Sophie Vimont

Rapid detection of the O25b-ST131 clone of Escherichia coli encompassing the CTX-M-15-producing strains

OBJECTIVES Recently, a CTX-M-15 extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli O25b-ST131 clone, belonging to the B2 phylogenetic group and with a high virulence potential, has been reported all over the world, representing a major public health problem. The present study was carried out to develop a rapid and simple detection assay that identifies members of this clone. METHODS A total of 627 E. coli isolates of which 373 produced an ESBL, collected across four continents, were screened using a O25b-ST131 clone allele-specific PCR for the pabB gene. RESULTS One hundred and forty-three ESBL isolates were found positive with the assay. These isolates were all of O25b type and, when studied by multilocus sequence typing (25 cases), were all of ST131. The O25b-ST131 clone was found to produce ESBLs other than CTX-M-15, specifically CTX-M-2, -3, -14, -27, -32 and -61 as well as TEM-24. This clone represents 3% of non-ESBL B2 isolates originating from urinary tract infections in Paris. CONCLUSIONS We have developed a PCR-based assay that easily identifies a clone with high likelihood of producing ESBLs, including CTX-M-15.

Emergence and Outbreaks of CTX-M {beta}-Lactamase-Producing Escherichia coli and Klebsiella pneumoniae Strains in a Tunisian Hospital

ABSTRACT Sixty-two isolates of Enterobacteriaceae (35 Escherichia coli and 27 Klebsiella pneumoniae isolates) producing CTX-M-type β-lactamases were collected between March 2000 and June 2003 in different wards of Charles Nicolle Hospital in Tunis (Tunisia). Sequencing identified the blaCTX-M-15 determinant in 55 isolates and blaCTX-M-16 in 7 isolates. The CTX-M-15-producing strains were isolated in several wards and consisted mainly of two successive clonal groups of E. coli and a major clonal group of K. pneumoniae. The second clonal group of E. coli belonged to phylogenetic group B2 and harbored more virulence factors than the first clonal group. Among the 22 transconjugants or electroporants obtained with selected E. coli and K. pneumoniae CTX-M-15-producing strains, a predominant plasmid restriction pattern was obtained with 17 isolates. The four CTX-M-16-producing strains of E. coli yielded the same pulsed-field gel electrophoresis (PFGE) pattern, while the three CTX-M-16-producing strains of K. pneumoniae yielded two different PFGE patterns. All of the CTX-M-16-producing isolates were recovered in the pediatric ward and had the same plasmid restriction pattern.

Hormonal control of the renal immune response and antibacterial host defense by arginine vasopressin.

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 κB 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.

The CTX-M-15-producing Escherichia coli clone O25b: H4-ST131 has high intestine colonization and urinary tract infection abilities.

Increasing numbers of pyelonephritis-associated uropathogenic Escherichia coli (UPEC) are exhibiting high resistance to antibiotic therapy. They include a particular clonal group, the CTX-M-15-producing O25b:H4-ST131 clone, which has been shown to have a high dissemination potential. Here we show that a representative isolate of this E. coli clone, referred to as TN03, has enhanced metabolic capacities, acts as a potent intestine- colonizing strain, and displays the typical features of UPEC strains. In a modified streptomycin-treated mouse model of intestinal colonization where streptomycin was stopped 5 days before inoculation, we show that TN03 outcompetes the commensal E. coli strains K-12 MG1655, IAI1, and ED1a at days 1 and 7. Using an experimental model of ascending UTI in C3H/HeN mice, we then show that TN03 colonized the urinary tract. One week after the transurethral inoculation of the TN03 isolates, the bacterial loads in the bladder and kidneys were significantly greater than those of two other UPEC strains (CFT073 and HT7) belonging to the same B2 phylogenetic group. The differences in bacterial loads did not seem to be directly linked to differences in the inflammatory response, since the intrarenal expression of chemokines and cytokines and the number of polymorphonuclear neutrophils attracted to the site of inflammation was the same in kidneys colonized by TN03, CFT073, or HT7. Lastly, we show that in vitro TN03 has a high maximum growth rate in both complex (Luria-Bertani and human urine) and minimum media. In conclusion, our findings indicate that TN03 is a potent UPEC strain that colonizes the intestinal tract and may persist in the kidneys of infected hosts.

TLR4 Facilitates Translocation of Bacteria across Renal Collecting Duct Cells

Uropathogenic Escherichia coli (UPEC) are the most frequent causes of urinary tract infections and pyelonephritis. Renal medullary collecting duct (MCD) cells are the intrarenal site to which UPEC strains prefer to adhere and initiate an inflammatory response, but the ability of UPEC strains to translocate across impermeant MCD cells has not been demonstrated definitively. Here, several UPEC strains adhered to the apical surface and translocated across confluent murine inner MCD cells grown on filters. UPEC strains expressing cytolytic and vacuolating cytotoxins disrupted the integrity of cell layers, whereas noncytolytic UPEC strains passed through the cell layers without altering tight junctions. Apical-to-basal transcellular translocation was dramatically reduced after extinction of Toll-like receptor 4 (TLR4) and the lipid raft marker caveolin-1 by small interfering RNA. Furthermore, disruption of lipid raft integrity by filipin III and methyl-beta-cyclodextrin significantly reduced both the transcellular translocation of UPEC across murine inner MCD cell layers and the stimulation of proinflammatory mediators. Bacterial translocation was also significantly reduced in primary cultures of TLR4-deficient mouse MCD cells compared with MCD cells from wild-type mice. Benzyl alcohol, an anesthetic that enhances membrane fluidity, favored the recruitment of caveolin-1 in lipid rafts and increased the translocation of UPEC across cultured TLR4-deficient MCD cells. These findings demonstrate that the transcellular translocation of UPEC strains across impermeant layers of MCD cells may occur through lipid rafts via a TLR4-facilitated process.

