VTechWorks Repository :: Browsing by Author "Falkinham, Joseph O. III"

Browsing by Author "Falkinham, Joseph O. III"

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Absence of Mycobacterium intracellulare and Presence of Mycobacterium chimaera in Household Water and Biofilm Samples of Patients in the United States with Mycobacterium avium Complex Respiratory Disease
Wallace, Richard J. Jr.; Iakhiaeva, Elena; Williams, Myra D.; Brown-Elliott, Barbara A.; Vasireddy, Sruthi; Vasireddy, Ravikiran; Lande, Leah; Peterson, Donald D.; Sawicki, Janet; Kwait, Rebecca; Tichenor, Wellington S.; Turenne, Christine; Falkinham, Joseph O. III (American Society for Microbiology, 2013-03-27)
Recent studies have shown that respiratory isolates from pulmonary disease patients and household water/biofilm isolates of Mycobacterium avium could be matched by DNA fingerprinting. To determine if this is true for Mycobacterium intracellulare, household water sources for 36 patients with Mycobacterium avium complex (MAC) lung disease were evaluated. MAC household water isolates from three published studies that included 37 additional MAC respiratory disease patients were also evaluated. Species identification was done initially using nonsequencing methods with confirmation by internal transcribed spacer (ITS) and/or partial 16S rRNA gene sequencing. M. intracellulare was identified by nonsequencing methods in 54 respiratory cultures and 41 household water/biofilm samples. By ITS sequencing, 49 (90.7%) respiratory isolates were M. intracellulare and 4 (7.4%) were Mycobacterium chimaera. In contrast, 30 (73%) household water samples were M. chimaera, 8 (20%) were other MAC X species (i.e., isolates positive with a MAC probe but negative with species-specific M. avium and M. intracellulare probes), and 3 (7%) were M. avium; none were M. intracellulare. In comparison, M. avium was recovered from 141 water/biofilm samples. These results indicate that M. intracellulare lung disease in the United States is acquired from environmental sources other than household water. Nonsequencing methods for identification of nontuberculous mycobacteria (including those of the MAC) might fail to distinguish closely related species (such as M. intracellulare and M. chimaera). This is the first report of M. chimaera recovery from household water. The study underscores the importance of taxonomy and distinguishing the many species and subspecies of the MAC.
Adherence and Biofilm Formation of Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus in household plumbing
Mullis, Summer (Virginia Tech, 2012-09-05)
Nontuberculous mycobacteria (NTM) are ubiquitous in the environment and found in drinking water distribution systems and household plumbing. They are opportunistic pathogens of humans, causing lung disease. Their ability to adhere and form biofilm is attributed to a waxy, lipid-rich, cell envelope. This highly hydrophobic envelope also contributes to the characteristic antibiotic-, chlorine-, and disinfectant- resistance of NTM. NTM in household plumbing reside primarily in biofilms and the ability to form biofilm has been linked to virulence. Shedding of cells from biofilm and the subsequent aerosolization of microorganisms through showerheads presents a significant public health risk, particularly to those individuals with associated risk factors. Three species of NTM, Mycobacterium avium, Mycobacterium intracellulare, and Mycobacterium abscessus, were examined for adherence and biofilm formation on surfaces common to household plumbing systems, including glass, copper, stainless steel, polyvinyl chloride, and galvanized steel. All experiments were conducted with sterile, Blacksburg tap water in a CDC Biofilm Reactor. Highest adherence was observed by M. avium on galvanized steel surfaces, reaching 15,100 CFU/cm2 surface at 6 hours incubation at room temperature. After 3 weeks incubation at room temperature, biofilm formation of M. avium was also highest on galvanized steel surfaces, reaching 14,000,000 CFU/cm2 surface. Lowest adherence was observed by M. abscessus on polyvinyl chloride (PVC) surfaces, reaching 40 CFU/cm2. Lowest biofilm formation was observed by M. intracellulare on glass surfaces, reaching 5,900 CFU/cm2. Surfaces, such as galvanized (zinc), on which high adherence and biofilm formation was observed, should be avoided in household plumbing systems of NTM patients and individuals at risk for developing NTM disease. Additionally, surfaces such as copper, harbor fewer NTM and may provide a safer alternative for household plumbing of NTM patients.
