Abstract A prospective cohort study was performed among travelers from the Netherlands to investigate the acquisition of carbapenemase-producing Enterobacteriaceae (CP-E) and extended-spectrum β-lactamase–producing Enterobacteriaceae (ESBL-E) and associated risk factors. Questionnaires were administered and rectal swabs were collected and tested before and after return. Of 370 travelers, 32 (8.6%) were colonized with ESBL-E before travel; 113 (30.5%) acquired an ESBL-E during travel, and 26 were still colonized 6 months after return. No CP-E were found. Independent risk factors for ESBL-E acquisition were travel to South and East Asia. Multilocus sequence typing showed extensive genetic diversity among Escherichia coli. Predominant ESBLs were CTX-M enzymes. The acquisition rate, 30.5%, of ESBL-E in travelers from the Netherlands to all destinations studied was high. Active surveillance for ESBL-E and CP-E and contact isolation precautions may be recommended at admission to medical facilities for patients who traveled to Asia during the previous 6 months.
We report detection of Panton-Valentine leukocidin-positive clonal complex 398 human-origin methicillin-resistant Staphylococcus aureus L2 in the Netherlands. This hypervirulent lineage originated in the Asia-Pacific Region and could become community-acquired in Europe after recurrent travel-related introductions. Genomic surveillance enables early detection to guide control measures and help limit spread of pathogens in urban settings.
SUMMARY X-linked agammaglobulinaemia (XLA) is an immunodeficiency caused by mutations in Bruton's tyrosine kinase (Btk) and is characterized by an almost complete arrest of B cell development. We analysed expression of Btk in B lymphoblastoid cell lines (BLCL) derived from four unrelated XLA patients. In one patient, with a 3.5 kb genomic deletion encompassing the first (untranslated) exon, mRNA levels and in vitro kinase activities were very low. The patient manifested a mild phenotype with a delayed onset of the disease. Another mutation, in which the intron 3 donor splice site is lost, was also associated with very low mRNA levels and an absence of detectable Btk protein. Patients with this mutation showed extensive heterogeneity of the immunological phenotype. In the BLCL of a third patient, with an Arg288 substitution in the SH2 domain, the mutation did not appear to affect the expression level, nor to abrogate in vitro phosphorylation activity. In the BLCL of the fourth patient, with an Arg28 mutation in the PH domain, tyrosine kinase activity in BTK precipitates appeared to be decreased compared with control BLCL.
Abstract X‐linked immunodeficiency with hyperimmunoglobulinemia M (XHM) reflects an impairment of the immunoglobulin (Ig) heavy (H) chain class switch of B lymphocytes from IgM to IgG and IgA. XHM is recessive; female carriers manifest normal IgG and IgA production. Due to random X chromosome inactivation in all somatic cells of females, about half of the lymphocytes of XHM carriers are not able to express an intact XHM gene. An intrinsic defect of the Ig H chain class switch mechanism in XHM B lymphocytes would thus lead to a skewed X chromosome inactivation pattern in the IgG‐ and IgA‐expressing B lymphocytes of female carriers. IgM‐, IgG‐ and IgA‐expressing B lymphoblastoid cells (BLC) were established by Epstein‐Barr virus transformation of peripheral blood mononuclear cells of two female XHM carriers. In an analysis of differential methylation of the polymorphic DXS255 loci, random X chromosome inactivation patterns were found in populations of T lymphocytes, in IgM‐expressing B lymphocytes and in IgG‐ or IgA‐expressing B lymphocytes. The heterogeneity of Ig H chain rearrangements and the Ig light chain usage in the IgA‐ or IgG‐expressing BLC clones that had inactivated the X chromosome which carries the intact XHM gene and in BLC clones with the homologous X chromosome inactivated were similar. The results indicated that the intrinsic Ig H chain class switch mechanism in XHM B lymphocytes is fully intact. We conclude that the XHM gene encodes a class switch inducer that is transferred to B lymphocytes.
X-linked agammaglobulinemia (XLA) is an inherited immunodeficiency disease in man, reflecting an arrest in differentiation of pre-B cells to mature B cell stages. The gene defective in XLA has been identified as a cytoplasmic protein tyrosine kinase, named btk (Bruton's tyrosine kinase). Here we report the characterization of mutations in the btk gene of five unrelated XLA families. Amplified products were generated from cDNA, cloned and sequenced. Three single point mutations and two small insertions were identified. One of the point mutations and the two insertions created stop codons that would lead to truncated btk proteins. In one XLA patient we found a single basepair substitution that altered the highly conserved Arg288 within the SH2 domain and would therefore abrogate interactions with substrate phosphotyrosines. In another XLA patient a single basepair substitution was observed that altered the conserved Arg28 residue in the N-terminal unique region of unknown function. This residue is also mutated in the xid mouse, which has a different, less severe, B cell deficiency. We conclude that a similar mutation in the btk gene leads in man to an almost complete arrest at an early stage of B cell differentiation, but in the mouse to only limited B cell abnormalities.
