A novel broth medium for enhanced growth of Francisella tularensis.

Francisella tularensis is a fastidious organism that requires a lengthy incubation time in liquid growth media for detection. The objective of this study was to develop a medium formulation using readily available supplements that enhanced early growth of F. tularensis. Francisella tularensis live vaccine strain was used to evaluate the growth responses for each of the media formulations tested. Growth in brain heart infusion broth supplemented with 2% Vitox, 10% Fildes and 1% histidine (BVFH) resulted in a significant increase in growth after 8 h incubation compared to other media formulations tested (P < 0·001). Virulent strains of F. tularensis grown in BVFH medium demonstrated similar enhanced early growth. Cell densities of 3·9-5·2 log10 CFU per ml were obtained after 24 h of growth in BVFH from a 1-2 cell ml-1 starting inoculum of the virulent Type A Schu4 strain, indicating the suitability of this medium in rapidly amplifying low starting titres of F. tularensis. Collectively, these results indicate that the novel formulation of the BVFH medium was capable of producing enhanced growth response for F. tularensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The need for rapid cultivation of Francisella tularensis is essential for detection and monitoring during natural outbreak events or intentional bioterrorism attacks. The addition of selected supplements into the base medium BHI (BVFH) developed in this study enhanced growth of F. tularensis Type A1, A2 and B strains compared to BHI alone. Growth of these organisms in BVFH will allow for improved response time should a natural or intentional contamination event occur.

Inactivation of bacterial biothreat agents in water, a review.

Water supplies and water distribution systems have been identified as potential targets for contamination by bacterial biothreat agents. Since the 2001 Bacillus anthracis bioterrorist attacks, additional efforts have been aimed at research to characterize biothreat organisms in regards to their susceptibility to disinfectants and technologies currently in use for potable water. Here, we present a review of research relevant to disinfection of bacteria with the potential to pose a severe threat to public health and safety, and their potential surrogates. The efficacy of chlorine, monochloramine, chlorine dioxide, and ultraviolet light to inactivate each organism in suspension is described. The complexities of disinfection under varying water conditions and when the organisms are associated with biofilms in distribution systems are discussed.

Use of acid treatment and a selective medium to enhance the recovery of Francisella tularensis from water.

Francisella tularensis has been associated with naturally occurring waterborne outbreaks and is also of interest as a potential biological weapon. Recovery of this pathogen from water using cultural methods is challenging due to the organism's fastidious growth requirements and interference by indigenous bacteria. A 15-min acid treatment procedure prior to culture on a selective agar was evaluated for recovery of F. tularensis from seeded water samples. Mean levels of reduction of virulent strains of F. tularensis subsp. holarctica and subsp. tularensis were less than 20% following acid treatment. The attenuated live vaccine strain (LVS) was less resistant to acid exposure. The acid treatment procedure coupled with plating on cystine heart agar with rabbit blood and antibiotics (CHARBab) allowed the isolation of F. tularensis seeded into five natural water samples.

Chlorine disinfection of Francisella tularensis.

AIMS: To determine the range of free available chlorine (FAC) required for disinfection of the live vaccine strain (LVS) and wild-type strains of Francisella tularensis. METHODS AND RESULTS: Seven strains of planktonic F. tularensis were exposed to 0·5 mg·l(-1) FAC for two pH values, 7 and 8, at 5 and 25°C. LVS was inactivated 2 to 4 times more quickly than any of the wild-type F. tularensis strains at pH 8 and 5°C. CONCLUSIONS: Free available chlorine residual concentrations routinely maintained in drinking water distribution systems would require up to two hours to reduce all F. tularensis strains by 4 log10. LVS was inactivated most quickly of the tested strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This work provides contact time (CT) values that are useful for drinking water risk assessment and also suggests that LVS may not be a good surrogate in disinfection studies.

Inactivation of spores of Bacillus anthracis Sterne, Bacillus cereus, and Bacillus thuringiensis subsp. israelensis by chlorination.

Three species of Bacillus were evaluated as potential surrogates for Bacillus anthracis for determining the sporicidal activity of chlorination as commonly used in drinking water treatment. Spores of Bacillus thuringiensis subsp. israelensis were found to be an appropriate surrogate for spores of B. anthracis for use in chlorine inactivation studies.

Low pressure ultraviolet studies for inactivation of Giardia muris cysts.