Aerosolization of Drinking Water Metals to Indoor Air and Assessment of Human Taste and Visual Thresholds for Manganese
Sain, Amanda Elizabeth (Virginia Tech, 2013-04-17)
Exposure to excess manganese via drinking water raises concerns due to potential for adverse neurological impacts, particularly in children. Manganese is ubiquitous in US groundwaters above the SMCL = 0.05 mg/L. Manganese is an essential nutrient, but exposures to elevated manganese have neurotoxic effects. Chapter 2 focuses on human senses\' ability to detect manganese in drinking water. Findings indicate human senses cannot be relied upon to detect excess Mn(II) in drinking water. Mn(IV) is easily visually detected, but cannot be tasted at 10 times the SMCL. Chapter 3 is an assessment the ability of an ultrasonic humidifier to expel drinking water impurities in aerosols. The quality of the water used to charge the humidifier reservoir affects the composition of elements in the aerosols and condensate. Findings indicate condensed humidifier aerosols contain 85% of elements present in the reservoir water for a variety of water types if there is no precipitation. Waters with high concentration of hardness or iron formed precipitates that decreased the concentrations of these metals in the aerosols causing variable results for other elements that were initially present at < 1mg/L in the charge water. This indicates that humidifiers could be a source of inhalation exposure for source water contaminants.
Antimicrobial Producing Bacteria as Agents of Microbial Population Dynamics
Tanner, Justin Rogers (Virginia Tech, 2010-10-27)
The need for new antibiotics has been highlighted recently with the increasing pace of emergence of drug resistant pathogens (MRSA, XDR-TB, etc.). Modification of existing antibiotics with the additions of side chains or other chemical groups and genomics based drug targeting have been the preferred method of drug development at the corporate level in recent years. These approaches have yielded few viable antibiotics and natural products are once again becoming an area of interest for drug discovery. We examined the antimicrobial "Red Soils" of the Hashemite Kingdom of Jordan that have historically been used to prevent infection and cure rashes by the native peoples. Antimicrobial producing bacteria were present in these soils and found to be the reason for their antibiotic activity. After isolation, these bacteria were found to excrete their antimicrobials into the liquid culture media which we could then attempt to isolate for further study. Adsorbent resins were employed to capture the antimicrobial compounds and then elute them in a more concentrated solution. As part of a drug discovery program, we sought a way to quickly characterize other soils for potential antibiotic producing bacteria. The community level physiologic profile was examined to determine if this approach would allow for a rapid categorizing of soils based on their probability of containing antimicrobial producing microorganisms. This method proved to have a high level of variability that could not be overcome even after mixing using a commercial blender. The role of these antimicrobial producing bacteria within their natural microbial community has largely been confined to microbe-plant interactions. The role of antimicrobial-producing microorganisms in driving the diversity of their community has not been a focus of considerable study. The potential of an antimicrobial-producing bacterium to act as a driver of diversity was examined using an artificial microbial community based in a sand microcosm. The changes in the microbial assemblage indicate that antimicrobial-producing bacteria may act in an allelopathic manner rather than in a predatory role.
Biosystematic Studies in Crepidotus and the Crepidotaceae (Basidiomycetes, Agaricales)
Aime, Mary Catherine (Virginia Tech, 2001-05-01)
Fungi of the Crepidotaceae are characterized by saprotrophic habit, filamentous cuticle, and brown-pigmented basidiospores that lack either a germ pore or plage. The majority of species belong to Crepidotus, distinguished by their pleurotoid basidiomata. Because of their diverse morphology, the presence of several conflicting classifications, and lack of data regarding the biology, phenotypic plasticity, or phylogeny of these fungi, the present study sought first to determine phylogenetic relationships among the different taxonomic groups as a basis for addressing other aspects of Crepidotus biology and evolution. Sequencing analyses show the Crepidotaceae is not monophyletic, and the family concept is revised. Crepidotus and its sister genus Simocybe are found to be monophyletic. At least nine phylogenetic lineages within Crepidotus were uncovered, although relationships between them could not be resolved. However, none of the previously proposed infrageneric classifications are reflective of phylogeny. Morphological, biological, and phylogenetic species concepts were compared within a single phylogenetic unit, termed the Sphaerula group, showing an unusual amount of phenotypic plasticity exists within species, and a taxonomic revision of these species proposed. Also reported are several unique or unusual aspects of Crepidotus biology, including presence of a prolonged latent period prior to basidiospore germination; spontaneous reversion of differentiated hymenial cells to vegetative growth; and the revelation that structures previously termed pleurocystidia are actually the expression of secondary growth from basidia. Results from mating system, culture, and type studies, reassessment of morphological characters traditionally applied to agaric taxonomy, and a revised life cycle for the Crepidoti are presented.
