Jacqueline Cattani

Performance Assessment of Three Commercial Assays for Direct Detection of Bacillus anthracis Spores

Bacillus anthracis , the cause of anthrax, has been used as a bioterrorism agent. Because the isolation and identification of B. anthracis by culture can take days, first response units (hazardous materials [HAZMAT], firemen, police, and hospital personnel) desire a quick and easy test that can be

Novel Sample Preparation Method for Safe and Rapid Detection of Bacillus anthracis Spores in Environmental Powders and Nasal Swabs

ABSTRACT Bacillus anthracis spores have been used as a biological weapon in the United States. We wanted to develop a safe, rapid method of sample preparation that provided safe DNA for the detection of spores in environmental and clinical specimens. Our method reproducibly detects B. anthracis in samples containing <10 spores.

Bacillus Anthracis Virulent Plasmid pX02 Genes Found in Large Plasmids of Two Other Bacillus Species

ABSTRACT In order to cause the disease anthrax, Bacillus anthracis requires two plasmids, pX01 and pX02, which carry toxin and capsule genes, respectively, that are used as genetic targets in the laboratory detection of the bacterium. Clinical, forensic, and environmental samples that test positive by PCR protocols established by the Centers for Disease Control and Prevention for B. anthracis are considered to be potentially B. anthracis until confirmed by culture and a secondary battery of tests. We report the presence of 10 genes (acpA, capA, capB, capC, capR, capD, IS1627, ORF 48, ORF 61, and repA) and the sequence for the capsule promoter normally found on pX02 in Bacillus circulans and a Bacillus species closely related to Bacillus luciferensis. Tests revealed these sequences to be present on a large plasmid in each isolate. The 11 sequences consistently matched to B. anthracis plasmid pX02, GenBank accession numbers AF188935.1, AE011191.1, and AE017335.3. The percent nucleotide identities for capD and the capsule promoter were 99.9% and 99.7%, respectively, and for the remaining nine genes, the nucleotide identity was 100% for both isolates. The presence of these genes, which are usually associated with the pX02 plasmid, in two soil Bacillus species unrelated to B. anthracis alerts us to the necessity of identifying additional sequences that will signal the presence of B. anthracis in clinical, forensic, and environmental samples.

Comparison of Methods for DNA Isolation from Food Samples for Detection of Shiga Toxin-Producing Escherichia coli by Real-Time PCR

ABSTRACT In this study, food samples were intentionally contaminated with Escherichia coli O157:H7, and then DNA was isolated by using four commercial kits. The isolated DNA samples were compared by using real-time PCR detection of the Shiga toxin genes. The four kits tested worked similarly.

Real-time PCR detection of the thermostable direct hemolysin and thermolabile hemolysin genes in a Vibrio parahaemolyticus cultured from mussels and mussel homogenate associated with a foodborne outbreak.

Molecular methods have become vital epidemiological tools in the detection and characterization of bacteria associated with a foodborne outbreak. We used both culture and real-time PCR to detect a Vibrio parahaemolyticus isolate associated with a foodborne outbreak. The outbreak occurred in July 2002 in Polk County, Florida, and originated at a Chinese buffet, with one person being hospitalized. The hospital isolated V. parahaemolyticus from the patient but destroyed the sample after diagnosis. From an onsite visit of the restaurant, food samples that possibly contributed to the outbreak were collected and sent to the Florida Department of Health, Tampa Branch Laboratory. Crab legs, crabsticks, and mussel samples were homogenized and incubated according to the Food and Drug Administration Bacteriological Analytical Manual culture protocol. Three sets of primers and a TaqMan probe were designed to target the tdh, trh, and tlh genes and used for real-time PCR. This study was successful in isolating V. parahaemolyticus from a mussel sample and detecting two of its genes (tdh and tlh) in food and pure culture by real-time PCR.

A survey of usage protocols of syndromic surveillance systems by state public health departments in the United States

OBJECTIVE To broadly describe current syndromic surveillance systems in use throughout the United States and to provide basic descriptive information on responses to syndromic system signals. METHODS Cross-sectional survey (telephone and e-mail) of state epidemiologists in all 50 states and the District of Columbia. RESULTS Forty-one states participated in the survey for a response rate of 80 percent. Thirty-three states (80%) had at least one syndromic surveillance system in addition to BioSense operating within the state. Every state with an urban area at highest risk of a terrorist attack reported monitoring syndromic surveillance data, and a states overall preparedness level was not related to the presence (or lack) of operational syndromic surveillance systems. The most common syndromic surveillance systems included BioSense (n = 20, 61%) and RODS (n = 13, 39%). Seventy-six percent of states with syndromic surveillance initiated investigations at the state level, 64 percent at the county level, and 45 percent at both the state and county levels. CONCLUSIONS The majority of states reported using syndromic surveillance systems, with greatest penetration in those at highest risk for a terrorist attack. Most states used multiple systems and had varied methods (central and local) of responding to alerts, indicating the need for detailed response protocols.

