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 Administration's 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.

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 state's 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.

Two-year study evaluating the potential loss of methicillin resistance in a methicillin-resistant Staphylococcus aureus culture collection.

A reported loss of mecA prompted us to monitor 360 cryostocked methicillin-resistant Staphylococcus aureus strains for stability. Concurrently, 14 well-characterized strains were stored in a Microbank preservation system and subjected to multiple freeze-thaw events. There were no significant declines in the methicillin-resistant populations with either method over a two-year period.

Molecular characterization of Vibrio parahaemolyticus strains associated with foodborne illness in Florida.

Vibrio parahaemolyticus is the leading cause of bacterial seafood-based illness in the United States. Real-time PCR, pandemic group-specific PCR, ribotyping, and multilocus sequence typing were used to characterize 30 strains of V. parahaemolyticus including 11 strains associated with foodborne outbreaks in Florida and 6 known pandemic strains. Thirteen strains were positive for four pandemic group-specific PCR markers, including 5 strains associated with outbreaks in Florida. Molecular typing methods were used to further define the pandemic status of these strains because the current PCR markers are not sufficient to identify pandemic isolates. Nine of the Florida strains clustered with a majority of the known pandemic strains, based on ribotyping patterns using PvuII, but no isolated pandemic branch was formed. Using multilocus sequence typing, it was determined that 14 strains possess a previously determined pandemic sequence type. This study identified 13 novel sequence types and seven to nine novel alleles for each locus. Furthermore, the results indicate that seven of the strains from recent foodborne outbreaks in Florida are pandemic strains, and that multilocus sequence typing was the most accurate molecular method to identify these strains.

Virtual digest identification of secondary enzymes for use in pulsed-field gel electrophoresis of Staphylococcus aureus.

Pulsed-field gel electrophoresis (PFGE) is currently the gold standard for methicillin-resistant Staphylococcus aureus (MRSA) typing but only one enzyme, SmaI, is currently used for restriction digest. We report the use of virtual digestion to identify enzymes for S. aureus PFGE. Two enzymes (EagI and SacII) were identified and successfully used to characterize two sets of S. aureus isolates, 12 USA300, and 14 additional MRSA isolates comprised of seven SmaI patterns. Phylogenetic analysis of patterns generated by all enzymes determined that the USA300 MRSAs are identical. In contrast, digestion with EagI or SacII resolved one to two band differences among three MRSA pattern sets that were not detected using SmaI. These results demonstrate that a second enzyme may detect differences in S. aureus isolates not detected by single enzyme digestion. However, because isolates differing by one to two bands are considered identical, such discrimination may not be clinically or epidemiologically relevant.

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.

National survey of Laboratory Response Network sentinel laboratory preparedness.

OBJECTIVE: The Laboratory Response Network (LRN) is the United States' laboratory system for detecting, confirming, and reporting potential bioterrorism agents. The first tier-sentinel laboratories-is composed principally of hospital-based laboratories and is tasked with ruling out potential biological threat agents in clinical specimens or the identification of suspicious specimens for further testing in higher tiers of the LRN system. The aim of the present study was to broadly describe preparedness of the first tier of the hospital LRN, the sentinel laboratories, with a specific focus on training, personnel, and communications. METHODS: A semistructured cross-sectional survey of US sentinel laboratories was designed and conducted. Hospitals with greater than 250 beds and an emergency department were considered eligible for inclusion. A geographically weighted sample of 201 hospitals was selected for inclusion. The survey was administered by telephone to the microbiology managers (or designees) at the selected hospitals. The survey contained questions related to drill frequency, proficiency survey participation, personnel training, personnel responsibilities, procedures for biological threat response, and overall confidence in preparedness. RESULTS: Overall, 179 hospitals (89.1%) identified themselves as sentinel laboratories and participated in the survey; 11.7% reported that they had had an emergency alert within the last 2 years. Although rates of internal drills were low (20.7%), participation in some form of bioterrorism proficiency evaluation was high (79.9%). In all, 83.8% of laboratories reported that they had personnel designated to coordinate response to acts of bioterrorism. More than 73% of respondents indicated that they had sufficient personnel, equipment, and training to respond to a biological terrorism event. By multivariate analysis, sentinel laboratories were 3.4 times more likely to feel confident that they had sufficient personnel, equipment, and training to respond to a biological terrorism event if they had designated personnel for bioterrorism roles. CONCLUSIONS: This pilot study of sentinel laboratory bioterrorism preparedness demonstrated that hospital laboratory personnel, training, and communication preparedness were not universal, despite designation as sentinel laboratories. A need for unified monitoring of sentinel laboratories exists, and efforts should be made to develop standardized metrics for sentinel laboratory preparedness.