Brucella abortus RB51 vaccine: Testing its Spectrum of Protective and Curative Characteristics
Contreras Rojas, Andrea Paz (Virginia Tech, 2004-07-30)
Brucella abortus (BA) are gram-negative, facultative intracellular bacteria that cause abortions in cattle and debilitating illness in humans. The US is now virtually free of bovine brucellosis, but the disease is endemic in wildlife. The official brucellosis vaccine in the US is strain RB51 (RB51). It elicits protective cell-mediated immunity (CMI) against BA infections. Mycobacterium avium subspecies paratuberculosis (MAP) causes paratuberculosis in ruminants. It is a slow growing intracellular parasite that requires CMI for its control, belongs to the genus Mycobacterium, and is closely related to M. avium avium (MA). Using RB51 as a vector that induces strong protective CMI may be useful to protect against MAP if it expresses MAP protective antigens. Therefore, MAP 85A and 35kDa proteins were expressed at low levels in RB51, and the immune responses elicited by these vaccines in BALB/c mice were evaluated. Strong anti-Brucella immunity was generated, but the anti-mycobacterial response was low. To evaluate protective efficacy, a BALB/c model using MA was developed. When mice were challenged with MA, protection was obtained in some experiments but was inconsistent. This may be due to the low expression of MAP antigens in RB51. Another objective was to evaluate the effect of an ongoing Brucella-infection on the efficacy of RB51 vaccination, and whether vaccination of already infected animals could have a curative effect. Mice acutely or chronically infected with virulent BA, rapidly cleared the RB51 vaccine organisms, but there was no significant decrease in the number of virulent BA. Brucella spp. have been developed as biological weapons, but there are no vaccines to protect humans. The development of a very attenuated protective vaccine is necessary to prevent human infections, as well as to protect wildlife. To generate such a vaccine, RB51 based vaccines were irradiated to render them non-replicative, but metabolically active. We demonstrated that in general, irradiated and non-irradiated RB51 vaccines remain protective at levels similar to those elicited by the live vaccines. Therefore, irradiation of strain RB51 is an effective means of attenuating the strain without affecting its protective characteristics, and could eventually be used as a vaccine for wildlife and humans.
Challenges of NTM Drug Development
Falkinham, Joseph O. III (Frontiers, 2018-07-18)
Discovery and development of antibiotics active against the environmental opportunistic non-tuberculous mycobacteria (NTM) have been retarded by innate antibiotic-resistance of NTM cells and methodological challenges in the laboratory. The basis for the innate resistance of NTM cells is its lipid rich outer membrane that results in hydrophobic cells and the outer membrane's impermeability, and the residence of NTM cells in phagocytic cells, and the slow growth and dormancy of NTM. Laboratory challenges include: the choice of species and strains for screening and measurement of anti-NTM activity, the high frequency colony switching between antibiotic-susceptible and resistant variants, the preference of NTM to adhere to surfaces and form biofilms, and the aggregation of NTM cells. Understanding these challenges can guide and inform our approaches to discovery and development of antibiotics with activity against NTM.
