Retrospective Evaluation of Infants Aged 1 to 60 Days with Residual Cerebrospinal Fluid (CSF) Tested Using the FilmArray Meningitis/Encephalitis (ME) Panel.

In pediatric practice it is common for infants under 2 months of age to undergo evaluation for sepsis when they are ill, often including lumbar puncture to assess for central nervous system (CNS) infection. The FilmArray Meningitis/Encephalitis (ME) panel is a newly approved test for rapid identification of CNS pathogens. Our objective was to study the epidemiology of CNS infection in young infants and the potential impact of rapid multiplex PCR on their care. A performance evaluation of the FilmArray ME panel was conducted from February 2014 to September 2014 at 11 sites. FilmArray ME panel results were compared to reference standards but not shared with providers. In our study, medical records for infants (aged 1 to 60 days) enrolled at three sites were reviewed for clinical, laboratory, and outcome data. A total of 145 infants were reviewed. The median age was 25 days. Most of the infants were hospitalized (134/145 [92%]) and received antibiotics (123/145 [85%]), and almost half (71/145 [49%]) received acyclovir. One infant had a bacterial pathogen, likely false positive, identified by the FilmArray ME panel. Thirty-six infants (25%) had a viral pathogen detected, including 21 enteroviruses. All infants with enteroviral meningitis detected by the FilmArray ME panel and conventional PCR were hospitalized, but 20% were discharged in less than 24 h when conventional PCR results became available. The FilmArray ME panel may play a role in the evaluation of young infants for CNS infection. Results may be used to guide management, possibly resulting in a decreased length of stay and less antimicrobial exposure for infants with low-risk viral infection detected.

Evaluation of the FilmArray Blood Culture Identification Panel: Results of a Multicenter Controlled Trial.

Sepsis is a major cause of morbidity, mortality, and increased medical expense. Rapid diagnosis improves outcomes and reduces costs. The FilmArray blood culture identification panel (BioFire Diagnostics LLC, Salt Lake City, UT), a highly multiplexed PCR assay, can identify 24 etiologic agents of sepsis (8 Gram-positive, 11 Gram-negative, and 5 yeast species) and three antimicrobial resistance genes (mecA, vanA/B, and blaKPC) from positive blood culture bottles. It provides results in about 1 h with 2 min for assay setup. We present the results of an eight-center trial comparing the sensitivity and specificity of the panel with those of the laboratories' standard phenotypic identification techniques, as well as with molecular methods used to distinguish Acinetobacter baumannii from other members of the A. calcoaceticus-A. baumannii complex and to detect antimicrobial resistance genes. Testing included 2,207 positive aerobic blood culture samples, 1,568 clinical and 639 seeded. Samples were tested fresh or were frozen for later testing within 8 h after the bottles were flagged as positive by an automated blood culture system. At least one organism was detected by the panel in 1,382 (88.1%) of the positive clinical specimens. The others contained primarily off-panel organisms. The panel reported multiple organisms in 81 (5.86%) positive clinical specimens. The unresolved blood culture identification sensitivity for all target detections exceeded 96%, except for Klebsiella oxytoca (92.2%), which achieved 98.3% sensitivity after resolution of an unavoidable phenotypic error. The sensitivity and specificity for vanA/B and blaKPC were 100%; those for mecA were 98.4 and 98.3%, respectively.

Multicenter evaluation of the BioFire FilmArray gastrointestinal panel for etiologic diagnosis of infectious gastroenteritis.

The appropriate treatment and control of infectious gastroenteritis depend on the ability to rapidly detect the wide range of etiologic agents associated with the disease. Clinical laboratories currently utilize an array of different methodologies to test for bacterial, parasitic, and viral causes of gastroenteritis, a strategy that suffers from poor sensitivity, potentially long turnaround times, and complicated ordering practices and workflows. Additionally, there are limited or no testing methods routinely available for most diarrheagenic Escherichia coli strains, astroviruses, and sapoviruses. This study assessed the performance of the FilmArray Gastrointestinal (GI) Panel for the simultaneous detection of 22 different enteric pathogens directly from stool specimens: Campylobacter spp., Clostridium difficile (toxin A/B), Plesiomonas shigelloides, Salmonella spp., Vibrio spp., Vibrio cholerae, Yersinia enterocolitica, enteroaggregative E. coli, enteropathogenic E. coli, enterotoxigenic E. coli, Shiga-like toxin-producing E. coli (stx1 and stx2) (including specific detection of E. coli O157), Shigella spp./enteroinvasive E. coli, Cryptosporidium spp., Cyclospora cayetanensis, Entamoeba histolytica, Giardia lamblia, adenovirus F 40/41, astrovirus, norovirus GI/GII, rotavirus A, and sapovirus. Prospectively collected stool specimens (n = 1,556) were evaluated using the BioFire FilmArray GI Panel and tested with conventional stool culture and molecular methods for comparison. The FilmArray GI Panel sensitivity was 100% for 12/22 targets and ≥94.5% for an additional 7/22 targets. For the remaining three targets, sensitivity could not be calculated due to the low prevalences in this study. The FilmArray GI Panel specificity was ≥97.1% for all panel targets. The FilmArray GI Panel provides a comprehensive, rapid, and streamlined alternative to conventional methods for the etiologic diagnosis of infectious gastroenteritis in the laboratory setting. The potential advantages include improved performance parameters, a more extensive menu of pathogens, and a turnaround time of as short as 1 h.

