The presence of viable but nonculturable bacteria in human clean-catch and mouse bladder-isolated urine specimens was investigated. Viable but nonculturable bacteria are alive but do not give rise to visible growth under nonselective growth conditions. Urine specimens obtained from human female volunteers with or without an active urinary tract infection were found to contain, on average, significantly more viable than culturable forms of bacteria. Additional support for the presence of viable but nonculturable cells in urine specimens considered sterile was obtained from examination of urine specimens obtained directly from the bladder of healthy mice. Because the viability assay used to study the viable but nonculturable condition is by necessity growth independent, and hence indirect, the accuracy of this assay that scores cells with intact cell membranes as being viable was studied. Greater than 95% of Escherichia coli cells exposed to lethal doses of UV irradiation were found to lose their membrane integrity within a day, a time frame similar to that used to examine urine specimens. These data suggest that viable but nonculturable cells can occur within regions of the urinary tract previously considered sterile.Urinary tract infections (UTIs) affect as many as 50% of women at least once during their lifetime (29, 32), and 25% of those who acquire a UTI will have another infection within the following 6 months (17). A UTI occurs when the urinary tract is infected with microorganisms, and uropathogenic Escherichia coli accounts for greater than 80% of all UTI cases (4, 30). One method of diagnosing a UTI is by culturing urine specimens; a threshold of 100,000 CFU/ml in clean-catch urine specimens is considered to indicate a UTI (4, 28). This threshold is not an absolute indicator, as both asymptomatic bacteriuria and patients with UTI symptoms having no culturable urine bacteria occur (29,32).Urine within the urinary tract is generally considered sterile (14). This conclusion is based upon a lack of culturable cells present in urine specimens obtained via clean-catch and catheterization methods. The presence of viable bacteria in the urine specimens of healthy patients would impact on hypotheses to explain recurrent UTIs as well as diagnostic procedures. Most recurrent UTIs result from reinfection; however, a higher percentage than would be expected by chance are caused by the index strain (6,18,26,27,41). The physical location and physiological status of index strain cells that remain after successful antibiotic therapy are unknown. Observations with a mouse model indicate that uropathogenic E. coli cells can remain in the urinary tract following antibiotic ther-

Involvement of the viable but nonculturable (VBNC) condition in recurrent urinary tract infections (UTIs) was investigated. VBNC bacteria are those which are alive but do not give rise to visible growth under nonselective growth conditions. Urine, bladder, and kidney samples collected over a 2-month period from BALB/c mice inoculated with the uropathogenic Escherichia coli strain J96 were examined to determine the level of culturable and viable bacteria. Urine from uninoculated mice was found to contain more viable than culturable bacteria. Inoculated mice had a transient increase in the level of culturable forms of the uropathogen in their urine, followed by a decrease to background levels; they also had multiple log higher levels of viable cells than culturable cells. The culturable pathogenic bacteria in mice that were inoculated and received antibiotic treatment dropped to undetectable levels within 1 week. At 2 out of 12 subsequent time points spanning an additional 65 days, culturable forms of the inoculated pathogenic bacteria were recovered. Polymerase chain reaction (PCR) analysis confirmed that DNA from the inoculated bacteria was present in a sample that yielded no culturable bacteria. These data indicate that the inoculated uropathogenic E. coli was not eliminated by antibiotic therapy, and suggest that these bacteria may escape detection by current standard culturability assays because they are VBNC.

The development of realistic risk models that predict the dissemination, dispersion and persistence of potential biothreat agents have utilized nonpathogenic surrogate organisms such as Bacillus atrophaeus subsp. globigii or commercial products such as Bacillus thuringiensis subsp. kurstaki. Comparison of results from outdoor tests under different conditions requires the use of genetically identical strains; however, the requirement for isogenic strains limits the ability to compare other desirable properties, such as the behavior in the environment of the same strain prepared using different methods. Finally, current methods do not allow long-term studies of persistence or reaerosolization in test sites where simulants are heavily used or in areas where B. thuringiensis subsp. kurstaki is applied as a biopesticide. To create a set of genetically heterogeneous yet phenotypically indistinguishable strains so that variables intrinsic to simulations (e.g., sample preparation) can be varied and the strains can be tested under otherwise identical conditions, we have developed a strategy of introducing small genetic signatures ("barcodes") into neutral regions of the genome. The barcodes are stable over 300 generations and do not impact in vitro growth or sporulation. Each barcode contains common and specific tags that allow differentiation of marked strains from wild-type strains and from each other. Each tag is paired with specific real-time PCR assays that facilitate discrimination of barcoded strains from wild-type strains and from each other. These uniquely barcoded strains will be valuable tools for research into the environmental fate of released organisms by providing specific artificial detection signatures.

In 2015, a laboratory of the United States Department of Defense (DoD) inadvertently shipped preparations of gamma-irradiated spores of Bacillus anthracis that contained live spores. In response, a systematic evidence-based method for preparing, concentrating, irradiating, and verifying the inactivation of spore materials was developed. We demonstrate the consistency of spore preparations across multiple biological replicates and show that two different DoD institutions independently obtained comparable dose-inactivation curves for a monodisperse suspension of B. anthracis spores containing 3 ϫ 10 10 CFU. Spore preparations from three different institutions and three strain backgrounds yielded similar decimal reduction (D 10 ) values and irradiation doses required to ensure sterility (D SAL ) to the point at which the probability of detecting a viable spore is 10 Ϫ6 . Furthermore, spores of a genetically tagged strain of B. anthracis strain Sterne were used to show that high densities of dead spores suppress the recovery of viable spores. Together, we present an integrated method for preparing, irradiating, and verifying the inactivation of spores of B. anthracis for use as standard reagents for testing and evaluating detection and diagnostic devices and techniques. IMPORTANCEThe inadvertent shipment by a U.S. Department of Defense (DoD) laboratory of live Bacillus anthracis (anthrax) spores to U.S. and international destinations revealed the need to standardize inactivation methods for materials derived from biological select agents and toxins (BSAT) and for the development of evidence-based methods to prevent the recurrence of such an event. Following a retrospective analysis of the procedures previously employed to generate inactivated B. anthracis spores, a study was commissioned by the DoD to provide data required to support the production of inactivated spores for the biodefense community. The results of this work are presented in this publication, which details the method by which spores can be prepared, irradiated, and tested, such that the chance of finding residual living spores in any given preparation is 1/1,000,000. These irradiated spores are used to test equipment and methods for the detection of agents of biological warfare and bioterrorism.