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.

Responding to a bioterrorism attack--one scenario: part 1.

This article continues the discussions introduced in an earlier article submitted to The Health Care Manager entitled "Epidemic Simulation for Syndromic Surveillance," wherein a format for analysis of the incidence of a bioterrorist attack was presented. This article outlines a simulation conducted as part of a federal grant award administered through the Center for Biological Defense at the University of South Florida. The disease entity simulated was an attack of anthrax introduced into the Central Florida region. The spread, effects, and eventual control of the disease entity are highlighted.

Responding to a bioterrorism attack-one scenario: part 2.

This article continues the discussions introduced in the earlier article submitted to The Health Care Manager that is titled Epidemic Simulation for Syndromic Surveillance, where a format for analysis of the incidence of a bioterrorist attack was presented. Part 2 of this series provides a discussion of the observed outcomes from the simulation techniques. This simulation was conducted as part of a federal grant award administered through the Center for Biological Defense at the University of South Florida. The disease entity simulated was an attack of anthrax introduced into the Central Florida region. The spread, effects, and eventual control of the disease entity are highlighted.

Epidemic simulation for syndromic surveillance.

This article reports on a project to develop a simulation-based test bed for the BioDefend Syndromic Surveillance System. BioDefend is a system that data mines syndrome reports from emergency rooms and so forth to produce early alerts of epidemic onset. An existing large-scale epidemic simulation will be adapted to provide synthetic reports of syndromes associated with extremely rare events such as pandemics and bioterrorism. The Spatiotemporal Epidemiological Modeler will be used as the basis of the test bed. Results from the much simpler Spatiotemporal Epidemiological Modeler simulation will be validated by comparison against results from the more complex Epidemiological Simulation System. These synthesized reports will be used to test BioDefend's ability to detect epidemic outbreaks and to evaluate its data-mining algorithm. The development of an optimal algorithm for processing syndrome reports to provide reliable epidemic early warnings is a difficult research problem that the test bed should help address.

Susceptibility of Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides and Bacillus thuringiensis to 24 antimicrobials using Sensititre automated microbroth dilution and Etest agar gradient diffusion methods.

OBJECTIVES: To examine susceptibilities of Bacillus anthracis and related species to 24 antimicrobials using and concurrently comparing two methods. METHODS: Twenty-four antimicrobials were tested against 95 isolates of the Bacillus cereus group including 18 B. anthracis, 42 B. cereus, 5 Bacillus mycoides, 5 Bacillus mycoides/pseudomycoides, 6 Bacillus pseudomycoides and 19 Bacillus thuringiensis to determine their MICs, MIC ranges, MIC50s and MIC90s with Etest and Sensititre at 30 and 35 degrees C for 18, 24 and 48 h. RESULTS: Both methods yielded near-identical results at both temperatures for all antimicrobials except trimethoprim/sulfamethoxazole. Resistance to trimethoprim/sulfamethoxazole in 97% (92/95) was not always evident until tests were incubated for 48 h at 30 degrees C. All B. anthracis isolates were susceptible to 22 antimicrobials and resistant to trimethoprim/sulfamethoxazole while three isolates were erythromycin-intermediate. Whereas the B. thuringiensis were resistant to the beta-lactams, two B. cereus, one B. mycoides, five B. pseudomycoides and two B. mycoides/pseudomycoides were susceptible. Three B. cereus were solely clindamycin-resistant. Of the seven erythromycin-intermediate or -resistant B. cereus, three were resistant to clindamycin and one was resistant to clarithromycin and clindamycin. One B. mycoides was intermediately resistant to quinupristin/dalfopristin and meropenem and one was clindamycin-resistant. All B. pseudomycoides were clindamycin-resistant with one quinupristin/dalfopristin-resistant. Two B. mycoides/pseudomycoides were intermediately resistant to quinupristin/dalfopristin and clindamycin and a third was intermediately resistant to clindamycin alone. All isolates were susceptible to chloramphenicol, ciprofloxacin, gatifloxacin, gentamicin, levofloxacin, linezolid, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline and vancomycin. CONCLUSIONS: This paper expands the list of therapeutic or prophylactic antimicrobials potentially effective against B. cereus group isolates using two testing methods that produced comparable results.

