Survival of ice nucleation-active and genetically engineered inactive strains of Pseudomonas syringae

The survival of ice nucleation-active (INA) and genetically engineered non-INA strains of Pseudomonas syringae was compared under starvation and freezing conditions. In starvation experiments, both strains displayed similar survival curves and recovery from starvation was nearly identical for the two strains. In freezing experiments, oat seedlings colonized by each strain were subjected to freezing temperatures. Populations of the INA strain increased 15-fold in the 18 hours after oat plants incurred frost damage at {dollar}-{dollar}5 and {dollar}-{dollar}12{dollar}\sp\circ{dollar}C. Plants colonized by the non-INA strain were undamaged at {dollar}-{dollar}5{dollar}\sp\circ{dollar}C and exhibited no changes in population size. As temperatures were lowered, plants colonized by the non-INA strain suffered increased frost damage concomitant with bacterial population increases. At {dollar}-{dollar}12{dollar}\sp\circ{dollar}C, both strains behaved identically. The data show a relationship between frost damage to plants and subsequent increased bacterial population size, indicating a potential competitive advantage for INA strains of P. syringae in mild freezing environments

Evaluation of the Biological Sampling Kit (BiSKit) for Large-Area Surface Sampling

Current surface sampling methods for microbial contaminants are designed to sample small areas and utilize culture analysis. The total number of microbes recovered is low because a small area is sampled, making detection of a potential pathogen more difficult. Furthermore, sampling of small areas requires a greater number of samples to be collected, which delays the reporting of results, taxes laboratory resources and staffing, and increases analysis costs. A new biological surface sampling method, the Biological Sampling Kit (BiSKit), designed to sample large areas and to be compatible with testing with a variety of technologies, including PCR and immunoassay, was evaluated and compared to other surface sampling strategies. In experimental room trials, wood laminate and metal surfaces were contaminated by aerosolization of Bacillus atrophaeus spores, a simulant for Bacillus anthracis, into the room, followed by settling of the spores onto the test surfaces. The surfaces were sampled with the BiSKit, a cotton-based swab, and a foam-based swab. Samples were analyzed by culturing, quantitative PCR, and immunological assays. The results showed that the large surface area (1 m2) sampled with the BiSKit resulted in concentrations of B. atrophaeus in samples that were up to 10-fold higher than the concentrations obtained with the other methods tested. A comparison of wet and dry sampling with the BiSKit indicated that dry sampling was more efficient (efficiency, 18.4%) than wet sampling (efficiency, 11.3%). The sensitivities of detection of B. atrophaeus on metal surfaces were 42 ± 5.8 CFU/m2 for wet sampling and 100.5 ± 10.2 CFU/m2 for dry sampling. These results demonstrate that the use of a sampling device capable of sampling larger areas results in higher sensitivity than that obtained with currently available methods and has the advantage of sampling larger areas, thus requiring collection of fewer samples per site

Characterization of Microbial Activity

The overall goal of this study is to investigate the phenomena that affect the fate and transport of radionuclides in the environment. The objective of this task, “Characterization of Microbial Activity”, is to develop a molecular biological method for the characterization of the microbial population indigenous to the Yucca Mountain Project site, with emphasis in detection and measurement of species or groups of microorganisms that could be involved in actinide and/or metal reduction, and subsurface transport. Subtasks consist of QA planning and preparation, and literature review. This task is part of a cooperative agreement between the UNLV Research Foundation and the U.S. Department of Energy (#DE-FC28-04RW12237) titled “Yucca Mountain Groundwater Characterization”

Prevalence and Antimicrobial Susceptibility of Methicillin-resistant Staphylococcus aureus in Pregnant Women and Their Newborns in Las Vegas, Nevada

