Increasing proportion of carbapenemase-producing Enterobacteriaceae and emergence of a MCR-1 producer through a multicentric study among hospital-based and private laboratories in Belgium from September to November 2015

Carbapenemase-producing Enterobacteriaceae (CPE) strains have been increasingly reported in Belgium. We aimed to determine the proportion of CPE among Enterobacteriaceae isolated from hospitalised patients and community outpatients in Belgium in 2015. For the hospitalised patients, the results were compared to a previous similar survey performed in the same hospitals in 2012. Twenty-four hospital-based and 10 private laboratories collected prospectively 200 non-duplicated Enterobacteriaceae isolates from clinical specimens. All isolates were screened locally by carbapenem disk diffusion using European Committee on Antimicrobial Susceptibility Testing methodology. Putative CPE strains with inhibition zone diameters below the screening breakpoints were referred centrally for confirmation of carbapenemase production. From September to November 2015, we found a proportion of clinical CPE of 0.55% (26/4,705) and of 0.60% (12/1,991) among hospitalised patients and among ambulatory outpatients respectively. Klebsiella pneumoniae (26/38) and OXA-48-like carbapenemase (28/38) were the predominant species and enzyme among CPE. One OXA-48-producing Escherichia coli isolated from a hospital was found carrying plasmid-mediated MCR-1 colistin resistance. Compared with the 2012 survey, we found a significant increased proportion of clinical CPE (0.55% in 2015 vs 0.25% in 2012; p = 0.02) and an increased proportion of hospitals (13/24 in 2015 vs 8/24 in 2012) with at least one CPE detected. The study results confirmed the concerning spread of CPE including a colistin-resistant MCR-1 producer in hospitals and the establishment of CPE in the community in Belgium

Prevalence and mechanisms of resistance to carbapenems in Enterobacteriaceae

Objectives: To determine the point prevalence of carbapenem-non-susceptible Enterobacteriaceae (CNSE) and carbapenemase-producing Enterobacteriaceae (CPE) isolates among hospitalized patients in Belgium. Methods: Twenty-four hospital-based laboratories prospectively collected 200 non-duplicated Enterobacteriaceae isolates from clinical specimens of hospitalized patients over a 2 month period. All isolates were screened locally for decreased susceptibility to carbapenem drugs using a disc diffusion method according to CLSI interpretative criteria. CNSE strains were referred centrally for confirmation of carbapenemase by phenotypic and molecular testing. Results: From February to April 2012, 158 of the 4564 screened Enterobacteriaceae isolates were categorized as non-susceptible to carbapenems, resulting in a point prevalence of CNSE of 3.5% (95% CI: 2.9%–4.2%; range per centre: 0.5%–8.5%). Of the 125 referred CNSE isolates, 11 Klebsiella pneumoniae isolates [OXA-48 (n=7), KPC type (n=3) and NDM type (n=1)], 1 OXA-48-positive Escherichia coli isolate and 1 KPC-positive Klebsiella oxytoca isolate were detected in eight hospitals. None of the 72 carbapenem-non-susceptible Enterobacter spp. isolates were confirmed as CPE. The minimal estimated point prevalence of CPE isolates was 0.28% (13/ 4564; 95% CI: 0.13%–0.44%) overall (range per centre: 0%–1.5%). Conclusions: Despite the overall low prevalence of CNSE found in this study, the detection of CPE isolates in one-third of the participating centres raises concerns and highly suggests the spread and establishment of CPE in Belgian hospitals

Evaluation of a new meropenem-EDTA double-ended Etest strip for the detection of the cfiA metallo-beta-lactamase gene in clinical isolates of Bacteroides fragilis.

Thirty-five Bacteroides fragilis clinical isolates with varying susceptibility to meropenem were analysed with a prototype of a double-ended Etest strip containing meropenem +/- EDTA, designed for the detection of the CfiA metallo-beta-lactamase. Phenotypic results obtained with this new Etest strip were related to the genotype and compared to the results of the Etest containing imipenem +/- EDTA. Whereas the Etest with imipenem +/- EDTA only allowed detection of isolates with high-level resistance (both MICs of imipenem and meropenem >32 mg/L), reflecting the possible underestimation of CfiA prevalence in B. fragilis, the Etest with meropenem +/- EDTA proved to be more accurate, particularly for isolates with low-level carbapenem resistance, suggesting its potential for broader detection of CfiA production

The GPAT4/6/8 clade functions in Arabidopsis root suberization nonredundantly with the GPAT5/7 clade required for suberin lamellae.

Lipid polymers such as cutin and suberin strengthen the diffusion barrier properties of the cell wall in specific cell types and are essential for water relations, mineral nutrition, and stress protection in plants. Land plant-specific glycerol-3-phosphate acyltransferases (GPATs) of different clades are central players in cutin and suberin monomer biosynthesis. Here, we show that the GPAT4/6/8 clade in Arabidopsis thaliana, which is known to mediate cutin formation, is also required for developmentally regulated root suberization, in addition to the established roles of GPAT5/7 in suberization. The GPAT5/7 clade is mainly required for abscisic acid-regulated suberization. In addition, the GPAT5/7 clade is crucial for the formation of the typical lamellated suberin ultrastructure observed by transmission electron microscopy, as distinct amorphous globular polyester structures were deposited in the apoplast of the gpat5 gpat7 double mutant, in contrast to the thinner but still lamellated suberin deposition in the gpat4 gpat6 gpat8 triple mutant. Site-directed mutagenesis revealed that the intrinsic phosphatase activity of GPAT4, GPAT6, and GPAT8, which leads to monoacylglycerol biosynthesis, contributes to suberin formation. GPAT5/7 lack an active phosphatase domain and the amorphous globular polyester structure observed in the gpat5 gpat7 double mutant was partially reverted by treatment with a phosphatase inhibitor or the expression of phosphatase-dead variants of GPAT4/6/8. Thus, GPATs that lack an active phosphatase domain synthetize lysophosphatidic acids that might play a role in the formation of the lamellated structure of suberin. GPATs with active and nonactive phosphatase domains appear to have nonredundant functions and must cooperate to achieve the efficient biosynthesis of correctly structured suberin

The Root Cap Cuticle: A Cell Wall Structure for Seedling Establishment and Lateral Root Formation.

