Nancy M. Garrett

Invasive Escherichia coli are a feature of Crohn's disease.

Crohns disease (CD) and ulcerative colitis (UC) are idiopathic inflammatory conditions of the gut. Our goal was to investigate if invasive Escherichia coli strains were present in patients with inflammatory bowel disease (IBD). Bacterial strains were isolated from biopsy material obtained from normal controls, and patients with a clinical diagnosis of CD and UC. Invasive bacteria were characterized by gentamicin protection assay and biochemical profiling (Api-20E). Strains were characterized by induction of cytokine expression in epithelial and macrophage cell cultures, measurement of epithelial barrier function, and confocal microscopy. Of all invasive bacterial strains in CD 98.9% were identified as E. coli as opposed to 42.1% in UC and 2.1% in normal controls. Epithelial invasion in vitro was significantly higher for CD-associated E. coli (8.4%, ±5.5 of initial inoculum (I/O)) in comparison to UC (2.5%, ±0.4 I/O), but highest for strains from inflamed CD tissue (11.3%, ±4.3 I/O). Both, CD and UC E. coli strains induced high mean TNF-α expression in macrophage cell lines (2604.8 pg/105 cells, ±447.4; 2,402.6 pg/105 cells, ±476.3, respectively), but concentrations were significantly higher for isolates from inflamed CD tissue (3071.3 pg/105 cells, ±226.0). Invasive E. coli from IBD tissue induced similar concentrations of interleukin (IL)-8 in epithelial cell cultures, but strains from inflamed CD tissue induced significantly less epithelial IL-8 (674.1 pg/105 cells, ±58.0 vs 920.5 pg/105 cells, ±94.6). IBD-associated E. coli strains significantly decreased transepithelial resistance, induced disorganization of F-actin and displacement of ZO-1, and E-cadherin from the apical junctional complex (AJC). In comparison to normal controls and UC, E. coli are more prevalent in CD, are highly invasive, and do not encode for known effector proteins. E. coli strains from IBD patients regulate cytokine expression and epithelial barrier function, two pathological features of IBD.

Biochemical, Serological, and Virulence Characterization of Clinical and Oyster Vibrio parahaemolyticus Isolates

ABSTRACT In this study, 77 clinical and 67 oyster Vibrio parahaemolyticus isolates from North America were examined for biochemical profiles, serotype, and the presence of potential virulence factors (tdh, trh, and type III secretion system [T3SS] genes). All isolates were positive for oxidase, indole, and glucose fermentation, consistent with previous reports. The isolates represented 35 different serotypes, 9 of which were shared by clinical and oyster isolates. Serotypes associated with pandemic strains (O1:KUT, O1:K25, O3:K6, and O4:K68) were observed for clinical isolates, and 7 (9%) oyster isolates belonged to serotype O1:KUT. Of the clinical isolates, 27% were negative for tdh and trh, while 45% contained both genes. Oyster isolates were preferentially selected for the presence of tdh and/or trh; 34% contained both genes, 42% had trh but not tdh, and 3% had tdh but not trh. All but 1 isolate (143/144) had at least three of the four T3SS1 genes examined. The isolates lacking both tdh and trh contained no T3SS2α or T3SS2β genes. All clinical isolates positive for tdh and negative for trh possessed all T3SS2α genes, and all isolates negative for tdh and positive for trh possessed all T3SS2β genes. The two oyster isolates containing tdh but not trh possessed all but the vopB2 gene of T3SS2α, as reported previously. In contrast to the findings of previous studies, all strains examined that were positive for both tdh and trh also carried T3SS2β genes. This report identifies the serotype as the most distinguishing feature between clinical and oyster isolates. Our findings raise concerns about the reliability of the tdh, trh, and T3SS genes as virulence markers and highlight the need for more-detailed pathogenicity investigations of V. parahaemolyticus.

Characterization of Toxigenic Vibrio cholerae from Haiti, 2010-2011

A virulent clone from Africa or southern Asia was likely introduced at a single time point.