Colonization and infection by resistant strains of Staphylococcus aureus are being reported in epidemic proportions. The goal of this study was to determine the local prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization in pregnant women in southern Nevada and how it correlates with colonization and infection of their neonates. Signed consent was obtained, and a brief questionnaire was administered by the medical staff to each pregnant woman to collect demographic data and pertinent medical, family and social history. Nasal and vaginal specimens were obtained from pregnant women at ≥35 weeks gestation, and nasal and umbilicus specimens were obtained from their newborns. Specimens were cultured onto two selective media for S. aureus and MRSA. Potential MRSA isolates were further evaluated for susceptibility to antibiotics. Specimens from 307 pregnant women and 174 neonates were collected, resulting in 172 mother-neonate paired specimens. A total of 278 questionnaires were received from study participants. MRSA prevalence in pregnant women was 1.0% and 0.3% for nasal and vaginal specimens, respectively. The MRSA prevalence in neonates was 0% and 0.6% for nasal and umbilical specimens, respectively. Four different antimicrobial susceptibility profiles were observed among the MRSA isolates. The results did not show transmission of MRSA from pregnant women to their newborns, or infections of newborns with MRSA. It is expected that the results of this study will inform future decisions on surveillance, treatment and prevention of MRSA infections in Nevada

Effectiveness of a Portable, Large-Area Ultraviolet Germicidal Device

Effective disinfection of the hospital environment is a key component in the prevention of healthcare-associated infections. The objective of this project was to evaluate the effectiveness of an ultraviolet germicidal device in reducing the concentrations of culturable bacteria on indoor surfaces. The ultraviolet germicidal device was installed and operated in four experimental trials conducted in a microbiology research chamber. Agar plates inoculated with known concentrations of two test microorganisms were placed on benches inside the chamber at two distances, 1.5 meters and 3.0 meters from the machine, for exposure times of 5 minutes, 10 minutes, and 20 minutes. With test agar plates directly exposed to ultraviolet radiation, percent reductions were all \u3e99.9% compared with the laboratory control plates. However, with indirect UV exposure, the edge of the plastic petri dishes provided some protection from the UV source, as indicated by the presence of colonies along the edge of the agar plates. Additional research will be conducted to further characterize the device for optimal use in surface decontamination and to determine its effectiveness in reducing airborne culturable bacterial concentrations

Surface, Water and Air Biocharacterization - A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft Environment

A Comprehensive Characterization of Microorganisms and Allergens in Spacecraft (SWAB) will use advanced molecular techniques to comprehensively evaluate microbes on board the space station, including pathogens (organisms that may cause disease). It also will track changes in the microbial community as spacecraft visit the station and new station modules are added. This study will allow an assessment of the risk of microbes to the crew and the spacecraft. Research Summary: Previous microbial analysis of spacecraft only identify microorganisms that will grow in culture, omitting greater than 90% of all microorganisms including pathogens such as Legionella (the bacterium which causes Legionnaires' disease) and Cryptosporidium (a parasite common in contaminated water) The incidence of potent allergens, such as dust mites, has never been systematically studied in spacecraft environments and microbial toxins have not been previously monitored. This study will use modern molecular techniques to identify microorganisms and allergens. Direct sampling of the ISS allows identification of the microbial communities present, and determination of whether these change or mutate over time. SWAB complements the nominal ISS environmental monitoring by providing a comparison of analyses from current media-based and advanced molecular-based technologies

Prevalence and Antimicrobial Agent Susceptibility of Methicillin-resistant Staphylococcus aureus in Healthy Pediatric Outpatients in Las Vegas

Colonization and infection by community-associated resistant strains of Staphylococcus aureus are being reported in epidemic proportions. The purpose of this study was to determine the local prevalence of methicillin-resistant Staphylococcus aureus (MRSA) colonization in children and to characterize the MRSA isolates in the laboratory with regard to antimicrobial agent susceptibility patterns, and the presence of the mecA and the Panton-Valentine leukocidin (PVL) genes. Nasal swabs were collected at two pediatric clinics from a total of 505 children during health maintenance visits. A brief questionnaire was administered to collect demographic data and pertinent medical, family, and social history. Samples were cultured onto 2 selective media for S. aureus and MRSA. Potential MRSA isolates were further evaluated by real-time polymerase chain reaction (PCR), and for susceptibility to eight antibiotics by disk diffusion. Culture results showed that MRSA was present in 15 of the 505 specimens (3.0%). Six different antimicrobial susceptibility profiles were observed among the MRSA isolates. PCR amplification results showed that all 15 MRSA isolates were positive for the presence of the mecA gene, and 10 MRSA isolates contained the PVL gene. Understanding local prevalence rates and the role of colonization in infection are needed to develop effective interventions to reduce MRSA infections

Determination of the Efficacy of Two Building Decontamination Strategies by Surface Sampling with Culture and Quantitative PCR Analysis