The Changing Pattern of Nontuberculous Mycobacterial Disease
Falkinham, Joseph O. III (Hindawi, 2003-01-01)
Nontuberculous mycobacteria are human opportunistic pathogens whose source of infection is the environment. These include both slow-growing (eg, Mycobacterium kansasii and Mycobacterium avium) and rapid-growing (eg, Mycobacterium abscessus and Mycobacterium fortuitum) species. Transmission is through ingestion or inhalation of water, particulate matter or aerosols, or through trauma. The historic presentation of pulmonary disease in older individuals with predisposing lung conditions and in children has been changing. Pulmonary disease in elderly individuals who lack the classic predisposing lung conditions is increasing. Pulmonary disease and hypersensitivity pneumonitis have been linked with occupational or home exposures to nontuberculous mycobacteria. There has been a shift from Mycobacterium scrofulaceum to M avium in children with cervical lymphadenitis. Further, individuals who are immunosuppressed due to therapy or HIV-infection are at a greatly increased risk for nontuberculous mycobacterial infection. The changing pattern of nontuberculous mycobacterial disease is due in part to the ability of these pathogens to survive and proliferate in habitats that they share with humans, such as drinking water. The advent of an aging population and an increase in the proportion of immunosuppressed individuals suggest that the prevalence of nontuberculous mycobacterial disease will increase.
Characterization of DNA-repair potential in deep subsurface bacteria challenged by UV light, hydrogen peroxide, and gamma radiation
Arrage, Andrew Anthony (Virginia Tech, 1991-08-07)
Subsurface bacterial isolates obtained through the DOE Subsurface Science Program were tested for resistance to UV light, gamma radiation and H₂0₂. Some deep subsurface bacteria were resistant to UV light, demonstrating ≥1.0% survival at fluences which resulted in a 0.0001% survival level of E. coli B. The percentage of UV resistant aerobic subsurface bacteria and surface soil bacteria were similar; 30.8% and 25.8% respectively. All of the microaerophilic subsurface isolates were UV sensitive as defined in this work; however, subsurface isolates demonstrated UV resistance levels similar to reference bacterial strains of the same Gram reaction. These results were not in agreement with the hypothesis that the resistance of an organism to UV is correlated with the amount of solar radiation in its natural habitat. Evidence for photoreactivation and the presence of an SOS-like mechanism was also detected in subsurface bacteria. The presence of UV resistance and photoreactivation in subsurface bacteria that have been shielded from solar radiation for millions of years may point to a limited rate of evolution in the deep subsurface environment. In subsurface bacteria, there was a relatedness between UV resistance and resistance to gamma radiation and H₂0₂ UV-resistant aerobic subsurface isolates were also gamma and H₂0₂- resistant compared to the microaerophilic isolates tested. Due to the similarities of bacterial responses to UV, H₂0₂ , and gamma radiation, either UV or H₂0₂ may be utilized to model the effects of ionizing radiation on bacterial cultures used for the bioremediation of organic and radioactive waste-containing environments.
Characterization of Mycobacterium avium cytoplasmic membrane proteins with an emphasis on the major cytoplasmic membrane protein
Carlisle, Glenn E. (Virginia Tech, 1991-05-16)
Proteins of the cytoplasmic membrane of Mycobacterium avium were investigated to identify those which were: (1)intrinsic or extrinsic, (2) attached to the cell wall, (3)surface accessible and (4) excreted. In addition sera containing anti-cytoplasmic membrane proteins were obtained and preliminary purification of the cytoplasmic membrane protein was attempted. The predominating cytoplasmic membrane protein of 31,000 daltons (MCMP) was found to be intrinsic, attached to the cell wall and possibly surface accessible. The MCMP was not excreted, even in media in which the MCMP is not found in the cytoplasmic membrane. Other cytoplasmic membrane proteins were also found to be intrinsic; a few were likely to be extrinsic based upon their separation from the membrane in sucrose gradients. Cytoplasmic membrane proteins of 66, 000, 115, 000 and 129 dalton were surface accessible as judged by I 125-Iodobead labeling. Antisera against the HCMP and other cytoplasmic membrane proteins was obtained and will be useful in further cytoplasmic membrane protein characterization. Acetone precipitation of a cytoplasmic membrane preparation was performed to partially purify the MCMP. The data from this study can be used for the development of serodiagnostic reagents for detecting mycobacterial infection.
