Application of QSARs and VFARs to the rapid risk assessment process at US EPA.

With continued development of new chemicals and genetically engineered microbes as potential agents for terrorism and industrial development, there is a great need for the continued development and application of quantitative structure activity relationships (QSARs) and virulence factor activity relationships (VFARs). Development and application of QSARs and VFARs will facilitate efficient and streamlined use of dwindling resources and assessment of risks associated with exposures to chemical and biological agents. To facilitate the continued development of QSARs and VFARs at US Environmental Protection Agency, a two day workshop was organized June 20-21, 2006, in Cincinnati, OH, USA. This article summarizes the workshop report by highlighting the importance of continued QSAR research, the current state of VFAR science, and the guidance provided to the National Homeland Security Research Center and National Risk Management Research Laboratory by an expert panel for the continued use and development of computational approaches.

Evaluating virulence of waterborne and clinical Aeromonas isolates using gene expression and mortality in neonatal mice followed by assessing cell culture's ability to predict virulence based on transcriptional response.

AIMS: To assess the virulence of Aeromonas spp. using two models, a neonatal mouse assay and a mouse intestinal cell culture. METHODS AND RESULTS: After artificial infection with a variety of Aeromonas spp., mRNA extracts from the two models were processed and hydridized to murine microarrays to determine host gene response. Definition of virulence was determined based on host mRNA production in murine neonatal intestinal tissue and mortality of infected animals. Infections of mouse intestinal cell cultures were then performed to determine whether this simpler model system's mRNA responses correlated to neonatal results and therefore be predictive of virulence of Aeromonas spp. Virulent aeromonads up-regulated transcripts in both models including multiple host defense gene products (chemokines, regulation of transcription and apoptosis and cell signalling). Avirulent species exhibited little or no host response in neonates. Mortality results correlated well with both bacterial dose and average fold change of up-regulated transcripts in the neonatal mice. CONCLUSIONS: Cell culture results were less discriminating but showed promise as potentially being able to be predictive of virulence. Jun oncogene up-regulation in murine cell culture is potentially predictive of Aeromonas virulence. SIGNIFICANCE AND IMPACT OF THE STUDY: Having the ability to determine virulence of waterborne pathogens quickly would potentially assist public health officials to rapidly assess exposure risks.

Natural atypical Listeria innocua strains with Listeria monocytogenes pathogenicity island 1 genes.

Identification of bona fide Listeria isolates into the six species of the genus normally requires only a few tests. Aberrant isolates do occur, but even then only one or two extra confirmatory tests are generally needed for identification to species level. We have discovered a hemolytic-positive, rhamnose and xylose fermentation-negative Listeria strain with surprising recalcitrance to identification to the species level due to contradictory results in standard confirmatory tests. The issue had to be resolved by using total DNA-DNA hybridization testing and then confirmed by further specific PCR-based tests including a Listeria microarray assay. The results show that this isolate is indeed a novel one. Its discovery provides the first fully documented instance of a hemolytic Listeria innocua strain. This species, by definition, is typically nonhemolytic. The L. innocua isolate contains all the members of the PrfA-regulated virulence gene cluster (Listeria pathogenicity island 1) of L. monocytogenes. It is avirulent in the mouse pathogenicity test. Avirulence is likely at least partly due to the absence of the L. monocytogenes-specific allele of iap, as well as the absence of inlA, inlB, inlC, and daaA. At least two of the virulence cluster genes, hly and plcA, which encode the L. monocytogenes hemolysin (listeriolysin O) and inositol-specific phospholipase C, respectively, are phenotypically expressed in this L. innocua strain. The detection by PCR assays of specific L. innocua genes (lin0198, lin0372, lin0419, lin0558, lin1068, lin1073, lin1074, lin2454, and lin2693) and noncoding intergenic regions (lin0454-lin0455 and nadA-lin2134) in the strain is consistent with its L. innocua DNA-DNA hybridization identity. Additional distinctly different hemolytic L. innocua strains were also studied.

Isolation and identification of nontuberculous mycobacteria from foods as possible exposure sources.