The root cap surrounding the tip of plant roots is thought to protect the delicate stem cells in the root meristem. We discovered that the first layer of root cap cells is covered by an electron-opaque cell wall modification resembling a plant cuticle. Cuticles are polyester-based protective structures considered exclusive to aerial plant organs. Mutations in cutin biosynthesis genes affect the composition and ultrastructure of this cuticular structure, confirming its cutin-like characteristics. Strikingly, targeted degradation of the root cap cuticle causes a hypersensitivity to abiotic stresses during seedling establishment. Furthermore, lateral root primordia also display a cuticle that, when defective, causes delayed outgrowth and organ deformations, suggesting that it facilitates lateral root emergence. Our results show that the previously unrecognized root cap cuticle protects the root meristem during the critical phase of seedling establishment and promotes the efficient formation of lateral roots

Evaluation of Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Identification of Nocardia Species▿

The identification of Nocardia species, usually based on biochemical tests together with phenotypic in vitro susceptibility and resistance patterns, is a difficult and lengthy process owing to the slow growth and limited reactivity of these bacteria. In this study, a panel of 153 clinical and reference strains of Nocardia spp., altogether representing 19 different species, were characterized by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). As reference methods for species identification, full-length 16S rRNA gene sequencing and phenotypical biochemical and enzymatic tests were used. In a first step, a complementary homemade reference database was established by the analysis of 110 Nocardia isolates (pretreated with 30 min of boiling and extraction) in the MALDI BioTyper software according to the manufacturer's recommendations for microflex measurement (Bruker Daltonik GmbH, Leipzig, Germany), generating a dendrogram with species-specific cluster patterns. In a second step, the MALDI BioTyper database and the generated database were challenged with 43 blind-coded clinical isolates of Nocardia spp. Following addition of the homemade database in the BioTyper software, MALDI-TOF MS provided reliable identification to the species level for five species of which more than a single isolate was analyzed. Correct identification was achieved for 38 of the 43 isolates (88%), including 34 strains identified to the species level and 4 strains identified to the genus level according to the manufacturer's log score specifications. These data suggest that MALDI-TOF MS has potential for use as a rapid (<1 h) and reliable method for the identification of Nocardia species without any substantial costs for consumables

Detection and characterization of class A extended-spectrum-{beta}-lactamase-producing Pseudomonas aeruginosa isolates in Belgian hospitals.

Objectives To investigate the presence of extended-spectrum beta-lactamases (ESBLs) among Pseudomonas aeruginosa clinical isolates referred to two Belgian reference laboratories. Methods Antibiograms were analysed for P. aeruginosa isolates referred between 2004 and 2008. Isolates resistant to ceftazidime (MIC > 8 mg/L) and with a positive double-disc synergy test between ceftazidime and clavulanate were serotyped and screened for the presence of ESBL-encoding genes. Genes encoding metallo-beta-lactamases (bla(MBL)) were sought by PCR in ESBL-producing isolates with positive imipenem/EDTA synergy tests. PFGE of SpeI-digested genomic DNA was used to compare isolates and selected strains were characterized by multilocus sequence typing. Results Forty-eight (2.2%) of 2150 P. aeruginosa isolates were confirmed as class A ESBL-producing isolates by molecular testing. bla(BEL) and bla(PER) alleles were detected, respectively, in 39 and 10 P. aeruginosa isolates originating from 16 hospitals (two isolates were simultaneously positive for BEL and PER). Fifteen of the isolates were found to co-produce ESBLs and VIM carbapenemases. These strains were pan-resistant and remained susceptible only to colistin (MICs </= 2 mg/L). The majority of the ESBL-producing isolates belonged to the same PFGE clone and were identified as ST235; serotype O11. Conclusions BEL enzymes were produced by 80% of P. aeruginosa isolates with phenotypic evidence of ESBL production. BEL or PER ESBLs co-existed with VIM carbapenemases in 15 isolates and caused outbreaks in four hospitals. Our data further highlight the epidemic potential of the international clone ST235, which may have acquired bla(BEL-1) gene cassettes from a yet unidentified local gene reservoir

Entérobactéries productrices de carbapénémases importées: contexte belge

Le programme de surveillance active des CPE en Belgique initié en 2012 a permis d&#039;estimer la proportion des cas de CPE liés à l&#039;étranger dans notre pays. Le présent article souligne les limites de cette surveillance (36% de données manquantes) et passe en revue d&#039;autres sources potentielles d&#039;importation de CPE, liées ou non à des contacts avec les soins de santé à l&#039;étranger.Il semble prudent de considérer aujourd&#039;hui comme à risque accru de portage de CPE (et d&#039;autres MDRO) tout patient ayant reçu des soins de santé dans un établissement à l&#039;étranger, quel que soit le pays considéré, même si il est probable que le niveau de risque puisse être très différent selon les zones géographiques considérées.</p