Characterization of the genes and gene products of the acetate-activating enzymes and a novel iron-sulfur flavoprotein from Methanosarcina thermophila strain TM-1
Latimer, Matthew T. (Virginia Tech, 1993-10-05)
The genes encoding the acetate kinase and phosphotransacetylase enzymes from Methanosarcina thermophila were isolated from a genomic library on a fifteen kilobase fragment The genes are located adjacent to one another, with the phosphotransacetylase gene (pta) directly upstream of the acetate kinase gene (ack). The two genes were sequenced, along with a third Open Reading Frame (designated orfY). The orfY gene appears to encode a novel protein whose physiological function has yet to be determined.
Characterization of the interaction of putrescine and the adenosine-3' ,5'-cyclic monophosphate-cAMP receptor protein complex in the regulation of the speC gene encoding ornithine decarboxylase in Escherichia coli
Busse, Leigh Anne (Virginia Tech, 1988-04-07)
Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to produce the diamine, putrescine, in the bacterium Escherichia coli. The speC gene encoding ODC has been shown to be subject to transcriptional repression by either putrescine or the cAMP- cAMP receptor protein (CRP) complex. To determine whether these regulatory modes are independent, the expression of ODC was determined by measuring the specific activity of ODC in crude extracts prepared from exponentially grown cultures of wild type ~ coli K-12 as well as in strains unable to synthesize cAMP (cya) and/or CRP (crp). 1-5 mM cAMP repressed ODe activity 22-38% in wild type, 57-66% in the cya strain, and only 7-18% in the ~ strains. 2-10 mM putrescine repressed ODe activity 30-32% in wild type, 48-49% in the cya strain, and 37-38% in the ~ strain. As putrescine repressed ODe activity in the absence of eRP protein (i.e. in a crp strain), putrescine-mediated repression of ODe appears to be independent of the repression of ODC by the cAMP-eRP complex. This conclusion was verified by demonstrating th t .oDC repression by putrescine and cAMP together was additive.
Cloning and characterization of glycogen synthase from Dictyostelium discoideum
Williamson, Brian (Virginia Tech, 1995)
In Dictyostelium, glycogen metabolism plays a major role in development. Undifferentiated cells contain stores of glycogen that are broken down and converted to structural components in differentiated cells. The enzyme that synthesizes this developmentally important pool of glycogen is glycogen synthase. I have cloned the entire coding and 1.3 kb of upstream noncoding region, of glycogen synthase, using PCR amplification and genomic library screening. In order to clone the 3’ portion of the gene it was necessary to develop a new technique, enrichment-PCR, that relies on the base composition of the Dictyostelium genome. Due to the high A+T content of the Dictyostelium genome, a polyT primer and a gene specific primer were used to amplify an unknown DNA fragment, flanking a known sequence. Analysis of the complete coding region showed that glycogen synthase possesses three introns that contain the consensus splice sites for Dictyostelium. The luciferase reporter gene was used to study the transcriptional regulation of glycogen synthase. I defined the cis-acting elements that are required for proper transcriptional regulation of glycogen synthase by using promoter/luciferase fusions of varying sizes. Using the luciferase reporter system a putative promoter element was identified. Additional luciferase constructs were made to identify the specific nucleotide involved in transcription of the glycogen synthase gene. Small defined deletions are often necessary for reporter gene analysis. We have developed a deletion cassette that can expand the functionality of any commonly-used vector. The deletion cassette confers the ability to make small specific sequential deletions of the DNA flanking the cassette. We have shown that this altered vector (pDNBL) now has the ability to create 2, 4, 5 or 9 bp deletions. A number of experimental approaches were taken to study the regulation of glycogen synthase. Homologous recombination was used to try to generate a glycogen synthase (-) cell line. In addition, I have constructed a green fluorescent protein (GFP) vector (pNV) based on the pVTL2 vector. This reporter gene is useful for monitoring the expression of a particular gene in vivo.