A variety of foods collected from local supermarkets and produce stands were examined as possible sources of nontuberculous mycobacterial exposure. Food samples were combined with sterile ultrapure water and manually shaken. To remove large particles, the suspensions were filtered through a sterile strainer, centrifuged, and the supernatants were discarded. The food pellets were stored at -75 degrees C. The pellets were treated with either oxalic acid or sodium hydroxide-sodium citrate solutions to reduce contamination by nonmycobacterial organisms. Decontaminated pellets were cultured on both Middlebrook 7H10C agar and Middlebrook 7H10C agar with supplemental malachite green. Plates were observed for growth at 2 and 8 weeks. Isolates demonstrating acid-fastness were identified to species using polymerase chain reaction and restriction enzyme analysis. Nontuberculous mycobacteria (NTM) were recovered from 25 of 121 foods. Six different species of NTM were isolated, the most predominant being Mycobacterium avium.

Occurrence of nontuberculous mycobacteria in environmental samples.

Nontuberculous mycobacteria (NTM) are a major cause of opportunistic infection in immunocompromised hosts. Because there is no evidence of person-to-person transmission and NTM have been found in drinking water, the environment is considered a likely source of infection. In this study the widespread occurrence of NTM was examined in drinking water, bottled water, and ice samples. A total of 139 samples were examined for NTM by a membrane filtration culture technique followed by PCR amplification and 16S rRNA sequence determination to identify the isolates. NTM were not detected in bottled water or cisterns but were detected in 54% of the ice samples and 35% of the public drinking-water samples from 21 states. The most frequently occurring isolate was M. mucogenicum (formerly referred to as an M. chelonae-like organism).

PCR comparison of Mycobacterium avium isolates obtained from patients and foods.

Mycobacterium avium is a cause of disseminated disease in AIDS patients. A need for a better understanding of possible sources and routes of transmission of this organism has arisen. This study utilized a PCR typing method designed to amplify DNA segments located between the insertion sequences IS1245 and IS1311 to compare levels of relatedness of M. avium isolates found in patients and foods. Twenty-five of 121 food samples yielded 29 mycobacterial isolates, of which 12 were M. avium. Twelve food and 103 clinical M. avium isolates were tested. A clinical isolate was found to be identical to a food isolate, and close relationships were found between two patient isolates and two food isolates. Relatedness between food isolates and patient isolates suggests the possibility that food is a potential source of M. avium infection. This study demonstrates a rapid, inexpensive method for typing M. avium, possibly replacing pulsed-field gel electrophoresis.

Comparison of large restriction fragments of Mycobacterium avium isolates recovered from AIDS and non-AIDS patients with those of isolates from potable water.

We examined potable water in Los Angeles, California, as a possible source of infection in AIDS and non-AIDS patients. Nontuberculous mycobacteria were recovered from 12 (92%) of 13 reservoirs, 45 (82%) of 55 homes, 31 (100%) of 31 commercial buildings, and 15 (100%) of 15 hospitals. Large-restriction-fragment (LRF) pattern analyses were done with AseI. The LRF patterns of Mycobacterium avium isolates recovered from potable water in three homes, two commercial buildings, one reservoir, and eight hospitals had varying degrees of relatedness to 19 clinical isolates recovered from 17 patients. The high number of M. avium isolates recovered from hospital water and their close relationship with clinical isolates suggests the potential threat of nosocomial spread. This study supports the possibility that potable water is a source for the acquisition of M. avium infections.

Confirmational Identification of Escherichia coli, a Comparison of Genotypic and Phenotypic Assays for Glutamate Decarboxylase and beta-d-Glucuronidase.

[This corrects the article on p. 3350 in vol. 62, PMID: 8795225.].

Effect of aerosolization on culturability and viability of gram-negative bacteria.

