Browsing by Author "Pyle, Barry H."

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Detection and source tracking of Escherichia coli, harboring intimin and Shiga toxin genes, isolated from the Little Bighorn River, Montana
(2014-09) Hamner, Steve; Broadaway, Susan C.; Berg, Ethan; Stettner, Sean; Pyle, Barry H.; Big Man, N.; Old Elk, J.; Eggers, Margaret J.; Doyle, John T.; Kindness, L.; Good Luck, B.; Ford, Tim E.; Camper, Anne K.
The Little Bighorn River flows through the Crow Indian Reservation in Montana. In 2008, Escherichia coli concentrations as high as 7,179 MPN/100 ml were detected in the river at the Crow Agency Water Treatment Plant intake site. During 2008, 2009, and 2012, 10 different serotypes of E. coli, including O157:H7, harboring both intimin and Shiga toxin genes were isolated from a popular swim site of the Little Bighorn River in Crow Agency. As part of a microbial source tracking study, E. coli strains were isolated from river samples as well as from manure collected from a large cattle feeding operation in the upper Little Bighorn River watershed; 23% of 167 isolates of E. coli obtained from the manure tested positive for the intimin gene. Among these manure isolates, 19 were identified as O156:H8, matching the serotype of an isolate collected from a river sampling site close to the cattle feeding area.
Detection of Pathogenic and Non-pathogenic Bacteria in Drinking Water and Associated Biofilms on the Crow Reservation, Montana, USA
(2018-07) Richards, Crystal L.; Broadaway, Susan C.; Eggers, Margaret J.; Doyle, John T.; Pyle, Barry H.; Camper, Anne K.; Ford, Tim E.
Private residences in rural areas with water systems that are not adequately regulated, monitored, and updated could have drinking water that poses a health risk. To investigate water quality on the Crow Reservation in Montana, water and biofilm samples were collected from 57 public buildings and private residences served by either treated municipal or individual groundwater well systems. Bacteriological quality was assessed including detection of fecal coliform bacteria and heterotrophic plate count (HPC) as well as three potentially pathogenic bacterial genera, Mycobacterium, Legionella, and Helicobacter. All three target genera were detected in drinking water systems on the Crow Reservation. Species detected included the opportunistic and frank pathogens Mycobacterium avium, Mycobacterium gordonae, Mycobacterium flavescens, Legionella pneumophila, and Helicobacter pylori. Additionally, there was an association between HPC bacteria and the presence of Mycobacterium and Legionella but not the presence of Helicobacter. This research has shown that groundwater and municipal drinking water systems on the Crow Reservation can harbor potential bacterial pathogens.
Effects of culture conditions and biofilm formation on the iodine susceptibility of legionella pneumophila
(1992-01) Cargill, Kari Lisa; Pyle, Barry H.; Sauer, R. L.; McFeters, Gordon A.
Alternatives to chlorination of water have been sought for reasons which include trihalomethane formation, possible bacterial regrowth, the high concentrations of chlorine required in certain circumstances, and the taste, odour and bodily irritation in chlorine-treated water. Electrolytically generated Cu and Ag ions at low levels, in addition to very low chlorine concentrations, have been suggested as an alternative to routine chlorination. We have examined the combination of Cu and Ag ions with low levels of iodine. Pseudomonas cepacia was grown either in rich medium or under nutrient restriction prior to disinfection. Survival of the organism and its ability to regrow after treatment as well as the effects of varying buffers, metal ion and iodine concentrations were determined. Low concentrations of metal ions (100 ppb Cu and 11 ppb Ag) and iodine (200 ppb) were more effective than either metal ions or iodine alone against Ps. cepacia grown on rich agar or in low nutrient buffer. After iodination, buffer-grown suspensions recovered to their original cell concentrations within 7 d. When Cu and Ag ions were used with or without iodine, regrowth was prevented. The results show that low concentrations of Cu and Ag in combination with iodine permit effective disinfection of bacteria after cultivation on either rich media or under nutrient restriction. These results, along with published data, suggest that the combination of these metals with halogenation may have applications in the disinfection of both recreational and potable water.
