Ascending urinary tract infection (UTI) and pyelonephritis caused by uropathogenic Escherichia coli (UPEC) are very common infections that can cause severe kidney damage. Collecting duct cells, the ...site of hormonally regulated ion transport and water absorption controlled by vasopressin, are the preferential intrarenal site of bacterial adhesion and initiation of inflammatory response. We investigated the effect of the potent V2 receptor (V2R) agonist deamino-8-D-arginine vasopressin (dDAVP) on the activation of the innate immune response using established and primary cultured collecting duct cells and an experimental model of ascending UTI. dDAVP inhibited Toll-like receptor 4-mediated nuclear factor kappaB activation and chemokine secretion in a V2R-specific manner. The dDAVP-mediated suppression involved activation of protein phosphatase 2A and required an intact cystic fibrosis transmembrane conductance regulator Cl- channel. In vivo infusion of dDAVP induced a marked fall in proinflammatory mediators and neutrophil recruitment, and a dramatic rise in the renal bacterial burden in mice inoculated with UPECs. Conversely, administration of the V2R antagonist SR121463B to UPEC-infected mice stimulated both the local innate response and the antibacterial host defense. These findings evidenced a novel hormonal regulation of innate immune cellular activation and demonstrate that dDAVP is a potent modulator of microbial-induced inflammation in the kidney.
Objectives To develop a rapid and reliable tool to detect by multiplex PCR assays the most frequently widespread β-lactamase genes encoding the OXA-1-like broad-spectrum β-lactamases, ...extended-spectrum β-lactamases (ESBLs), plasmid-mediated AmpC β-lactamases and class A, B and D carbapenemases. Methods Following the design of a specific group of primers and optimization using control strains, a set of six multiplex PCRs and one simplex PCR was created. An evaluation of the set was performed using a collection of 31 Enterobacteriaceae strains isolated from clinical specimens showing a resistance phenotype towards broad-spectrum cephalosporins and/or cephamycins and/or carbapenems. Direct sequencing from PCR products was subsequently carried out to identify β-lactamase genes. Results Under optimized conditions, all positive controls confirmed the specificity of group-specific PCR primers. Except for the detection of carbapenemase genes, multiplex and simplex PCR assays were carried out using the same PCR conditions, allowing assays to be performed in a single run. Out of 31 isolates selected, 22 strains produced an ESBL, mostly CTX-M-15 but also CTX-M-1 and CTX-M-9, SHV-12, SHV-5, SHV-2, TEM-21, TEM-52 and a VEB-type ESBL, 6 strains produced a plasmid-mediated AmpC β-lactamase (five DHA-1 and one CMY-2) and 3 strains produced both an ESBL (two SHV-12, one CTX-M-15) and a plasmid-mediated AmpC β-lactamase (DHA-1). Conclusions We report here the development of a useful method composed of a set of six multiplex PCRs and one simplex PCR for the rapid screening of the most frequently encountered β-lactamases. This method allowed direct sequencing from the PCR products.
Two closely related Enterobacter aerogenes isolates presented a new identical aac(6')-Ib-cr genetic environment, including IS26. One isolate showed lower MICs of ciprofloxacin, norfloxacin, ...tobramycin, and amikacin and decreased expression of aac(6')-Ib-cr, which might be related to a 12-bp deletion causing a displacement of the –10 box upstream of the aac(6')-Ib-cr gene.
Objectives: We studied eight imipenem-resistant isolates of Klebsiella pneumoniae involved in an outbreak in a French teaching hospital. Methods: The eight isolates were recovered from clinical ...specimens or rectal swabs. Antibiotic susceptibilities were determined using standard agar diffusion and dilution methods including synergy tests. PFGE was used to study the relatedness of isolates. Genes encoding β-lactamases were characterized by transfer assays, specific amplification and cloning. Results: The eight isolates were closely related by PFGE analysis and highly related to a K. pneumoniae strain from Greece. They were highly resistant to β-lactams, including aztreonam and imipenem (MIC ≥32 mg/L), and were positive by the imipenem-EDTA disc synergy test. Isolates were also resistant to aminoglycosides, newer quinolones and sulfamethoxazole, and showed an intermediate level of resistance to tetracycline. VIM-1 and SHV-5 β-lactamases were revealed in all isolates by PCR. The analysis of plasmid contents of Escherichia coli DH10B electroporants expressing the VIM-1 β-lactamase or the SHV-5 β-lactamase confirmed that the two enzymes were coded by two different plasmids. The blaVIM-1 gene was part of a class 1 integron that also included aac6, dhfrI and aadA genes and was similar to those reported from strains isolated in Greece. Conclusions: This study confirms the potential risk of spread of multiresistant bacteria with international transfer of patients.
We report on the first detection of an AmpC-type Ambler class C 1 (ACC-1) β-lactamase in Citrobacter freundi isolated from a patient also harboring ACC-1-producing Escherichia coli and Klebsiella ...pneumoniae. We propose a simple cefoxitin-based double-disk synergy test (DDST) for the specific detection of ACC-1 in members of the family Enterobacteriaceae, including natural AmpC producers, in association with a cloxacillin-based DDST as a first-line AmpC-type β-lactamase screening test.
Class C β-lactamases or cephalosporinases can be classified into two functional groups (1, 1e) with considerable molecular variability (≤20% sequence identity). These enzymes are mostly encoded by ...chromosomal and inducible genes and are widespread among bacteria, including Proteobacteria in particular. Molecular identification is based principally on three catalytic motifs (
SXSK,
YXN,
KTG), but more than 70 conserved amino-acid residues (≥90%) have been identified, many close to these catalytic motifs. Nevertheless, the identification of a tiny, phylogenetically distant cluster (including enzymes from the genera
,
, and
) has raised questions about the possible existence of a C2 subclass of β-lactamases, previously identified as serine hydrolases. In a context of the clinical emergence of extended-spectrum AmpC β-lactamases (ESACs), the genetic modifications observed
and
(point mutations, insertions, or deletions) during the evolution of these enzymes have mostly involved the Ω- and H-10/R2-loops, which vary considerably between genera, and, in some cases, the conserved triplet
YXN. Furthermore, the conserved deletion of several amino-acid residues in opportunistic pathogenic species of Acinetobacter, such as A. baumannii, A. calcoaceticus, A. pittii and A. nosocomialis (deletion of residues 304-306), and in Hafnia alvei and H. paralvei (deletion of residues 289-290), provides support for the notion of natural ESACs. The emergence of higher levels of resistance to β-lactams, including carbapenems, and to inhibitors such as avibactam is a reality, as the enzymes responsible are subject to complex regulation encompassing several other genes (
R,
D,
G, etc.). Combinations of resistance mechanisms may therefore be at work, including overproduction or change in permeability, with the loss of porins and/or activation of efflux systems.
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