Estimations of the bacterial content of air can be more easily made now than a decade ago, with colony formation the method of choice for enumeration of airborne bacteria. However, plate counts are subject to error because bacteria exposed to the air may remain viable yet lose the ability to form colonies, i.e., they become viable but nonculturable. If airborne bacteria exhibit this phenomenon, colony formation data will significantly underestimate the bacterial populations in air samples. The objective of the study reported here was to determine the effect of aerosolization on viability and colony-forming ability of Serratia marcescens, Klebsiella planticola, and Cytophaga allerginae. A collision nebulizer was used to spray bacterial suspensions into an aerosol chamber, after which duplicate samples were collected in all-glass impingers over a 4-h period. Humidity was maintained at ca. 20 to 25%, and temperature was maintained at 20 to 22 degrees C for each of two replicate trials per microorganism. Viability was determined by using a modified direct viable count method, employing nalidixic acid or aztreonam and p-iodonitrotetrazolium violet (INT). Cells were stained with acridine orange and observed by epifluorescence microscopy to enumerate total and viable cells. Viable cells were defined as those elongating in the presence of antibiotic and/or reducing INT. CFU were determined by plating on tryptic soy agar and R2A agar. It was found that culture techniques did not provide an adequate description of the bacterial burdens of indoor air (i.e., less than 10% of the aerosolized bacteria were capable of forming visible colonies). It is concluded that total cell count procedures provide a better approximation of the number of bacterial cells in air and that procedures other than plate counting are needed to enumerate bacteria in aerosol samples, especially if the public health quality of indoor air is to be estimated.

Confirmational identification of Escherichia coli, a comparison of genotypic and phenotypic assays for glutamate decarboxylase and beta-D-glucuronidase.

Genotypic and phenotypic assays for glutamate decarboxylase (GAD) and beta-D-glucuronidase (GUD) were compared for their abilities to detect various strains of Escherichia coli and to discriminate among other bacterial species. Test strains included nonpathogenic E. coli, three major groups of diarrheagenic E. coli, three other non-coli Escherichia species, and various other gram-negative and -positive bacteria found in water. The genotypic assays were performed with hybridization probes generated by PCR amplification of 670- and 623-bp segments of the gadA/B (GAD) and uidA (GUD) genes, respectively. The GAD enzymes catalyze the alpha-decarboxylation of L-glutamic acid to yield gamma-aminobutyric acid and carbon dioxide, which are detected in the phenotypic assay by a pH-sensitive indicator dye. The phenotypic assay for GUD involves the transformation of 4-methylumbelliferyl-beta-D-glucuronide to the fluorogenic compound 4-methylumbelliferone. The GAD phenotypic assay detected the majority of the E. coli strains tested, whereas a number of these strains, including all representatives of the O157:H7 serotype and several nonpathogenic E. coli strains, gave negative results in the GUD assay. Both phenotypic assays detected some but not all strains from each of the four Shigella species. A strain of Citrobacter freundii was also detected by the GUD assay but not by the GAD assay. All E. coli and Shigella strains were detected with both the gadA/B and uidA probes. A few Escherichia fergusonii strains gave weak hybridization signals in response to both probes at 65 degrees C but not at 68 degrees C. None of the other bacterial species tested were detected by either probe. These results were consistent with previous reports which have indicated that the GAD phenotypic assay detects a wider range of E. coli strains than does the GUD assay and is also somewhat more specific for this species. The genotypic assays for the two enzymes were found to be equivalent in both of these respects and superior to both of the phenotypic assays in terms of the range of E. coli strains and isolates detected.

Comparison of methods for detection and enumeration of airborne microorganisms collected by liquid impingement.