Common Features of Opportunistic Premise Plumbing Pathogens
Falkinham, Joseph O. III (MDPI, 2015-04-24)
Recently it has been estimated that the annual cost of diseases caused by the waterborne pathogens Legionella pneumonia, Mycobacterium avium, and Pseudomonas aeruginosa is $500 million. For the period 2001–2012, the estimated cost of hospital admissions for nontuberculous mycobacterial pulmonary disease, the majority caused by M. avium, was almost $1 billion. These three waterborne opportunistic pathogens are normal inhabitants of drinking water—not contaminants—that share a number of key characteristics that predispose them to survival, persistence, and growth in drinking water distribution systems and premise plumbing. Herein, I list and describe these shared characteristics that include: disinfectant-resistance, biofilm-formation, growth in amoebae, growth at low organic carbon concentrations (oligotrophic), and growth under conditions of stagnation. This review is intended to increase awareness of OPPPs, identify emerging OPPPs, and challenge the drinking water industry to develop novel approaches toward their control.
Construction and Use of a Transposon for Identification of Essential Genes in Mycobacteria
Riggs, Sarah Danielle (Virginia Tech, 2011-04-18)
The continuing emergence of multi-drug resistant Mycobacterium tuberculosis is threatening the ability to treat tuberculosis (TB) worldwide. The development of new anti-TB drugs requires new approaches and new drug targets. In this study, a mariner-based transposon, TnQuoVadis, was constructed to identify essential genes as potential drug targets. This transposon has an outward-facing anhydrotetracycline (ATc)-inducible promoter at each end. A mutant with TnQuoVadis inserted upstream of an essential gene may display normal growth in the presence of ATc, but exhibit no growth or severely diminished growth in the absence of ATc. TnQuoVadis was placed onto a vector with a temperature sensitive replication origin for more efficient mutagenesis of mycobacteria. In a preliminary genetic screen using the model organism Mycobacterium smegmatis, 13 mutants with ATc-dependent growth were identified. Identification of the insertion sites by cloning and sequencing indicated that there were nine genetic loci containing transposon insertions upstream of essential gene candidates in M. smegmatis. Further analysis of these genes indicated that many were previously known essential in both M. smegmatis and M. tuberculosis. These results demonstrate that TnQuoVadis and its delivery system can be utilized for the identification of essential genes in mycobacteria
Construction and Use of Transposon MycoTetOP(2) for Isolation of Conditional Mycobacteria Mutants
Riggs-Shute, Sarah D.; Falkinham, Joseph O. III; Yang, Zhaomin (2020-01-21)
Mycobacteria are unique in many aspects of their biology. The development of genetic tools to identify genes critical for their growth by forward genetic analysis holds great promises to advance our understanding of their cellular, physiological and biochemical processes. Here we report the development of a novel transposon, MycoTetOP(2), to aid the identification of such genes by direct transposon mutagenesis. This mariner-based transposon contains nested anhydrotetracycline (ATc)-inducible promoters to drive transcription outward from both of its ends. In addition, it includes the Escherichia coli R6K gamma origin to facilitate the identification of insertion sites. MycoTetOP(2) was placed in a shuttle plasmid with a temperature-sensitive DNA replication origin in mycobacteria. This allows propagation of mycobacteria harboring the plasmid at a permissive temperature. The resulting population of cells can then be subjected to a temperature shift to select for transposon mutants. This transposon and its delivery system, once constructed, were tested in the fast-growing model Mycobacterium smegmatis and 13 mutants with ATc-dependent growth were isolated. The identification of the insertion sites in these mutants led to nine unique genetic loci with genes critical for essential processes in both M. smegmatis and Mycobacterium tuberculosis. These results demonstrate that MycoTetOP(2) and its delivery vector provide valuable tools for the studies of mycobacteria by forward genetics.