Rapid Identification of Vibrio parahaemolyticus by Whole-Cell Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry

ABSTRACT Vibrio parahaemolyticus is a pathogenic marine bacterium that is the main causative agent of bacterial seafood-borne gastroenteritis in the United States. An increase in the frequency of V. parahaemolyticus-related infections during the last decade has been attributed to the emergence of an O3:K6 pandemic clone in 1995. The diversity of the O3:K6 pandemic clone and its serovariants has been examined using multiple molecular techniques including multilocus sequence analysis, pulsed-field gel electrophoresis, and group-specific PCR analysis. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has become a powerful tool for rapidly distinguishing between related bacterial species. In the current study, we demonstrate the development of a whole-cell MALDI-TOF MS method for the distinction of V. parahaemolyticus from other Vibrio spp. We identified 30 peaks that were present only in the spectra of the V. parahaemolyticus strains examined in this study that may be developed as MALDI-TOF MS biomarkers for identification of V. parahaemolyticus. We detected variation in the MALDI-TOF spectra of V. parahaemolyticus strains isolated from different geographical locations and at different times. The MALDI-TOF MS spectra of the V. parahaemolyticus strains examined were distinct from those of the other Vibrio species examined including the closely related V. alginolyticus, V. harveyi, and V. campbellii. The results of this study demonstrate the first use of whole-cell MALDI-TOF MS analysis for the rapid identification of V. parahaemolyticus.

Re-evaluation, optimization, and multilaboratory validation of the PulseNet-standardized pulsed-field gel electrophoresis protocol for Listeria monocytogenes.

The PulseNet Methods Development and Validation Laboratory began a re-evaluation of the standardized pulsed-field gel electrophoresis (PFGE) protocols with the goal of optimizing their overall performance and robustness. Herein, we describe a stepwise evaluation of the PulseNet-standardized PFGE protocol for Listeria monocytogenes that led to the modification of several steps which significantly improved the overall appearance and reproducibility of the resulting PFGE data. These improvements included the following: (1) reducing the cell suspension concentration, (2) increasing lysozyme incubation temperature from 37 degrees C to 56 degrees C, and (3) decreasing the number of units of restriction enzymes AscI and ApaI. These changes were incorporated into a proposed protocol that was evaluated by 16 PulseNet participating laboratories, including 2 international participants. Results from the validation study indicated that the updated L. monocytogenes protocol is more robust than the original PulseNet-standardized protocol established in 1998 and this resulted in the official adoption of the new protocol into the PulseNet system in the spring of 2008. The modifications not only represent an improvement to the protocol but also describe procedural improvements that could be potentially applied to the PFGE analysis of other Gram-positive organisms.

Toxigenic Vibrio cholerae O1 in Water and Seafood, Haiti

During the 2010 cholera outbreak in Haiti, water and seafood samples were collected to detect Vibrio cholerae. The outbreak strain of toxigenic V. cholerae O1 serotype Ogawa was isolated from freshwater and seafood samples. The cholera toxin gene was detected in harbor water samples.

US Outbreak of Human Salmonella Infections Associated With Aquatic Frogs, 2008–2011

OBJECTIVE: Although amphibians are known Salmonella carriers, no such outbreaks have been reported. We investigated a nationwide outbreak of human Salmonella Typhimurium infections occurring predominantly among children from 2008 to 2011. METHODS: We conducted a matched case-control study. Cases were defined as persons with Salmonella Typhimurium infection yielding an isolate indistinguishable from the outbreak strain. Controls were persons with recent infection with Salmonella strains other than the outbreak strain and matched to cases by age and geography. Environmental samples were obtained from patients’ homes; traceback investigations were conducted. RESULTS: We identified 376 cases from 44 states from January 1, 2008, to December 31, 2011; 29% (56/193) of patients were hospitalized and none died. Median patient age was 5 years (range <1–86 years); 69% were children <10 years old (253/367). Among 114 patients interviewed, 69 (61%) reported frog exposure. Of patients who knew frog type, 79% (44/56) reported African dwarf frogs (ADF), a type of aquatic frog. Among 18 cases and 29 controls, illness was significantly associated with frog exposure (67% cases versus 3% controls, matched odds ratio 12.4, 95% confidence interval 1.9–infinity). Environmental samples from aquariums containing ADFs in 8 patients’ homes, 2 ADF distributors, and a day care center yielded isolates indistinguishable from the outbreak strain. Traceback investigations of ADFs from patient purchases converged to a common ADF breeding facility. Environmental samples from the breeding facility yielded the outbreak strain. CONCLUSIONS: ADFs were the source of this nationwide pediatric predominant outbreak. Pediatricians should routinely inquire about pet ownership and advise families about illness risks associated with animals.