Bacterial agents and cell components can be spread as bioaerosols, producing infections and asthmatic problems. This study compares four methods for the detection and enumeration of aerosolized bacteria collected in an AGI-30 impinger. Changes in the total and viable concentrations of Pseudomonas fluorescens in the collection fluid with respect to time of impingement were determined. Two direct microscopic methods (acridine orange and BacLight) and aerodynamic aerosol-size spectrometry (Aerosizer) were employed to measure the total bacterial cell concentrations in the impinger collection fluid and the air, respectively. These data were compared with plate counts on selective (MacConkey agar) and nonselective (Trypticase soy agar) media, and the percentages of culturable cells in the collection fluid and the bacterial injury response to the impingement process were determined'. The bacterial collection rate was found to be relatively unchanged during 60 min of impingement. The aerosol measurements indicated an increased amount of cell fragments upstream of the impinger due to continuous bacterial nebulization. Some of the bacterial clusters, present in the air upstream of the impinger, deagglomerated during impingement, thus increasing the total bacterial count by both direct microscopic methods. The BacLight staining technique was also used to determine the changes in viable bacterial concentration during the impingement process. The percentage of viable bacteria, determined as a ratio of BacLight live to total counts was only 20% after 60 min of sampling. High counts on Trypticase soy agar indicated that most of the injured cells could recover. On the other hand, the counts from the MacConkey agar were very low, indicating that most of the cells were structurally damaged in the impinger. The comparison of data on the percentage of injured bacteria obtained by the traditional plate count with the data on percentage of nonviable bacteria obtained by the BacLight method showed good agreement.

Recovery of Cyclospora organisms from patients with prolonged diarrhea.

A newly recognized protozoan human parasite, Cyclospora has been incriminated as the cause of prolonged diarrhea. Five patients had episodes of diarrhea accompanied by nausea, weight loss, and/or low-grade fever for 10-45 days. Multiple fecal samples fixed in sodium acetate-acetic acid-formalin contained spherical organisms measuring 8-10 microns in diameter; a modified concentration technique was used to detect them. The sediment was examined by direct microscopy and autofluorescence, and the identification was confirmed by acid-fast stain. All patients had visited either Mexico or Thailand. The presence of Cyclospora organisms in these patients shows that these can be etiologic agents of traveler's diarrhea in both immunocompetent and immunocompromised hosts. Fecal specimens from patients with unexplained diarrhea should be routinely examined for their presence.

New medium for the simultaneous detection of total coliforms and Escherichia coli in water.

A new membrane filter agar medium (MI agar) containing a chromogen, indoxyl-beta-D-glucuronide, and a fluorogen, 4-methylumbelliferyl-beta-D-galactopyranoside, was developed to simultaneously detect and enumerate Escherichia coli and total coliforms (TC) in water samples on the basis of their enzyme activities. TC produced beta-galactosidase, which cleaved 4-methylumbelliferyl-beta-D-galactopyranoside to form 4-methylumbelliferone, a compound that fluoresced under longwave UV light (366 nm), while E. coli produced beta-glucuronidase, which cleaved indoxyl-beta-D-glucuronide to form a blue color. The new medium TC and E. coli recoveries were compared with those of mEndo agar and two E. coli media, mTEC agar and nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide, using natural water samples and spiked drinking water samples. On average, the new medium recovered 1.8 times as many TC as mEndo agar, with greatly reduced background counts (< or = 7%). These differences were statistically significant (significance level, 0.05). Although the overall analysis revealed no statistically significant difference between the E. coli recoveries on MI agar and mTEC agar, the new medium recovered more E. coli in 16 of 23 samples (69.6%). Both MI agar and mTEC agar recovered significantly more E. coli than nutrient agar supplemented with 4-methylumbelliferyl-beta-D-glucuronide. Specificities for E. coli, TC, and noncoliforms on MI agar were 95.7% (66 of 69 samples), 93.1% (161 of 173 samples), and 93.8% (61 of 65 samples), respectively. The E. coli false-positive and false-negative rates were both 4.3%. This selective and specific medium, which employs familiar membrane filter technology [corrected] to analyze several types of water samples, is less expensive than the liquid chromogen and fluorogen media and may be useful for compliance monitoring of drinking water.

Genotoxic activity detected in soils from a hazardous waste site by the Ames test and an SOS colorimetric test.