The efficacy of currently available decontamination strategies for the treatment of indoor furnishings contaminated with bioterrorism agents is poorly understood. Efficacy testing of decontamination products in a controlled environment is needed to ensure that effective methods are used to decontaminate domestic and workplace settings. An experimental room supplied with materials used in office furnishings (i.e., wood laminate, painted metal, and vinyl tile) was used with controlled dry aerosol releases of endospores of Bacillus atrophaeus (“Bacillus subtilis subsp. niger,” also referred to as BG), a Bacillus anthracis surrogate. Studies were performed using two test products, a foam decontaminant and chlorine dioxide gas. Surface samples were collected pre- and posttreatment with three sampling methods and analyzed by culture and quantitative PCR (QPCR). Additional aerosol releases with environmental background present on the surface materials were also conducted to determine if there was any interference with decontamination or sample analysis. Culture results indicated that 105 to 106 CFU per sample were present on surfaces before decontamination. After decontamination with the foam, no culturable B. atrophaeus spores were detected. After decontamination with chlorine dioxide gas, no culturable B. atrophaeus was detected in 24 of 27 samples (89%). However, QPCR analysis showed that B. atrophaeus DNA was still present after decontamination with both methods. Environmental background material had no apparent effect on decontamination, but inhibition of the QPCR assay was observed. These results demonstrate the effectiveness of two decontamination methods and illustrate the utility of surface sampling and QPCR analysis for the evaluation of decontamination strategies

Characterization of microbial populations in the subsurface

This task is part of a cooperative agreement between the UNLV Research Foundation and the U.S. Department of Energy (#DE-FC28-04RW12237) titled “Yucca Mountain Groundwater Characterization”. The work was conducted in the Harry Reid Center for Environmental Studies, Microbiology Division of the University of Nevada, Las Vegas from October 1, 2004 to September 30, 2006. The overall goal of this research was to investigate the phenomena that affect the fate and transport of radionuclides in the environment. The purpose of this task (ORD-RF-01), “Characterization of Microbial Activity”, was to develop a molecular biological method for the characterization of the microbial population indigenous to the Yucca Mountain Project site, with emphasis in detection and measurement of species or groups of microorganisms that could be involved in actinide and/or metal reduction, and subsurface transport. To quantify and characterize the microbial populations, including microorganisms that may be viable but are not currently physiologically active, a molecular biological approach was utilized to amplify and detect microbial DNA present in the subsurface. This approach, termed polymerase chain reaction (PCR), results in the amplification of DNA sequences that are unique to the groups of microorganisms of interest. Quantitative PCR (QPCR) assays were developed and used for the measurement of subsurface microbial populations. The protocols were evaluated in laboratory tests involving representative microbial species and genera, and tested by assaying available subsurface samples previously collected from the Yucca Mountain Project site. Other subtasks included Quality Assurance (QA) planning and preparation, and a literature review. This work was subject to the Nevada System of Higher Education (NSHE) QA Program requirements

Development of a New Polymerase Chain Reaction Assay for the Rapid Detection of the Oral Pathogenic Bacterium, Selenomonas Noxia

Background In recent studies, periodontal health has been linked to being overweight and/or obese. Among common oral bacteria, Selenomonas noxia has been implicated in converting periodontal health to disease, and Selenomonas species have also been found in gastric ulcers. The objective of this study was to develop and validate a quantitative polymerase chain reaction (qPCR) assay for the specific and rapid detection of S. noxia. Methods Two oligonucleotide primer pairs and one probe were designed and tested to determine optimal amplification signal with three strains of S. noxia. The PCR assay was tested against fourteen non-target organisms, including closely related oral Selenomonads, one phylogenetically closely related bacterium, and two commonly isolated oral bacteria. Results One of the primer sets was more sensitive at detecting the target organism and was selected for optimization and validation experiments. The designed primers and probe amplified the target organism with 100 % specificity. PCR inhibition was observed with an internal positive control, and inhibition was resolved by diluting the DNA extract. Conclusions The qPCR assay designed in this study can be used to specifically detect S. noxiain the clinical setting and in future research involving the enhanced detection of S. noxia. The assay can also be used in epidemiological studies for understanding the role of S. noxia in disease processes including, but not limited to, oral health and obesity of infectious origin