AIMS: The research was initiated to confirm earlier ultraviolet (u.v.) light inactivation studies performed on Giardia cysts using excystation as the viability indicator. Following this, a comparison of in vitro excystation and animal infectivity was performed for assessing cyst viability after exposure to low-pressure u.v. irradiation. METHODS AND RESULTS: Cysts of Giardia muris were inactivated using a low-pressure u.v. light source. Giardia muris was employed as a surrogate for the human pathogen Giardia lamblia. Cyst viability was determined by both in vitro excystation and animal infectivity. Cyst doses were counted using a flow cytometer for the animal infectivity experiments. Using in vitro excystation as the viability indicator, fluences as high as approximately 200 mJ cm(-2) did not prevent some cysts from excysting, thus verifying earlier work. Using animal infectivity, u.v. fluences of 1.4, 1.9 and 2.3 mJ cm(-2) yielded log10 reductions ranging from 0.3 to >or= 4.4. CONCLUSIONS: Results indicate that in vitro excystation is not a reliable indicator of G. muris cyst viability after u.v. disinfection. Very low doses of u.v. light rendered G. muris cysts non-infective in the mouse model employed. SIGNIFICANCE AND IMPACT OF THE STUDY: Data presented represent the only complete u.v. inactivation curve for G. muris. This research provides evidence that u.v. can be an effective barrier against Giardia spp. in the treatment of drinking water supplies.

Growth and recovery of selected gram-negative bacteria in reconditioned wastewater.

Previous reports indicate that Escherichia coli O157:H7, Salmonella spp., and Vibrio cholerae can grow in nutrient-limited, reconditioned wastewater over the temperature range of 4 to 46 degrees C when the biological oxygen demand of this water is <2, while its coliform growth response (CGR) is >2. In the current study, we investigated the growth response of Vibrio parahaemolyticus, Shigella spp., Vibrio vulnificus, and Pseudomonas aeruginosa in water samples with a CGR of >2 over the temperature range of 4 to 50 degrees C. Both the nonselective media, tryptic soy agar, and the selective media used to identify the pathogen were used for their recovery. The selective media were thiosulfate-citrate-bile-sucrose (TCBS), MacConkey agar (MAC), and Pseudomonas isolation agar (PIA) for the Vibrio, Shigella, and Pseudomonas spp., respectively. V. parahaemolyticus numbers declined rapidly after surviving for 6 days under the nutrient-limiting growth conditions. Shigella spp. did not grow but survived for >28 days at 4 to 25 degrees C. V. vulnificus grew over the narrow temperature range of 12 to 21 degrees C and survived for >21 days at the higher and lower temperature ranges. P. aeruginosa survived and grew during the 14-day test period at 13 to 35 degrees C. Recovery on the nonselective agar gave statistically (P > 0.05) higher numbers than the respective selective media commonly used for these pathogens. These results indicate that caution should be used in attempting direct recoveries using selective media of the four gram-negative bacteria species used in this study from the nutrient-limited water environment.

Short communication: survival of Escherichia coli O157:H7 in dairy cattle drinking water.

Cattle drinking water from two dairy farms was used in a study to determine the survival characteristics of the bacterial pathogen Escherichia coli O157:H7 and wild-type E. coli. The E. coli O157:H7 inoculum consisted of a consortium of isolates obtained from dairy cattle. Fresh manure was used as the source for the wild-type E. coli. In the water source from farm 1 the pathogens were present at both 5 and 15 degrees C during the 16-d duration of the study. In the water source from farm 2, the pathogens were detected at 5 degrees C through d 8 and through d 4 at 15 degrees C. The fecal indicator, wild-type E. coli, was always present when the pathogens were present.

Escherichia coli: the best biological drinking water indicator for public health protection.

Public health protection requires an indicator of fecal pollution. It is not necessary to analyse drinking water for all pathogens. Escherichia coli is found in all mammal faeces at concentrations of 10 log 9(-1), but it does not multiply appreciably in the environment. In the 1890s, it was chosen as the biological indicator of water treatment safety. Because of method deficiencies, E. coli surrogates such as the 'fecal coliform' and total coliforms tests were developed and became part of drinking water regulations. With the advent of the Defined Substrate Technology in the late 1980s, it became possible to analyse drinking water directly for E. coli (and, simultaneously, total coliforms) inexpensively and simply. Accordingly, E. coli was re-inserted in the drinking water regulations. E. coli survives in drinking water for between 4 and 12 weeks, depending on environmental conditions (temperature, microflora, etc.). Bacteria and viruses are approximately equally oxidant-sensitive, but parasites are less so. Under the conditions in distribution systems, E. coli will be much more long-lived. Therefore, under most circumstances it is possible to design a monitoring program that permits public health protection at a modest cost. Drinking water regulations currently require infrequent monitoring which may not adequately detect intermittent contamination events; however, it is cost-effective to markedly increase testing with E. coli to better protect the public's health. Comparison with other practical candidate fecal indicators shows that E. coli is far superior overall.

A spore counting method and cell culture model for chlorine disinfection studies of Encephalitozoon syn. Septata intestinalis.