Factors affecting the determination of respiratory activity on the basis of cyanoditolyl tetrazolium chloride reduction with membrane filtration
(1995-12) Pyle, Barry H.; Broadaway, Susan C.; McFeters, Gordon A.
Isolation of potentially pathogenic Escherichia coli O157:h7 from the Ganges River
(2007-02) Hamner, Steve; Broadaway, Susan C.; Mishra, Veer B.; Tripathi, Anshuman; Mishra, Rajesh K.; Pulcini, Elinor D.; Pyle, Barry H.; Ford, Tim E.
Escherichia coli serotype O157:H7 was detected among bacteria collected from the Ganges River. O157:H7 isolates tested positive for stx1, stx2, and eae gene sequences. Identification of potentially pathogenic isolates from extensively used source water indicates that O157:H7 may be a significant but as yet underacknowledged public health concern in India.
Optimizing the growth of stressed Helicobacter pylori
(2011-02) Richards, Crystal L.; Buchholz, B. J.; Ford, Tim E.; Broadaway, Susan C.; Pyle, Barry H.; Camper, Anne K.
Helicobacter pylori is a gram-negative bacterium that colonizes the human stomach and is responsible for causing gastric ulcers. H. pylori is known to become stressed and nonculturable after exposure to unfavorable conditions. In this study, we enhanced previously published resuscitation procedures, characterized conditions under which stressed H. pylori can be recovered, and formulated a selective and differential resuscitation medium.Results showed that a specialized broth supplemented with trace minerals and lysed human erythrocytes and serum is required for the recovery of nonculturable H. pylori. The type of stress was an important factor in the efficacy of resuscitation, with cells exposed to atmospheric oxygen more readily resuscitated than nutrient-deprived cells. After resuscitation, culturable cells were recovered from previously nonculturable oxygen stressed cells (24 and 72 h of exposure) and nonculturable nutrient deprived cells (24 h of exposure). The length of time the cells were exposed to the stress was also an important factor in the recovery of stressed H. pylori. RNA levels were quantified and transcription of the cell division related gene, cdrA (HP0066), was assessed by qRT-PCR. The low levels of RNA detected in stressed cells, after resuscitation, support the idea that a small population of viable cells may be responsible for the colonies recovered on solid agar. The modification of the resuscitation broth into a selective and differential slant culture medium also allowed the recovery of stressed H. pylori. The methods presented here highlight the benefits and limitations of using human blood products for recovering nonculturable H. pylori.
Physiological assessment of bacteria using fluorochromes
(1995-01) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
Physiological assessment of bacteria using fluorochromes
(1995-01) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
Physiological methods to study biofilm disinfection
(1995-10) McFeters, Gordon A.; Yu, Feipeng Philip; Pyle, Barry H.; Stewart, Philip S.
This report reviews the development of a rapidin situ approach to study the physiological responses of bacteria within biofilms to disinfectants. One method utilized direct viable counts (DVC) to assess the disinfection efficacy when thin biofilms were exposed to chlorine or monochloramine. Results obtained using the DVC method were one log higher than plate count (PC) estimates of the surviving population after disinfection. Other methods incorporated the use of fluorogenic stains, a cryotomy technique to yield thin (5-μm) sections of biofilm communities and examination by fluorescence microscopy. The fluorogenic stains used in this approach included 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), which indicates cellular electron transport activity and Rhodamine 123, which responds specifically to proton motive force. The use of these stains allowed the microscopic discrimination of physiologically active bacteria as well as heterogeneities of active cells within thicker biofilms. The results of experiments using these techniques with pure culture and binary population biofilms on stainless steel coupons indicated biocidal activity of chlorine-based disinfectants occurred initially at the bulk-fluid interface of the communities and progressed toward the substratum. This approach provided a unique opportunity to describe the spatial response of bacteria within biofilms to antimicrobial agents and address mechanisms explaining their comparative resistance to disinfection in a way that has not been possible using traditional approaches. Results obtained using this alternative approach were also consistently higher than PC data following disinfection. These observations suggest that traditional methods involving biofilm removal and bacterial enumeration by colony formation overestimate biocide efficacy. Hence the alternative approach described here more accurately indicates the ability of bacteria surviving disinfection to recover and grow as well as demonstrate spatial heterogeneities in cellular physiological activities within biofilms.