Ten soil samples from a hazardous waste site were compared for their genotoxic activity by the Ames test (Salmonella reverse mutation assay) and a modified SOS colorimetric test. Polynuclear aromatic hydrocarbons known to produce frameshift mutations were found in high levels in the soils. Salmonella typhimurium TA98, sensitive to frameshift mutations, was selected as the Ames tester strain. Escherichia coli K12 PQ37 (sulA::lacZ) was the SOS tester strain. Organic extracts were prepared from the soil samples by Soxhlet extraction. One set of the soil samples was extracted with methylene chloride and a second set with cyclohexane. Two criteria from reproducible dose-related increases in response to the soil were used to compare the positive responses: 1) the concentrations required for doubling responses and 2) a minimum concentration required to produce statistically significant increases from background controls. Analysis of variance indicated that with S9 mix, Ames and SOS results were similar for the same soils and solvent extractions. However, without S9 mix, the SOS test was significantly more sensitive than the Ames test to the genotoxins extracted from the soils. Both the Ames and SOS tests detected lower concentrations of genotoxins in methylene chloride than in cyclohexane extracts. The simplicity of the method, reduction in expenses, and results within 1 working day all contribute to the advantages of the SOS test.

Virulence characteristics of clinical and environmental isolates of Vibrio vulnificus.

Twenty-four randomly selected clinical and environmental Vibrio vulnificus isolates were tested for virulence in iron-overloaded mice (250 mg of iron dextran per kg of body weight). The log10 50% lethal doses of 17 isolates were lower by greater than or equal to 3.5 log10 units in iron-overloaded mice than in control mice. These isolates were classified as virulent. The 50% lethal doses of these virulent isolates were also lower in mice that were immunosuppressed by treatment with cyclophosphamide (150 mg/kg). Four of the seven isolates initially classified as avirulent were virulent in mice that were simultaneously iron overloaded and immunosuppressed. These isolates were classified as moderately virulent. The remaining three isolates were avirulent under all conditions. The incidence of virulent strains among clinical and environmental isolates did not differ. The virulent isolates produced high titers of hemolysin, were resistant to inactivation by serum complement, produced phenolate siderophore, and utilized transferrin-bound iron. The moderately virulent isolates differed from the virulent isolates only in their increased sensitivity to inactivation by serum complement. The avirulent isolates differed from those of the other two classes in their inability to either produce significant amounts of phenolate siderophore or utilize transferrin-bound iron. A modified agar plate diffusion method for transferrin-bound iron utilization was developed to differentiate the two classes of virulent isolates from the avirulent isolates in vitro.

Comparison of the Salmonella (Ames) test, umu tests, and the SOS Chromotests for detecting genotoxins.

The limits of detection of 10 genotoxins representing 7 chemical classes with varying structures and modes of action were compared using the Ames test (Salmonella plate-incorporation test) with 2 tester strains, 2 standard colorimetric methods (the umu test and SOS Chromotest), and modifications of the umu and SOS Chromotests developed during the course of this study. The purpose of the study was to determine the sensitivity and reproducibility of each of the six methods. The sensitivities of the methods were compared using two criteria: the concentrations required for doubling responses, and the minimum concentrations required to produce statistically significant increases from background controls. The Ames test with strains TA98 and TA100 was ranked as the most sensitive method more often than the others, but the results indicated that the umu tests were statistically equivalent to the Ames test. The original SOS Chromotest kit method was highly sensitive in detecting the direct acting genotoxins, but neither SOS test was as sensitive as the other methods in detecting indirect acting genotoxins. The umu microtiter plate test is the least expensive of the assays and would be the most suitable for screening large numbers of environmental samples.

A shigellosis outbreak traced to commercially distributed shredded lettuce.

In the period August 30-October 7, 1986, 347 persons in adjacent west Texas counties (Ector and Midland) contracted culture-confirmed Shigella sonnei gastroenteritis. A case-control study showed an increased risk of acquiring shigellosis in Ector County with eating at outlets of fast-food Restaurant A, and in Midland County with eating at Restaurant B or C. A second case-control study, of persons who had eaten at Ector County outlets of Restaurant A, showed an increased risk of acquiring shigellosis with eating foods containing shredded lettuce and tomatoes, which were served together (odds ratio = 68.8; 95% confidence interval 8.5-293.1). All implicated restaurants received shredded lettuce produced at one lettuce-shredding plant; two implicated restaurants did not receive tomatoes from the lot delivered to other implicated restaurants. The lettuce-shredding plant distributed shredded lettuce and intact lettuce; restaurants that received only intact lettuce were not involved in the outbreak. Investigation at the lettuce-shredding plant suggested that a food handler might have been the source of contamination and that the method of processing might have allowed cross-contamination to occur. In the laboratory, the outbreak strain of S. sonnei multiplied rapidly on shredded lettuce at 22 C and survived on refrigerated shredded lettuce for at least seven days. This outbreak, one of the largest outbreaks of Shigella infections in the United States in the last decade, indicates that a large, geographically widespread shigellosis outbreak can result from contaminated shredded lettuce that is distributed commercially.