Bacillus acidiceler sp. nov., isolated from a forensic specimen, containing Bacillus anthracis pX02 genes.

Research at the Center for Biological Defense identified plasmid-borne forms of Bacillus anthracis pXO2 genes in a Gram-positive, endospore-forming rod, isolated from a forensic specimen considered a credible threat of harbouring anthrax. Conventional, commercial and molecular-based methods indicated that the isolate (CBD 119(T)) was not B. anthracis and considered not to be a member of the Bacillus cereus group. Based on the 16S rRNA gene sequence similarities, strain CBD 119(T) was most closely related to Bacillus luciferensis LMG 18422(T) (99.3 %). Phenotyping and fatty acid methyl ester analysis of the isolate were conducted alongside B. luciferensis JCM 12212(T). The major cellular fatty acids (anteiso-C(15 : 0), iso-C(15 : 0), and >7 iso or anteiso forms) supported inclusion of the isolate in the genus Bacillus. Strain CBD 119(T) was inconsistent with B. luciferensis JCM 12212(T) for 18 of 96 traits evaluated including motility, degree of endospore-driven swelling and pH optimum; the two were linked by fatty acid methyl ester analysis as separate but closely related species. DNA-DNA relatedness between strain CBD 119(T) and B. luciferensis JCM 12212(T) resulted in less than 20 % hybridization. The results of biochemical and physiological characterization, chemotaxonomic analysis and DNA-DNA hybridization differentiated strain CBD 119(T) both phenotypically and genotypically from the only species with validly published name with greater than 97 % 16S rRNA gene sequence similarity. The isolate has an accelerated doubling time when grown in aerated broth at pH 5.9 relative to that at pH 7.1. Therefore, it is proposed that strain CBD 119(T) represents a novel species, Bacillus acidiceler sp. nov. The type strain is strain CBD 119(T) (=NRRL B-41736(T)=DSM 18954(T)).

Community-associated methicillin-resistant Staphylococcus aureus epidemic clone USA300 in isolates from Florida and Washington.

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.

Bacillus anthracis virulent plasmid pX02 genes found in large plasmids of two other Bacillus species.

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.

Use of two selective media and a broth motility test can aid in identification or exclusion of Bacillus anthracis.

During the anthrax attack of 2001, the Florida Department of Health (FDOH) Bureau of Laboratories in Tampa received hundreds of isolates suspected of being Bacillus anthracis. None were confirmed to be B. anthracis since most isolates were motile and not even in the Bacillus cereus group. Although the sentinel laboratories now send fewer isolates to FDOH laboratories, should another attack occur the number of isolates submitted would likely increase dramatically, and this upsurge would seriously challenge personnel who are expected to be busy examining an increased number of environmental samples. We examined two selective and differential growth media and alternative motility methods that could be used to streamline the processing of suspicious isolates. Of 60 isolates previously sent to the FDOH laboratory, 56 were endospore-forming gram-positive rods and only 7 grew on mannitol-egg yolk-polymyxin B agar and/or the Anthracis chromogenic agar. Microscopic observation of early-log-phase growth (2 to 3 h) in a shaking broth was the best method to detect motility in 40 isolates that appeared nonmotile in the motility media investigated. One of these growth media and microscopic examination of shaken broth cultures can be used to show that an isolate is not B. anthracis before expensive molecular and antibody-based tests are performed. By doing so, costs could be reduced and analysis time shortened.

Novel sample preparation method for safe and rapid detection of Bacillus anthracis spores in environmental powders and nasal swabs.

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.

Comparison of methods for DNA isolation from food samples for detection of Shiga toxin-producing Escherichia coli by real-time PCR.

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.