Presenilin/gamma-secretase cleaves CD46 in response to Neisseria infection.

CD46 is a type I transmembrane protein with complement and T cell regulatory functions in human cells. CD46 has signaling and receptor properties in immune and nonimmune cells, many of which are dependent on the expression of cytoplasmic tail (cyt) isoforms cyt1 or cyt2. Little is known about how cyt1 and cyt2 mediate cellular responses. We show that CD46-cyt1 and CD46-cyt2 are substrates for presenilin/gamma-secretase (PS/gammaS), an endogenous protease complex that regulates many important signaling proteins through proteolytic processing. PS/gammaS processing of CD46 releases immunoprecipitable cyt1 and cyt2 tail peptides into the cell, is blocked by chemical inhibitors, and is prevented in dominant negative presenilin mutant cell lines. Two human pathogens, Neisseria gonorrhoeae and Neisseria meningitidis, stimulate PS/gammaS processing of CD46-cyt1 and CD46-cyt2. This stimulation requires type IV pili and PilT, the type IV pilus retraction motor, implying that mechanotransduction plays a role in this event. We present a model for PS/gammaS processing of CD46 that provides a mechanism by which signals are transduced via the cyt1 and cyt2 tails to regulate CD46-dependent cellular responses. Our findings have broad implications for understanding the full range of CD46 functions in infection and noninfection situations.

Characterization of DNA-binding specificity and analysis of binding sites of the Pseudomonas aeruginosa global regulator, Vfr, a homologue of the Escherichia coli cAMP receptor protein.

Vfr, a global regulator of Pseudomonas aeruginosa virulence factors, is a homologue of the Escherichia coli cAMP receptor protein, CRP. Vfr is 91% similar to CRP and maintains many residues important for CRP to bind cAMP, bind DNA, and interact with RNA polymerase at target promoters. While vfr can complement an E. coli crp mutant in beta-galactosidase production, tryptophanase production and catabolite repression, crp can only complement a subset of Vfr-dependent phenotypes in P. aeruginosa. Using specific CRP binding site mutations, it is shown that Vfr requires the same nucleotides as CRP for optimal transcriptional activity from the E. coli lac promoter. In contrast, CRP did not bind Vfr target sequences in the promoters of the toxA and regA genes. Footprinting analysis revealed Vfr protected sequences upstream of toxA, regA, and the quorum sensing regulator lasR, that are similar to but significantly divergent from the CRP consensus binding sequence, and Vfr causes similar DNA bending to CRP in bound target sequences. Using a preliminary Vfr consensus binding sequence deduced from the Vfr-protected sites, Vfr target sequences were identified upstream of the virulence-associated genes plcN, plcHR, pbpG, prpL and algD, and in the vfr/orfX, argH/fimS, pilM/ponA intergenic regions. From these sequences the Vfr consensus binding sequence, 5'-ANWWTGNGAWNY : AGWTCACAT-3', was formulated. This study suggests that Vfr shares many of the same functions as CRP, but has specialized functions, at least in terms of DNA target sequence binding, required for regulation of a subset of genes in its regulon.

SepZ/EspZ is secreted and translocated into HeLa cells by the enteropathogenic Escherichia coli type III secretion system.

Enteropathogenic Escherichia coli (EPEC) is a major bacterial cause of infantile diarrhea in developing countries and is the prototype for a group of gastrointestinal pathogens causing characteristic attaching and effacing (A/E) histopathology on intestinal epithelia. A/E pathogens utilize a type III secretion system (TTSS), encoded by the locus of enterocyte effacement (LEE) pathogenicity island, to deliver effector proteins into host cells. Here, we investigate sequence divergence of the LEE-encoded SepZ protein and identify it as a TTSS-secreted and -translocated molecule. SepZ is hypervariable among A/E pathogens, with sequences sharing between 60 to 81% amino acid identity with SepZ of EPEC. A SepZ-CyaA fusion was secreted and translocated into HeLa cells in a TTSS-dependent manner. Additionally, we determined that the first 20 amino acids of SepZ were sufficient to direct its translocation. In contrast to previous studies suggesting a role in invasion and the structure and/or regulation of the TTSS, we found that SepZ does not mediate uptake of EPEC into host cells or affect translocation and tyrosine phosphorylation of the translocated intimin receptor. Immunohistochemistry reveals that, after an extended HeLa cell infection, accumulated SepZ can be detected beneath the site of bacterial attachment in a subset of pedestal regions. To indicate its newly identified status as a translocated effector protein, we propose to rename SepZ as EspZ.

Enteropathogenic Escherichia coli EspG disrupts microtubules and in conjunction with Orf3 enhances perturbation of the tight junction barrier.