Identification of a Chromosome-Borne Expanded-Spectrum Class A β-Lactamase from Erwinia persicina

ABSTRACT From whole-cell DNA of an enterobacterial Erwinia persicina reference strain that displayed a penicillinase-related antibiotic-resistant phenotype, a β-lactamase gene was cloned and expressed in Escherichia coli. It encoded a clavulanic-acid-inhibited Ambler class A β-lactamase, ERP-1, with a pI value of 8.1 and a relative molecular mass of ca. 28 kDa. ERP-1 shared 45 to 50% amino acid identity with the most closely related enzymes, the chromosomally encoded enzymes from Citrobacter koseri, Kluyvera ascorbata, Kluyvera cryocrescens, Klebsiella oxytoca, Proteus vulgaris, Proteus penneri, Rahnella aquatilis, Serratia fonticola, Yersinia enterocolitica, and the plasmid-mediated enzymes CTX-M-8 and CTX-M-9. The substrate profile of the noninducible ERP-1 was similar to that of these β-lactamases. ERP-1 is the first extended-spectrum β-lactamase from an enterobacterial species that is plant associated and plant pathogenic.

Flagellin/TLR5 signalling activates renal collecting duct cells and facilitates invasion and cellular translocation of uropathogenic Escherichia coli

Uropathogenic Escherichia coli (UPEC) colonizing kidneys is the main cause of acute pyelonephritis. TLR5 that senses flagellin was shown to be highly expressed in the bladder and to participate in host defence against flagellated UPEC, although its role in kidneys still remains elusive. Here we show that TLR5 is expressed in renal medullary collecting duct (MCD) cells, which represent a preferential site of UPEC adhesion. Flagellin, like lipopolysaccharide, stimulated the production of the chemoattractant chemokines CXCL1 and CXCL2, and subsequent migration capacity of neutrophils in cultured wild‐type (WT) and Tlr4−/− MCDs, but not in Tlr5−/− MCDs. UPEC can translocate across intact MCD layers without altering tight junctions. Strikingly, the invasion capacity and transcellular translocation of the UPEC strain HT7 were significantly lower in Tlr5−/− than in WT MCDs. The non‐motile HT7ΔfliC mutant lacking flagellin also exhibited much lower translocation capacities than the HT7 isolates. Finally, Tlr5−/− kidneys exhibited less infiltrating neutrophils than WT kidneys one day after the transurethral inoculation of HT7, and greater delayed renal bacterial loads in the day 4 post‐infected Tlr5−/− kidneys. Overall, these findings indicate that the epithelial TLR5 participates to renal antibacterial defence, but paradoxically favours the translocation of UPEC across intact MCD cell layers.

Universal versus targeted additional contact precautions for multidrug-resistant organism carriage for patients admitted to an intensive care unit

HIGHLIGHTSAn isolation‐targeted screening is non‐inferior to universal screening‐isolation.This strategy provides similar rates of acquired MDRO colonization or infection.It could be appropriate in some ICU and allow consumption of fewer resources.A hospital stay of more than 5 days is associated with MDRO carriage on admission.The choice of risk factors may influence the suitability of such a strategy. Background: Although additional contact precautions (ACPs) are routinely used to reduce cross‐transmission of multidrug‐resistant organisms (MDROs), the relevance of isolation precautions remains debated. We hypothesized that the collection of recognized risk factors for MDRO carriage on intensive care unit (ICU) admission might be helpful to target ACPs without increasing MDRO acquisition during ICU stays, compared with universal ACPs. Materials and Methods: This is a sequential single‐center observational study performed in consecutive patients admitted to a French medical and surgical ICU. During the first 6‐month period, screening for MDRO carriage and ACPs were performed in all patients. During the second 6‐month period, screening was maintained, but ACP use was guided by the presence of at least 1 defined risk factor for MDRO. Results: During both periods, 33 (10%) and 30 (10%) among 327 and 297 admissions were, respectively, associated with a positive admission MDRO carriage. During both periods, a second screening was performed in 147 (45%) and 127 (43%) patients. Altogether, the rate of acquired MDRO (positive screening or clinical specimen) was similar during both periods (10% [n = 15] and 11.8% [n = 15], respectively; P = .66). Conclusions: The results of our study contribute to support the safety of an isolation‐targeted screening policy on ICU admission compared with universal screening and isolation regarding the rate of ICU‐acquired MDRO colonization or infection.