The microsporidia have recently been recognized as a group of pathogens that have potential for waterborne transmission; however, little is known about the effects of routine disinfection on microsporidian spore viability. In this study, in vitro growth of Encephalitozoon syn. Septata intestinalis, a microsporidium found in the human gut, was used as a model to assess the effect of chlorine on the infectivity and viability of microsporidian spores. Spore inoculum concentrations were determined by using spectrophotometric measurements (percent transmittance at 625 nm) and by traditional hemacytometer counting. To determine quantitative dose-response data for spore infectivity, we optimized a rabbit kidney cell culture system in 24-well plates, which facilitated calculation of a 50% tissue culture infective dose (TCID(50)) and a minimal infective dose (MID) for E. intestinalis. The TCID(50) is a quantitative measure of infectivity and growth and is the number of organisms that must be present to infect 50% of the cell culture wells tested. The MID is as a measure of a system's permissiveness to infection and a measure of spore infectivity. A standardized MID and a standardized TCID(50) have not been reported previously for any microsporidian species. Both types of doses are reported in this paper, and the values were used to evaluate the effects of chlorine disinfection on the in vitro growth of microsporidia. Spores were treated with chlorine at concentrations of 0, 1, 2, 5, and 10 mg/liter. The exposure times ranged from 0 to 80 min at 25 degrees C and pH 7. MID data for E. intestinalis were compared before and after chlorine disinfection. A 3-log reduction (99.9% inhibition) in the E. intestinalis MID was observed at a chlorine concentration of 2 mg/liter after a minimum exposure time of 16 min. The log(10) reduction results based on percent transmittance-derived spore counts were equivalent to the results based on hemacytometer-derived spore counts. Our data suggest that chlorine treatment may be an effective water treatment for E. intestinalis and that spectrophotometric methods may be substituted for labor-intensive hemacytometer methods when spores are counted in laboratory-based chlorine disinfection studies.

Recovery and survival of Escherichia coli O157:H7 in reconditioned pork-processing wastewater.

The pathogen Escherichia coli O157:H7 has been recovered from various water sources and food samples. The growth potential of this bacterium in nutrient-limited, reconditioned wastewater from a pork-processing plant was determined over a temperature range of 4 to 46 degrees C. Even though the biological oxygen demand of the wastewater was <2 mg/liter, results of bioassays for assimilable organic carbon and the coliform growth response of the water suggested that sufficient nutrients were present to support limited bacterial growth. A three-strain mixture of E. coli O157:H7 grew over the temperature range of 10.2 to 29.4 degrees C. Bioassays appear to be a good indicator of the ability of this wastewater to support growth of this pathogen. Statistically higher levels of bacterial growth (P < 0.05) were detected on a nonselective medium (tryptic soy agar) than on a selective medium (sorbitol-MacConkey agar), suggesting that stress or injury of the bacterium occurs when the organism is exposed to the nutrient-limited conditions of the wastewater. These results indicate that E. coli O157:H7 can survive and grow in this particular nutrient-limited wastewater, suggesting a potential hazard if this water becomes contaminated with this pathogen.

Electrophoretic mobilities of Escherichia coli O157:H7 and wild-type Escherichia coli strains.

The electrophoretic mobilities (EPMs) of a number of Escherichia coli O157:H7 and wild-type E. coli strains were measured. The effects of pH and ionic strength on the EPMs were investigated. The EPMs of E. coli O157:H7 strains differed from those of wild-type strains. As the suspension pH decreased, the EPMs of both types of strains increased.

Chlorine inactivation of Escherichia coli O157:H7.

We analyzed isolates of Escherichia coli O157:H7 (which has recently caused waterborne outbreaks) and wild-type E. coli to determine their sensitivity to chlorination. Both pathogenic and nonpathogenic strains were significantly reduced within 1 minute of exposure to free chlorine. Results indicate that chlorine levels typically maintained in water systems are sufficient to inactivate these organisms.

Isolation of Arcobacter butzleri from ground water.

Arcobacter butzleri was isolated from a contaminated ground water source. These organisms, previously designated as aerotolerant Campylobacter, were capable of surviving in the ground water environment. Specific DNA probes were used to characterize the isolates in the initial identification and survival studies. Arcobacter butzleri was found to be sensitive to chlorine inactivation.

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.].

Inactivation of Helicobacter pylori by chlorination.

Three strains of Helicobacter pylori were studied to determine their resistance to chlorination. The organisms were readily inactivated by free chlorine and should therefore be controlled by disinfection practices normally employed in the treatment of drinking water.

Vibrio cholerae O1 can assume a chlorine-resistant rugose survival form that is virulent for humans.

Vibrio cholerae can shift to a "rugose" colonial morphology associated with expression of an amorphous exopolysaccharide that promotes cell aggregation. Flow cytometric studies indicated that up to 3% of particles in rugose cultures represented aggregates of >5 bacterial cells. Rugose variants of our test strains displayed resistance to killing by chlorine, with viable cells persisting for >30 min in 2 mg/L free chlorine; strains also showed resistance to killing by complement-mediated serum bactericidal activity. Six volunteers fed 10(6) cfu of a rugose variant of V. cholerae O1 El Tor Inaba N16961 developed symptoms typical of cholera, with a mean diarrheal stool volume of 2.2 L (range, 1.4-4.3). Isolates recovered from the stool of infected volunteers retained the rugose phenotype. The data suggest that rugose strains cause human disease. The role of these strains in the epidemiology of cholera remains to be determined.

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.