Experimental evidence for enteropathogenicity in Aeromonas veronii.

Eleven ornithine-positive strains of Aeromonas (9 A. veronii and 2 provisionally classified as Aeromonas species ornithine positive) were tested for ability to cause fluid accumulation in the rabbit ileal loop. All eight beta-hemolytic strains caused fluid accumulation. Gel diffusion analysis revealed that the A. veronii beta-hemolysin was serologically related to the A. hydrophila beta-hemolysin, a known enterotoxic molecule. The biological activity of the A. veronii hemolysin was neutralized by antiserum to A. hydrophila hemolysin. One of three strains that were not beta-hemolytic caused fluid accumulation but only when the ileal loops were inoculated with live cultures. These results suggest that A. veronii is a potential enteropathogen that can cause diarrhea by means of a cell-freed enterotoxin (beta-hemolysin) or by a second mechanism that requires the presence of whole cells.

Pathogenicity test for Listeria monocytogenes using immunocompromised mice.

The lethality of Listeria isolates was determined with normal adult mice and mice that were immunocompromised by treatment with 20 mg of carrageenan per kg. The mean 50% lethal doses (LD50s) of the pathogenic isolates were significantly lower (alpha = 0.05) in the immunocompromised mice than in the untreated mice, with an average reduction of 5.8 log10 units. In contrast, the mean LD50s of the nonpathogenic isolates were lower in the immunocompromised mice by an average of only 0.4 log10 unit, a difference that was not significant (alpha = 0.05). When immunocompromised mice were used, the LD50s of pathogenic Listeria monocytogenes isolates were lower than those of nonpathogenic L. innocua and L. seeligeri isolates by greater than or equal to 6 log10 units and lower than those of nonpathogenic L. ivanovii isolates by greater than or equal to 4 log10 units. Pathogenic L. monocytogenes isolates could be distinguished from nonpathogenic isolates by their ability to cause deaths in immunocompromised mice in 3 days at a dose of approximately 10(4) CFU per mouse. An alternative procedure using iron-overloaded mice failed to effectively differentiate pathogenic Listeria isolates.

Melanogenesis in murine B16 cells exposed to Aeromonas hydrophila cytotoxic enterotoxin.

Specific markers (growth, melanogenesis) of B16 murine melanoma cells in culture were used as indicators of toxin production by Aeromonas hydrophila. Cytotonic enterotoxinlike activity (inhibited growth, raised tyrosinase activity, and melanin accumulation) occurred at cytotoxic end points of purified beta-hemolysin and several culture filtrates. Antihemolysin rabbit serum inhibited this activity. A hemolysin-neutralized culture filtrate concentrate (10X) failed to elevate tyrosinase relative to untreated and cholera toxin treated controls. Similar dilution profiles using Chinese hamster ovary cells showed limited cell extension only at cytotoxic end points with antihemolysin inhibiting this activity. Cytotoxicity of Chinese hamster ovary cells and B16 cells was proportional to hemolytic activity, with B16 cells showing about 100-fold greater sensitivity on a per cell basis. Cell culture cytotoxicity attributed to beta-hemolysin correlated with reactivity in rabbit ileal loop assays. The ADP-ribosyl transferase activity of concentrated (10X) A. hydrophila culture filtrates and fractions thereof was negative. Apparently sublethal doses of A. hydrophila beta-hemolysin can nonspecifically stimulate cyclic adenosine monophosphate mediated events in melanoma and Chinese hamster ovary cell assays, producing lower activities than cholera toxin with shorter lag times.