Community-Associated Methicillin-Resistant Staphylococcus aureus Epidemic Clone USA300 in Isolates from Florida and Washington

ABSTRACT We examined 299 methicillin-resistant, community-associated Staphylococcus aureus isolates from Florida and Washington State for the presence of the USA300 epidemic clone. Pulsed-field gel electrophoresis demonstrated the epidemic clone in 43% of our S. aureus strains and in isolates from both states. The majority of the USA300 isolates (88%) were from wound infections.

Framework for the development of response protocols for public health syndromic surveillance systems: case studies of 8 US states.

OBJECTIVES To describe current syndromic surveillance system response protocols in health departments from 8 diverse states in the United States and to develop a framework for health departments to use as a guide in initial design and/or enhancement of response protocols. METHODS Case study design that incorporated in-depth interviews with health department staff, textual analysis of response plans, and a Delphi survey of syndromic surveillance response experts. RESULTS All 8 states and 30 of the 33 eligible health departments agreed to participate (91% response rate). Fewer than half (48%) of surveyed health departments had a written response protocol, and health departments reported conducting in-depth investigations on fewer than 15% of syndromic surveillance alerts. A convened panel of experts identified 32 essential elements for inclusion in public health protocols for response to syndromic surveillance system alerts. CONCLUSIONS Because of the lack of guidance, limited resources for development of response protocols, and few examples of syndromic surveillance detecting previously unknown events of public health significance, health departments have not prioritized the development and refinement of response protocols. Systems alone, however, are not effective without an organized public health response. The framework proposed here can guide health departments in creating protocols that will be standardized, tested, and relevant given their goals with such systems.

Comparison of antibiotic susceptibility profiles and molecular typing patterns of clinical and environmental Salmonella enterica serotype Newport.

The genus Salmonella is composed of more than 2,400 serotypes, many of which cause enteric diseases in humans and animals. Several Salmonella serotypes are multidrug resistant, and there is evidence of the clonal spread of these strains from animals to humans. Salmonella enterica serotype Newport is one of the serotypes that increasingly present a multidrug-resistant phenotype. Source tracking and antibiotic resistance testing are important considerations for identifying the outbreak strain. The first goal of this study was to examine the antibiotic susceptibility patterns of clinical and environmental Salmonella Newport isolates from various geographic locations and to compare the discriminatory ability of two DNA fingerprinting techniques. The second goal was to determine whether the antibiotic resistance profiles and typing patterns correlated. Thirty Salmonella Newport isolates, including environmental and human clinical strains, were subjected to pulsed-field gel electrophoresis (PFGE), ribotyping, and antibiotic susceptibility testing. Eighty percent of the isolates showed total or intermediate resistance to one or more drugs; 75% of the isolates were multidrug resistant. Ribotyping with the EcoRI enzyme and PFGE with the XbaI enzyme each divided the isolates into 14 groups. Cluster analysis based on antibiotic susceptibility patterns generated 23 profiles. The susceptible and resistant isolates were not differentiated on the basis of either of the molecular typing techniques. Hence, no correlation was observed between the antibiotic resistance profiles and the DNA subtyping patterns. In conclusion, ribotyping is as discriminatory as PFGE and, when used in combination with antibiotic resistance profiles, provides a powerful tool for the source tracking of Salmonella Newport.

An accelerated method for isolation of Salmonella enterica serotype Typhimurium from artificially contaminated foods, using a short preenrichment, immunomagnetic separation, and xylose-lysine-desoxycholate agar (6IX method).

Rapid isolation of Salmonella from food is essential for faster typing and source tracking in an outbreak. The objective of this study was to investigate a rapid isolation method that would augment the standard U.S. Food and Drug Administrations Bacteriological Analytical Manual (BAM) method. Food samples with low microbial load, including egg salad and ice cream, moderately high-microbial-load tomatoes, and high-microbial-load ground beef were intentionally inoculated with 2 to 48 CFU of Salmonella enterica serotype Typhimurium. The samples were preenriched in buffered peptone water for 6 h, and then selectively concentrated by immunomagnetic separation and plated for isolation on xylose-lysine-desoxycholate agar: the 6IX method. Salmonella Typhimurium was presumptively identified from approximately 97% of the low-microbial-load and moderately high-microbial-load samples by the 6IX method 2 days before the BAM standard method for isolation of Salmonella. In 49% of the beef samples, Salmonella Typhimurium was presumptively identified 1 or 2 days earlier by the 6IX method. Given the inocula used, our data clearly indicated that for most of the food samples tested, with the exception of ground beef, Salmonella Typhimurium could be isolated two laboratory days earlier with the 6IX method compared with the BAM method. In conclusion, this 6IX method may expedite Salmonella isolation and, therefore, has the potential to accelerate strain tracking for epidemiological analysis in a foodborne outbreak.