EspG, a secreted effector of enteropathogenic Escherichia coli (EPEC), as well as its homologue Orf3, has been shown to disrupt microtubules (MTs) in fibroblasts and non-polarized epithelial cells. The roles of MTs and the effects of MT disruption in these cell types differ significantly. The aim of this study was to investigate the effects of EspG on polarized, host target intestinal epithelial cells. Immunofluorescent labelling of tubulin showed that EPEC caused progressive fragmentation and loss of the MT network in cells harbouring attached organisms. Immunoblots of proteins extracted from EPEC-infected cells showed a corresponding loss of alpha-tubulin. Type III secretion system (TTSS)-deficient strains had no effect on MT suggesting TTSS dependence. Mutation of espG, but not espF or map, ablated EPEC's effects on MTs for up to 6 h. Ectopic expression of EspG in HeLa cells caused MT disruption. While deletion of espG alone had no effect on the EPEC-induced decrease in transepithelial electrical resistance (TER), mutation of both espG and orf3 significantly delayed the kinetics of this response. Complementation of the double mutant with espG alone restored the kinetics of TER drop to that of wild type. Herein, we describe a previously unrecognized phenotype for the EPEC effectors EspG and Orf3.

Characterization of the second long polar (LP) fimbriae of Escherichia coli O157:H7 and distribution of LP fimbriae in other pathogenic E. coli strains.

A second region containing five genes homologous to the long polar fimbrial operon of Salmonella enterica serovar Typhimurium is located in the chromosome of enterohemorrhagic Escherichia coli (EHEC) O157:H7. A non-fimbriated E. coli K-12 strain carrying the cloned EHEC lpf (lpf2) genes expressed thin fibrillae-like structures on its surface and displayed reduced adherence to tissue culture cells. Neither mutation in the lpfA2 gene in either the parent or lpfA1 mutant strains showed an effect in adherence or in the formation of A/E lesions on HeLa cells. lpfA2 isogenic mutant strains adhere to Caco-2 cells almost as well as the wild-type at 5 h, but they were deficient in adherence at early time points. A collection of diarrheagenic E. coli strains were investigated for the presence of lpfA1 and lpfA2 and results showed that these genes are present in specific serogroups which are phylogenetically related. Our results suggest that LP fimbriae 2 may contribute to the early stages of EHEC adhesion and that genes encoding the major LP fimbrial subunits are present in a small group of EHEC and EPEC serotypes.

fleQ, the gene encoding the major flagellar regulator of Pseudomonas aeruginosa, is sigma70 dependent and is downregulated by Vfr, a homolog of Escherichia coli cyclic AMP receptor protein.

The flagellar transcriptional regulator FleQ appears to be the highest-level regulator in the hierarchical regulatory cascade of flagellar biogenesis in Pseudomonas aeruginosa. Except for the posttranslational downregulation of FleQ activity by FleN, an antiactivator, not much is known about the regulation of the fleQ gene or its gene product. Some FleQ homologs in other bacterial species either are positively regulated by another regulator (e.g., CtrA, the master regulator regulating FlbD in Caulobacter crescentus) or are expressed from a sigma70-dependent promoter (e.g., FlgR of Helicobacter pylori). In this study we demonstrated that Vfr, an Escherichia coli CRP homolog known to function as an activator for various genes, including lasR, regA, and toxA, in P. aeruginosa, is capable of repressing fleQ transcription by binding to its consensus sequence in the fleQ promoter. In a DNase I footprint assay, purified Vfr protected the sequence 5'-AATTGACTAATCGTTCACATTTG-3'. When this putative Vfr binding site in the fleQ promoter was mutated, Vfr was unable to bind the fleQ promoter fragment and did not repress fleQ transcription effectively. Primer extension analysis of the fleQ transcript revealed two transcriptional start sites, t1 and t2, that map within the Vfr binding site. A putative -10 region (TAAAAT) for the t2 transcript, with a five-of-six match with the E. coli sigma70 binding consensus, overlaps with one end of the Vfr binding site. A 4-bp mutation and an 8-bp mutation in this -10 region markedly reduced the activity of the fleQ promoter. The same mutations led to the disappearance of the 203-nucleotide fleQ transcript in an in vitro transcription assay. Vfr probably represses fleQ transcription by binding to the Vfr binding site in the fleQ promoter and preventing the sigma factor from binding to the -10 region to initiate transcription.

Antibiotic-resistant cell-detaching Escherichia coli strains from Nigerian children.

The properties of 23 cell-detaching Escherichia coli strains that were isolated from stool specimens in Nigeria are described. Common properties of the strains included the presence of genes encoding alpha-hemolysin (100%), pyelonephritis-associated pili (100%), and cytotoxic necrotizing factor 1 (70%) as well as lactose negativity (70%) and multiple antibiotic resistance (74%). Antibiotic resistance was shown in most cases to be transferable and associated with the presence of class 1 integrons. Phenotypic properties and pulsed-field gel electrophoresis analysis demonstrated that the majority of the strains, particularly multiply resistant, lactose-negative O4:H40 strains, were closely related. Multiply-resistant cell-detaching E. coli strains may represent an important reservoir for antibiotic resistance genes.