Critical factors controlling regrowth of opportunistic pathogens in premise plumbing
Wang, Hong (Virginia Tech, 2013-03-28)
Opportunistic pathogens (e.g., Legionella pneumophila, Mycobacterium avium complex, Acanthamoeba polyphaga, Pseudomonas aeruginosa) residing in human-made water systems, particularly premise plumbing, are now the primary source of water-borne disease in developed countries. The prevention and control of opportunistic pathogens is a new challenge in premise plumbing due to the limited knowledge concerning the factors driving their occurrence and regrowth mechanisms, and also the complexity of premise plumbing conditions. The goal of this study is to identify key factors governing occurrence of opportunistic pathogens in drinking water distribution systems, particularly premise plumbing, via field investigations and lab-scale experiments. A molecular survey of three opportunistic pathogens (L. pneumophila, M. avium, P. aeruginosa), related groups (Legionella and mycobacteria) and two amoeba hosts (Acanthamoeba spp. and Hartmanella vermiformis) was performed in two real-word chloraminated drinking water distribution systems using quantitative polymerase chain reaction (q-PCR). A high occurrence of Legionella (" 69.0%) and mycobacteria (100%), lower occurrence of L. pneumophila (" 20%) and M. avium (" 33.3%), and rare detection of Pseudomonas aeruginosa (" 13.3%) was observed in both systems. Hartmanella vermiformis was more prevalent than Acanthamoeba. Three-minute flushing resulted in reduced gene copies of Legionella, mycobacteria, H. vermiformis and 16S rRNA genes (P<0.05) and distinct microbial community structure in postflushing water, implying strong regrowth potential of opportunistic pathogens in premise pluming. In order to examine the influence of pipe material, disinfectant type, and water age on occurrence and persistence of the target microorganisms, triplicate simulated distribution systems (SDSs) comparing iron, cement and PVC pipe materials were fed either chlorinated or chloraminated tap water, and were sampled at water ages ranging from 1d to 5.7d. q-PCR quantification of target microorganisms in both biofilm and bulk water revealed that Legionella, mycobacteria, P. aeruginosa and both amoebas naturally colonized the six SDSs, but L. pneumophila and M. avium were not detected. Disinfectant type and dose have the strongest influence on the microbiota. Disinfectant decay was noted with water age, particularly in chloraminated SDSs (due to nitrification), generally resulting in increased microbial detection frequencies and densities with water age. Influence of pipe material became apparent at water ages corresponding to low disinfectant residual. Natural colonization of Legionella spp., Mycobacterium spp., Acanthamoeba spp., H. vermiformis and M. avium was also observed in biofilms from five annular reactors, which were used to investigate effects of prior granular activated carbon (GAC) biofiltration and disinfectant type (chlorine, chloramine) on opportunistic pathogens under premise plumbing conditions. GAC pre-treatment effectively reduced total organic carbon (TOC). In most cases, total bacteria and opportunistic pathogens were higher in undisinfected annular reactors, but the levels were not proportional to the level of GAC pre-treatment/TOC. Chlorine was more effective for controlling mycobacteria and Acanthamoeba, whereas chloramine was more effective for controlling Legionella. Both chlorine and chloramine effectively reduced M. avium and H. vermiformis numbers. Pyrosequencing of 16S rRNA genes in biofilms revealed a significant effect of GAC pre-treatment and disinfectant type on the microbial community structure. Overall, the study provides insights to critical factors triggering proliferation of opportunistic pathogens in drinking water systems. Knowledge gained from this study can assist in formulating practical guidance for drinking water utilities and water consumers in terms of opportunistic pathogen prevention and control.
Desiccation-Tolerance of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium chimaera, Mycobacterium abscessus and Mycobacterium chelonae
Falkinham, Joseph O. III; Williams, Myra D. (MDPI, 2022-04-13)
Desiccation-tolerance of cells of four strains of Mycobacterium chimaera and individual strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, and Mycobacterium chelonae were measured by two methods. The survival of water-acclimated cells both in filter paper and on the surface of stainless-steel coupons were measured. In filter paper at 40% relative humidity at 25 °C, survival of patient isolates of M. avium and M. chimaera cells was 28% and 34% after 21 days of incubation, whereas it was 100% for the Sorin 3T isolate of M. chimaera. On stainless-steel biofilms after 42 days of incubation at 40% relative humidity at 25 °C, survival of water-acclimated cells of M. intracellulare was above 100%, while M. chelonae cells did not survive beyond 21 days, and survival of water-acclimated cells of M. avium and M. abscessus was 18% and 14%, respectively. On stainless-steel coupons, survival of patient and Sorin 3T isolates of M. chimaera was quite similar, specifically between 14% and 28% survival, after 42 days of incubation at 40% relative humidity at 25 °C. The experiments would support the hypothesis that some nontuberculous mycobacterial species are relatively desiccation-tolerant, whereas others are not. Further, long-term survival of the two M. chimaera strains is consistent with the presence of that species in Sorin 3T heater-coolers shipped throughout the world.