Abstract X‐linked agammaglobulinemia (XLA) is an immunodeficiency disease in man, resulting from an arrest in early B cell differentiation. The gene defective in XLA has recently been identified and encodes a cytoplasmic protein tyrosine kinase, named Bruton's tyrosine kinase (btk) , essential for cell differentiation and proliferation at the transition from pre‐B to later B cell stages. In this study we investigated btk expression by Northern blotting experiments in a series of human (precursor‐) B cell lines, acute lymphoblastic leukemias and plasmacytomas, btk was found to be already expressed in very early stages of B cell differentiation, even prior to immunoglobulin (Ig) heavy (H) or light (L) chain gene rearrangements. Transcripts were also detected at the pre‐B cell stage and in mature B cells, irrespective of the Ig H chain class expressed. Approximately at the transition from mature B cells to plasma cells, expression of the btk gene is down‐regulated. In addition, the btk gene was found to be expressed in myeloid cell lines and acute myeloid leukemias. btk expression in myeloid cells is probably not a prerequisite for myeloid differentiation, since myeloid cells in XL A patients seem not to be affected. No btk expression was found in T‐lineage cells. The btk expression profile, i.e. from early precursor‐B cell stages preceding Ig rearrangement up to mature B cells, supports the hypothesis that the XLA defect resides in a critical step of B cell development which is independent of the Ig gene recombination machinery.
Abstract B lymphoblastoid cell lines (BLCL), established from bone marrow and peripheral blood mononuclear cells from two severe combined immunodeficiency (SCID) patients, manifested a complete absence of genomic rearrangements of the immunoglobulin (Ig) heavy (H) and light (L) chain loci. The BLCL contained germ‐line transcripts of the Ig x region locus of approximately 1.2 kilobases (kb). By cDNA cloning and sequence analysis the transcripts were shown to consist of a C x segment, a J x 1 gene segment, 160 base pairs (bp) of J x 1 5′ intervening sequence, containing the heptamer/nonamer recombination recognition sequences and at the 5′ end a 523‐bp segment designated human x °, The first 206 bp of this 5′ segment were homologous to the reported murine x ° region. Genomic restriction mapping and DNA sequence analysis demonstrated that the human x ° segment is located approximately 4 kb upstream of J x 1. The x ° segment contains a putative promoter region with an OCT2 binding site, and has a splice donor site to accomplish splicing to an acceptor site 160 bp upstream of J x 1. Expression of the x ° gene segment was found in BLCL derived from normal fetal bone marrow, in which both Ig x loci were in the germ‐line configuration. These findings indicate that the described transcripts are not only present in SCID, but also in normal developing pre‐B lymphocytes. The expression of germ‐line Ig x L chain transcripts may be associated with the locus becoming accessible to gene rearrangement.
To characterize the mechanisms of fluoroquinolone and cephalosporin resistance in Enterobacteriaceae from a Dutch teaching hospital in 2008. We sequenced gyrA, gyrB, parC and parE. The presence of plasmid-encoded genes qnrA, qnrB, qnrS, aac(6′)-Ib, qepA, blaTEM, blaSHV,blaOXA, blaCTX-M and blaAmpC was studied by PCR. Escherichia coli isolates were further characterized by AFLP and multilocus sequence typing (MLST). In total, 49 E. coli, 16 Klebsiella pneumoniae and 3 Enterobacter cloacae isolates were investigated. Mutations in gyrA were found in all E. coli isolates. Forty-five (92%) E. coli isolates carried at least one point mutation in parC. Most E. coli isolates (59%) also carried mutations in parE, of which I529L was the most prevalent. I529L was unequivocally associated with E. coli sequence type (ST) 131. This single-nucleotide polymorphism (SNP) was later also found in eight out of nine ST131 strains from another collection. Twenty-nine E. coli isolates carried extended-spectrum β-lactamase (ESBL) genes, predominantly blaCTX-M-15. In E. coli, aac(6′)-Ib-cr was the predominant plasmid-mediated resistance mechanism, whereas in K. pneumoniae qnr genes were found mostly. In K. pneumoniae isolates, qnr and aac(6′)-Ib-cr co-occurred with ESBL genes (n = 13; blaCTX-M and blaSHV) and/or blaAmpC (n = 3; blaDHA-1). E. coli ST131 was the predominant clone, which accumulated a high number of chromosomal mutations. The I529L SNP in parE was a signature of most, but not all, ST131 strains. In contrast to E. coli, fluoroquinolone resistance mechanisms were predominantly plasmid-encoded in K. pneumoniae.