Rapid direct methods for enumeration of specific, active bacteria in water and biofilms
(1998-12) McFeters, Gordon A.; Pyle, Barry H.; Lisle, John T.; Broadaway, Susan C.
Conventional methods for detecting indicator and pathogenic bacteria in water may underestimate the actual population due to sublethal environmental injury, inability of the target bacteria to take up nutrients and other physiological factors which reduce bacterial culturability. Rapid and direct methods are needed to more accurately detect and enumerate active bacteria. Such a methodological advance would provide greater sensitivity in assessing the microbiological safety of water and food. The principle goal of this presentation is to describe novel approaches we have formulated for the rapid and simultaneous detection of bacteria plus the determination of their physiological activity in water and other environmental samples. The present version of our method involves the concentration of organisms by membrane filtration or immunomagnetic separation and combines an intracellular fluorochrome (CTC) for assessment of respiratory activity plus fluorescent-labelled antibody detection of specific bacteria. This approach has also been successfully used to demonstrate spatial and temporal heterogeneities of physiological activities in biofilms when coupled with cryosectioning. Candidate physiological stains include those capable of determining respiratory activity, membrane potential, membrane integrity, growth rate and cellular enzymatic activities. Results obtained thus far indicate that immunomagnetic separation can provide a high degree of sensitivity in the recovery of seeded target bacteria (Escherichia coli O157:H7) in water and hamburger. The captured and stained target bacteria are then enumerated by either conventional fluorescence microscopy or ChemScank, a new instrument that is very sensitive and rapid. The ChemScanR laser scanning instrument (Chemunex, Paris, France) provides the detection of individual fluorescently labelled bacterial cells using three emission channels in less than 5 min. A high degree of correlation has been demonstrated between results obtained with the ChemScan and traditional plate counts of mixed natural bacterial populations in water. The continuing evolution of these methods will be valuable in the rapid and accurate analysis of environmental samples.
Specific and rapid enumeration of viable but non-culturable and viable-culturable gram-negative bacteria using flow cytometry
(2010-06) Khan, Mohiuddin M. T.; Pyle, Barry H.; Camper, Anne K.
An issue of critical concern in microbiology is the ability to detect viable but non-culturable (VBNC) and viable-culturable (VC) cells by methods other than existing approaches. Culture methods are selective and underestimate the real population and other options (direct viable count and double-staining method using epifluorescence microscopy and inhibitory substance influenced molecular methods) are also biased and time consuming. A rapid approach that reduces selectivity, decreases bias from sample storage and incubation, and reduces assay time is needed. Flow cytometry is a sensitive analytical technique that can rapidly monitor physiological states of bacteria. This report outlines a method to optimize staining protocols and the flow cytometer (FCM) instrument settings for the enumeration of VBNC and VC bacterial cells within 70 min. Experiments were performed using the FCM to quantify VBNC and VC Escherichia coli O157:H7, Pseudomonas aeruginosa, Pseudomonas syringae, and Salmonella typhimurium after staining with different fluorescent probes: SYTO 9, SYTO 13, SYTO 17, SYTO 40 and propidium iodide (PI). The FCM data were compared with specific standard nutrient agar to enumerate the number of cells in different states. By comparing results from cultures at late log phase, 1 to 64% of cells were non-culturable, 40 to 98% were culturable, and 0.7 to 4.5% had damaged cell-membranes and were therefore theoretically dead. Data obtained using four different gram-negative bacteria exposed to heat and stained with PI also illustrates the usefulness of the approach for the rapid and unbiased detection of dead vs. live organisms.