Michele B. Parsons

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.

Drug-Resistance Mechanisms in Vibrio cholerae O1 Outbreak Strain, Haiti, 2010

To increase understanding of drug-resistant Vibrio cholerae, we studied selected molecular mechanisms of antimicrobial drug resistance in the 2010 Haiti V. cholerae outbreak strain. Most resistance resulted from acquired genes located on an integrating conjugative element showing high homology to an integrating conjugative element identified in a V. cholerae isolate from India.

Field evaluation of Crystal VC® Rapid Dipstick test for cholera during a cholera outbreak in Guinea‐Bissau

Objectives  To evaluate performance characteristics and ease of use of the new commercially available Crystal VC® Rapid Dipstick (VC) test (Span Diagnostics, India) for Vibrio cholerae O1 and O139.

Burden of typhoid and paratyphoid fever in a densely populated urban community, Dhaka, Bangladesh

BACKGROUND We conducted blood culture surveillance to estimate the incidence of typhoid and paratyphoid fever among urban slum residents in Dhaka, Bangladesh. METHODS Between January 7, 2003 and January 6, 2004, participants were visited weekly to detect febrile illnesses. Blood cultures were obtained at the clinic from patients with fever (≥38°C). Salmonella isolates were assayed for antimicrobial susceptibility. RESULTS Forty Salmonella Typhi and eight Salmonella Paratyphi A were isolated from 961 blood cultures. The incidence of typhoid fever was 2.0 episodes/1000 person-years, with a higher incidence in children aged<5 years (10.5/1000 person-years) than in older persons (0.9/1000 person-years) (relative risk=12, 95% confidence interval (CI) 6.3-22.6). The incidence of paratyphoid fever was 0.4/1000 person-years without variation by age group. Sixteen S. Typhi isolates were multidrug-resistant (MDR). All S. Paratyphi isolates were pan-susceptible. The duration of fever among patients with an MDR S. Typhi infection was longer than among patients with non-MDR S. Typhi (16±8 vs. 11±4 days, p=0.02) and S. Paratyphi (10±2 days, p=0.04) infections. CONCLUSIONS Typhoid fever is more common than paratyphoid fever in the urban Bangladeshi slum; children<5 years old have the highest incidence. Multidrug resistance is common in S. Typhi isolates and is associated with prolonged illness. Strategies for typhoid fever prevention in children aged<5 years in Bangladesh, including immunization, are needed.

An Outbreak of Enterotoxigenic Escherichia coli Associated with Sushi Restaurants in Nevada, 2004

BACKGROUND In August and November 2004, 2 clusters of diarrhea cases occurred among patrons of 2 affiliated sushi restaurants (sushi restaurant A and sushi restaurant B) in Nevada. In August 2004, a stool sample from 1 ill sushi restaurant A patron yielded enterotoxigenic Escherichia coli (ETEC). In December 2004, we investigated a third cluster of diarrhea cases among sushi restaurant B patrons. METHODS We defined a case as diarrhea in a person who ate at sushi restaurant B from 3 December through 13 December 2004. Control subjects were individuals who dined with case patients but did not become ill. Duplex polymerase chain reaction was used to detect genes coding for heat-stable and heat-labile enterotoxins of ETEC. RESULTS One-hundred thirty patrons of sushi restaurant B reported illness; we enrolled 36 case patients and 29 control subjects. The diarrhea-to-vomiting prevalence ratio among patients was 4.5. Illness was associated with consumption of butterfly shrimp (estimated odds ratio, 7.2; 95% confidence interval, 1.1 to infinity). The implicated food was distributed to many restaurants, but only sushi restaurant B patrons reported diarrhea. We observed poor food-handling and hand hygiene practices at sushi restaurant B. Stool samples from 6 of 7 ill patrons and 2 of 27 employees who denied illness yielded ETEC. CONCLUSIONS ETEC was identified as the etiologic agent of a large foodborne outbreak at a sushi restaurant in Nevada. Poor food-handling practices and infected foodhandlers likely contributed to this outbreak. Although ETEC is a well-documented cause of domestic foodborne outbreaks, few laboratories can test for it. Earlier recognition of ETEC infections may prevent subsequent outbreaks from occurring.