Insights into the environmental reservoir of pathogenic Vibrio parahaemolyticus using comparative genomics

Vibrio parahaemolyticus is an aquatic halophilic bacterium that occupies estuarine and coastal marine environments, and is a leading cause of seafood-borne food poisoning cases. To investigate the environmental reservoir and potential gene flow that occurs among V. parahaemolyticus isolates, the virulence-associated gene content and genome diversity of a collection of 133 V. parahaemolyticus isolates were analyzed. Phylogenetic analysis of housekeeping genes, and pulsed-field gel electrophoresis, demonstrated that there is genetic similarity among V. parahaemolyticus clinical and environmental isolates. Whole-genome sequencing and comparative analysis of six representative V. parahaemolyticus isolates was used to identify genes that are unique to the clinical and environmental isolates examined. Comparative genomics demonstrated an O3:K6 environmental isolate, AF91, which was cultured from sediment collected in Florida in 2006, has significant genomic similarity to the post-1995 O3:K6 isolates. However, AF91 lacks the majority of the virulence-associated genes and genomic islands associated with these highly virulent post-1995 O3:K6 genomes. These findings demonstrate that although they do not contain most of the known virulence-associated regions, some V. parahaemolyticus environmental isolates exhibit significant genetic similarity to clinical isolates. This highlights the dynamic nature of the V. parahaemolyticus genome allowing them to transition between aquatic and host-pathogen states.

Shifts in Geographic Distribution and Antimicrobial Resistance during a Prolonged Typhoid Fever Outbreak — Bundibugyo and Kasese Districts, Uganda, 2009–2011

Background Salmonella enterica serovar Typhi is transmitted by fecally contaminated food and water and causes approximately 22 million typhoid fever infections worldwide each year. Most cases occur in developing countries, where approximately 4% of patients develop intestinal perforation (IP). In Kasese District, Uganda, a typhoid fever outbreak notable for a high IP rate began in 2008. We report that this outbreak continued through 2011, when it spread to the neighboring district of Bundibugyo. Methodology/Principal Findings A suspected typhoid fever case was defined as IP or symptoms of fever, abdominal pain, and ≥1 of the following: gastrointestinal disruptions, body weakness, joint pain, headache, clinically suspected IP, or non-responsiveness to antimalarial medications. Cases were identified retrospectively via medical record reviews and prospectively through laboratory-enhanced case finding. Among Kasese residents, 709 cases were identified from August 1, 2009–December 31, 2011; of these, 149 were identified during the prospective period beginning November 1, 2011. Among Bundibugyo residents, 333 cases were identified from January 1–December 31, 2011, including 128 cases identified during the prospective period beginning October 28, 2011. IP was reported for 507 (82%) and 59 (20%) of Kasese and Bundibugyo cases, respectively. Blood and stool cultures performed for 154 patients during the prospective period yielded isolates from 24 (16%) patients. Three pulsed-field gel electrophoresis pattern combinations, including one observed in a Kasese isolate in 2009, were shared among Kasese and Bundibugyo isolates. Antimicrobial susceptibility was assessed for 18 isolates; among these 15 (83%) were multidrug-resistant (MDR), compared to 5% of 2009 isolates. Conclusions/Significance Molecular and epidemiological evidence suggest that during a prolonged outbreak, typhoid spread from Kasese to Bundibugyo. MDR strains became prevalent. Lasting interventions, such as typhoid vaccination and improvements in drinking water infrastructure, should be considered to minimize the risk of prolonged outbreaks in the future.

Multistate outbreak of human Salmonella Typhimurium infections linked to live poultry from agricultural feed stores and mail-order hatcheries, United States 2013

Live poultry-associated salmonellosis is an emerging public health issue in the United States. Public and animal health officials collaborated to investigate one of the largest (356 cases, 39 states) of these outbreaks reported to date. A case was defined as illness in a person infected with the outbreak strain of Salmonella Typhimurium with illness onset between 1 March and 22 October 2013. The median patient age was seven years (range: < 1–87 years); 58% of ill persons were children ≤ 10 years, 51% were female, 25% were hospitalized; 189 (76%) of 250 patients reported live poultry exposure in the week before illness; and 149 (95%) of 157 reported purchasing live poultry from agricultural feed stores. Traceback investigations identified 18 live poultry sources, including 16 mail-order hatcheries. Environmental sampling was conducted at two mail-order hatcheries. One (2.5%) of 40 duplicate samples collected at one hatchery yielded the outbreak strain. Live poultry are an important source of human salmonellosis, particularly among children, highlighting the need for educational campaigns and comprehensive interventions at the mail-order hatchery and agricultural feed store levels. Prevention and control efforts depend on a One Health approach, involving cooperation between public and animal health officials, industry, health professionals, and consumers.