resistance. Genes known to be associated with glycopeptide resistance (vraSR, yvqF, SA1703, graRS, walKR, and rpoB) were PCR sequenced; no de novo mutations were observed upon beta-lactam exposure. To determine whether trfA, a gene encoding a glycopeptide resistance factor, was essential in the selection of h-VISA upon beta-lactam pressure, a trfA-knockout strain was generated by allelic replacement. Indeed, beta-lactam exposure of this mutated strain showed no capacity to induce vancomycin resistance. In conclusion, these results showed that beta-lactam antibiotics at subinhibitory concentrations can induce intermediate vancomycin resistance in vitro. This induction required an intact trfA locus. Our results suggest that prior use of beta-lactam antibiotics can compromise vancomycin efficacy in the treatment of MRSA infections

Detection of Staphylococcus aureus delta-toxin production by whole-cell MALDI-TOF mass spectrometry.

International audienceThe aim of the present study was to detect the Staphylococcus aureus delta-toxin using Whole-Cell (WC) Matrix Assisted Laser Desorption Ionization-Time-of-Flight (MALDI-TOF) mass spectrometry (MS), correlate delta-toxin expression with accessory gene regulator (agr) status, and assess the prevalence of agr deficiency in clinical isolates with and without resistance to methicillin and glycopeptides. The position of the delta-toxin peak in the mass spectrum was identified using purified delta-toxin and isogenic wild type and mutant strains for agr-rnaIII, which encodes delta-toxin. Correlation between delta-toxin production and agr RNAIII expression was assessed by northern blotting. A series of 168 consecutive clinical isolates and 23 unrelated glycopeptide-intermediate S. aureus strains (GISA/heterogeneous GISA) were then tested by WC-MALDI-TOF MS. The delta-toxin peak was detected at 3005±5 Thomson, as expected for the naturally formylated delta toxin, or at 3035±5 Thomson for its G10S variant. Multivariate analysis showed that chronicity of S. aureus infection and glycopeptide resistance were significantly associated with delta-toxin deficiency (p = 0.048; CI 95%: 1.01-10.24; p = 0.023; CI 95%: 1.20-12.76, respectively). In conclusion, the S. aureus delta-toxin was identified in the WC-MALDI-TOF MS spectrum generated during routine identification procedures. Consequently, agr status can potentially predict infectious complications and rationalise application of novel virulence factor-based therapies

Delta-toxin peak in spectra from clinical strains.

<p>Whole-Cell Matrix Assisted Laser Desorption Ionization – Time-of-Flight mass spectrometry analysis of 6 representative clinical strains from the collection of 168. BE1103 3028 and BE1046 1395 are two delta-toxin positive strains showing 3005±5 Thomson (Th) peak; BE1104 4293 and BE1050 5040 are 2 strains expressing a mutated G10S delta-toxin; they exhibited no peak at 3005±5 Th but an additional peak at 3035±5 Th; BE1106 5397 and BE1048 2354 are two delta-toxin negative strains showing no peak at 3005±5 Th or at 3035±5 Th. Settings of the mass spectrometer are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040660#pone-0040660-g001" target="_blank">Figure 1</a>.</p

Multivariate analysis of <i>accessory gene regulator</i> status for 154 infecting isolates.

a<p><i>agr</i>: accessory gene regulator;</p>b<p>OR: odd ratio;</p>c<p>CI: confidence interval;</p>d<p>GISA: glycopeptide intermediate <i>Staphylococcus aureus</i> strains - hGISA: heterogeneous GISA;</p>e<p>Reference;</p>f<p>χ² (1 degree of freedom)  =  -2 log likelihood (model 2 - model 1)  = 0.608; <i>p</i> = 0.413;</p>g<p>MSSA: methicillin-sensitive <i>Staphylococcus aureus</i>;</p>h<p>MRSA: methicillin-resistant <i>Staphylococcus aureus.</i></p

Determination of delta-toxin status of clinical strains.

a<p>Th: Thomson.</p>b<p>MALDI-TOF MS: Matrix Assisted Laser Desorption Ionization Time-Of-Flight mass spectrometry.</p>c<p><i>hld</i>: gene encoding delta-toxin.</p>d<p>NT: not tested.</p>e<p>G10S: mutated delta-toxin with glycine 10 replaced by a serine.</p>f<p>WT: wild type delta-toxin.</p>g<p>MALDI/TOF-TOF MS: Matrix Assisted Laser Desorption Ionization Time-Of-Flight/Time-Of-Flight mass spectrometry.</p

Northern blot analysis of <i>accessory gene regulator</i>-RNAIII.

<p>Lane 1, positive control isolate (delta-toxin producer); lanes 2 to 18 correspond to the 17 delta-toxin negative clinical isolates. 5S rRNA was used as loading control.</p

Identification of the delta-toxin peak using purified delta-toxin and isogenic strains.

<p>Purified delta-toxin from wild-type <i>Staphylococcus aureus</i> (500 ng) or strains mutated in the <i>accessory gene regulator (agr)</i> locus were spotted on the target and analysed in the Matrix Assisted Laser Desorption Ionization – Time-of-Flight mass spectrometer with a mass-to-charge ratio (<i>m</i>/<i>z</i>) range of 2800 to 3200 Thomson (Th). Setting were as follows: mass range 2000–20000 Th; laser power 80 Volts; pulse extraction 8330 Th; number of laser fires per sample 500; noise cut off 10 mVolts with a minimum resolution of 300; Auto quality mode activated. The settings of the detector were according to the linear mode with a positive source of 20000 Th and a negative pulsed extraction at 2100 Th. The arrow in panel A indicates delta-toxin; additional peaks of 3024, 3040 and 3056 Th correspond to contaminants resulting from the purification of the toxin from <i>S. aureus</i> culture supernatant. Isogenic strains were: RN6390 (delta positive strain) RN6911 (full <i>agr knock-out</i>, delta negative strain) and LUG 950 (<i>rna</i>III <i>knock-out</i>, delta negative strain).</p