Evaluation and Validation of a PulseNet Standardized Pulsed-Field Gel Electrophoresis Protocol for Subtyping Vibrio parahaemolyticus: an International Multicenter Collaborative Study

ABSTRACT The pandemic spread of Vibrio parahaemolyticus is an international public health issue. Because of the outbreak potential of the organism, it is critical to establish an internationally recognized molecular subtyping protocol for V. parahaemolyticus that is both rapid and robust as a means to monitor its further spread and to guide control measures in combination with epidemiologic data. Here we describe the results of a multicenter, multicountry validation of a new PulseNet International standardized V. parahaemolyticus pulsed-field gel electrophoresis (PFGE) protocol. The results are from a composite analysis of 36 well-characterized V. parahaemolyticus isolates from six participating laboratories, and the isolates represent predominant serotypes and various genotypes isolated from different geographic regions and time periods. The discriminatory power is very high, as 34 out of 36 sporadic V. parahaemolyticus strains tested fell into 34 distinguishable PFGE groups when the data obtained with two restriction enzymes (SfiI and NotI) were combined. PFGE was further able to cluster members of known pandemic serogroups. The study also identified quality measures which may affect the performance of the protocol. Nonadherence to the recommended procedure may lead to high background in the PFGE gel patterns, partial digestion, and poor fragment resolution. When these quality measures were implemented, the PulseNet V. parahaemolyticus protocol was found to be both robust and reproducible among the collaborating laboratories.

Transmission risk factors and treatment of pediatric shigellosis during a large daycare center-associated outbreak of multidrug resistant Shigella sonnei: implications for the management of shigellosis outbreaks among children.

Background: Shigellosis outbreaks in daycare centers result in substantial disease and economic burdens in the United States. The emergence of multidrug resistant Shigella strains raises questions regarding control of transmission within daycare centers and treatment for children. From May to October 2005, 639 Shigella sonnei cases were reported in northwest Missouri, mostly among persons exposed to daycare centers. Methods: We conducted a case-control investigation among licensed daycare centers (LDCs) in northwest Missouri to determine transmission risk factors, tested isolates for antimicrobial resistance, and described treatment practices. Case LDCs had secondary attack rates of shigellosis ≥2% (range, 2%–25%) and control LDCs ≤2% (range, 0%–1.3%). We interviewed LDC staff and performed on-site inspections. Thirty-one outbreak isolates were tested for antimicrobial resistance. We interviewed physicians and reviewed health department outbreak-related treatment data. Results: We enrolled 18 case and 21 control LDCs. LDCs with ≥1 sink in every room (odds ratio [OR]: 0.1; 95% confidence interval [CI]: 0.02–0.5) or a diapering station in every room (OR: 0.1; 95% CI: 0.01–0.6) were less likely to be case-LDCs. Resistance to ampicillin and trimethoprim-sulfamethoxazole was found in 90% of the outbreak strains. Among 210 children treated with antimicrobial agents, azithromycin was used in 92 (44%) while a fluoroquinolone was used in 11 (5%) children. Conclusions: During a large daycare center-associated shigellosis outbreak, strains were highly resistant to ampicillin and trimethoprim-sulfamethoxazole. Children were frequently treated with azithromycin and occasionally fluoroquinolones. Appropriate handwashing and diapering infrastructure are necessary to minimize spread of shigellosis within daycare centers, and could reduce use of antimicrobial agents.

Clinical Characteristics and Molecular Subtyping of Vibrio vulnificus Illnesses, Israel

The genetically distinct biotype 3 has penetrated Israeli freshwaters and is causing severe illness in persons who handle tilapia or carp.