Kristen J. Kanack

Multicenter Evaluation of the BioFire FilmArray Gastrointestinal Panel for Etiologic Diagnosis of Infectious Gastroenteritis

ABSTRACT 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 (stx 1 and stx 2) (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.

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.

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 α‐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 EPECs 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.

SepZ/EspZ Is Secreted and Translocated into HeLa Cells by the Enteropathogenic Escherichia coli Type III Secretion System

ABSTRACT 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.

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

ABSTRACT 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 α-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.

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

Retrospective Evaluation of Infants Aged 1-60 Days With Residual CSF Tested Using the FilmArray® Meningitis/Encephalitis (ME) Panel

ABSTRACT 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.

Vibrio cholerae Detection by the FilmArray® Gastrointestinal (GI) Panel

Abstract Background The purpose of this study was to investigate reported false positive FilmArray GI Panel detections of V. cholerae in clinical patient samples. The gold standard for recovery of V. cholerae is routine stool culture. The FilmArray GI Panel contains two sensitive PCR assays for the detection of V. cholerae; one targets the gyrB gene for genus-level Vibrio identification (focused on detection of V. parahaemolyticus, V. vulnificus, and V. cholerae) and the second targets the toxR gene for specific identification of V. cholerae. Methods Clinical samples with discordant V. cholerae detections were requested from source laboratories and then tested on the FilmArray GI Panel. Amplicons from positive samples were extracted, sequenced, and assessed using BLAST search. Results A total of 14 samples were tested during this study. Three re-tested samples were reported positive for V. cholerae. One sample was confirmed as V. cholerae by the presence of V. cholerae specific gyrB and toxR gene sequences. A second sample was confirmed to be either Vibrio alginolyticus or Vibrio diabolicus by gyrB gene sequence and possessed a 100% homolog to the V. cholerae specific toxR gene. The third sample was confirmed to contain two species of Vibrio: V. cholerae and V. parahaemolyticus. This sample was also confirmed to have the V. cholerae specific toxR gene sequence. The remaining 11 samples resulted in a negative V. cholerae result upon retesting. Conclusion The investigation confirmed that the FilmArray GI Panel can detect low level V. cholerae organism not recovered by culture. Studies have shown that isolating V. cholerae through specialized media can prove difficult. V. cholerae causing vibriosis can be food-borne or acquired through exposure to affected bodies of water, marine wildlife, or seafood. We found two rare cases, one with the non-cholerae Vibrio species possessing a V. cholerae specific homolog to the toxR regulon and the one novel detection of a co-infection involving two Vibrio species. These data suggest that there will be an increase in Vibrio detections as molecular methods are more sensitive than culture and become much more common for gastrointestinal pathogen testing. Disclosures K. Clarke, BioFire Diagnostics, LLC: Employee, Salary; 
 M. Rogatcheva, BioFire Diagnostics, LLC: Employee, Salary; A. Demogines, BioFire Diagnostics, LLC: Employee, Salary; D. Henderson, BioFire Diagnostics, LLC: Employee, Salary; D. Saif, BioFire Diagnostics, LLC: Employee, Salary; K. Kanack, BioFire Diagnostics, LLC: Employee, Salary