Development and evaluation of a colorimetric coliphage assay detection system
Ijzerman, M. Marian (Virginia Tech, 1993-07-14)
A Colorimetric Coliphage Assay Detection System (CCADS) that is composed of a Liquid Colorimetric Presence-Absence (LCPA) method and a Colorimetric Agar-Based (CAB) method was developed to overcome the limitations imposed by the Standard Methods for the Examination of Water and Wastewater agar-based coliphage method (APHA method). Both CCADS methods are based on the induction of β-galactosidase in Escherichia coli and the release of the enzyme through a lytic cell infection. The released enzyme then cleaves a chromogenic substrate which produces a colored reaction product. The CCADS was evaluated against the APHA method under laboratory conditions using a common sewage coliphage strain as a model (American Type Culture Collection- 13706-B2), and under field conditions using water samples collected from four different sources. During the laboratory evaluation, both the LCPA and CAB methods were found to be superior in coliphage detection to the APHA method because: 1) the LCPA and CAB methods were easier to read and interpret than the APHA method, 2) the LCPA and CAB methods were not subject to false positive results, 3) the LCPA method theoretically detected fewer coliphage particles than the APHA method, and 4) the CAB method detected roughly twice the number of coliphage particles than the APHA method. During the field evaluation, the results indicated: 1) the LCP A method was as reliable as either the CAB or APHA methods in coliphage detection, 2) the LCP A and CAB methods were easier to read and interpret than the APHA method, 3) neither the LCPA method nor the CAB method were subject to false positive results, 4) the CAB method detected more coliphages than the APHA method under conditions of high fecal pollution, but both methods performed equally well in coliphage detection under conditions of low fecal contamination, and 5) the LCPA and CAB methods were equally as sensitive in coliphage detection as the APHA method. Finally, the coliphage group proved to be a useful indicator of fecal pollution in nonpotable water supplies that exhibited a high degree of fecal pollution, whereas they were not shown to be useful indicators in potable water supplies that exhibited low levels of fecal contamination. The lack of coliphage detection sensitivity under conditions of low fecal contamination does not appear to be method limited, but rather the result of inefficiencies in processing environmental samples using the concentration methods currently available.
Development of a Rapid Coliphage Assat
Stanek, James Emmett (Virginia Tech, 1997-01-24)
A rapid coliphage detection assay (RCDA), based on the phage-induced release of b-galactosidase from cells of Escherichia coli (Ijzerman, M., J.O. Falkinham III and C. Hagedorn. (1993) [A liquid, colorimetric presence-absence coliphage detection method. J. Virol. Meth. 45:229-234] was modified to reduce the number of steps required to perform the assay, remove the need for specialized media and buffers, reduce the volumes required, and simplify growth and reaction conditions. Tolerances of the assay were defined at each step of the assay. The number of steps has been reduced from 12 to 7. The b-galactosidase reaction buffer was eliminated. Culture volumes were reduced from 25 ml to 5 ml and reaction volumes were reduced from 10 ml to 0.5 ml. Optimal growth conditions were 37 o C with orbital shaking at 200 rpm, a one hour subculture time and an incubation of subculture with water sample for two hours. Color development occurred at 37 o C in 30 minutes. The changes and modifications of the assay increased the ease of its performance without sacrificing the ability of the assay to detect as few as two phage particles per sample. By understanding the tolerances of the assay, technical support representatives of companies producing kits modeled after the assay will be prepared to answer questions from customers concerning possible kit failures or user error.

Browsing by Author "Falkinham, Joseph O. III"

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