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E. coli diversity in livestock manures


Cook, K.L., Bolster, C.H., Ayers, K.A., Reynolds, D.N. 2011. Escherichia coli diversity in livestock manures and agriculturally impacted stream waters. Current Microbiology. 63(5):439–449.


Escherichia coli (E. coli) is a dominant intestinal commensal organism, an important fecal indicator bacterium (FIB), a pathogen and a target for microbial source tracking (MST). Strain level differences (genotypic and phenotypic) influence E. coli fate and transport and therefore have important implications for its validity as an FIB and for MST. The goals of this study were to (1) determine the diversity of E. coli in manures from livestock and stream-water samples taken following dry and wet weather events; (2) determine the profile of virulence-associated genes and; (3) evaluate the effect of strain level differences on the attachment and transport of E. coli. To evaluate diversity, 1346 E. coli isolates were obtained from three livestock species and seventeen stream-water samples. We found that many E. coli strains isolated from water sources had DNA fingerprints that were significantly different than those from stream-water in a predominantly agricultural area. Furthemore, significant differences were also seen between E. coli isolates from stream-water samples taken following wet and dry weather events. Wide diversity in the attachment efficiency of E. coli isolates from different sources occurred and those differences corresponded with the occurrence of virulence factors often correlated with adhesion. These findings underscore the genetic variation inherent to this important indicator organism. The influence of diversity on genetic exchange and the concomitant effect on the organisms’ fitness and adaptation to in situ environmental conditions require further investigation. The resultant issues for purposes of modeling, source tracking and risk assessment require careful consideration in future research studies.

Transport behaviors of E. coli

Bolster, C.H., Cook, K.L., Marcus, I.M., Haznedaroglu, B.Z., Walker, S.L. 2010. Correlating Transport Behavior with Cell Properties for Eight Porcine Escherichia coli Isolates. Environmental Science and Technology. 44(13):5008–5014.


Infiltration of fecal material into the subsurface can result in the contamination of ground water supplies by pathogenic microorganisms such as bacteria, viruses, and protozoa, thereby posing a threat to public health. To assess whether a ground water source is at risk for fecal contamination, agencies responsible for monitoring water supplies generally test for the presence of nonpathogenic as indicator organisms. One of the most commonly used indicator organisms in ground water systems is E coli. To improve our understanding of the mechanisms controlling E. coli movement in the environment, we conducted a set of transport studies to better understand the factors that control E. coli attachment to sediment surfaces. For quartz sand we found that E .coli attachment, and thus transport, was controlled by the surface charge of the cell. For Fe-coated sand a mild correlation between cell width and attachment was observed. These findings will help improve our understanding of the mechanisms controlling E .coli in the environment.

Broiler litter application and E. coli

Sistani, K.R., Torbert III, H.A., Way, T.R., Bolster, C.H., Pote, D.H., Warren, J.G. 2009. Broiler Litter Application Method and Runoff Timing Effects on Nutrient and Escherichia coli Losses from Tall Fescue Pasture. Journal of Environmental Quality. 38(3):1216-1223


Over two-thirds of the total U.S. Broiler chicken production is located in the southeastern United States, which is a major segment of the farm economy in the region. Poultry litter is generally surface applied to pastures and hay fields year-round to supply plant nutrients, particularly nitrogen (N) and phosphorus (P). Runoff from litter-applied land has the potential to transport nutrients and pathogenic microorganisms to nearby surface water. Proper land application of animal manure is critical to water quality in watersheds with significant livestock numbers. With regard to permanent pasture systems, inability to incorporate waste materials leads to increased nutrient concentration such as phosphorus, nitrogen, copper, and zinc and pathogenic microorganisms near the soil surface. Currently, broadcasting is a common method of litter application on soil in many parts of U.S. The objectives of this study were (i) to compare the effect of broiler litter application method on nutrient and E. coli losses in runoff from tall fescue pasture in the Appalachian Plateau, and (ii) to determine the impact of antecedent time (time between litter application and the first runoff event) on nutrient and E. coli losses. Inorganic N and E. coli concentrations in runoff were significantly greater from broadcast litter application than subsurface litter application, while fertilizer treatment had runoff with greater NH4-N but smaller NO3-N than litter application treatments. The loss of total phosphorus, NO3-N, and total suspended solids from broadcast litter application was 83.5%, 64%, and 68% greater than subsurface litter application, respectively. About 81% of the runoff total phosphorus concentration was in the form of dissolved reactive phosphorus for both litter-application methods.

Methods to reduce pathogen loads following application of broiler litter

Brooks, J.P., McLaughlin, M.R., Adeli, A., Miles, D.M. 2012. The effect of poultry manure application rate and AlCl3 treatment on bacterial fecal indicators in runoff. Journal of Water and Health. 10:619-628.


Land application of poultry litter is a beneficial method of using this fertilizer source. It can provide high levels of N and organic C. However, the protection of water sources following land application of any fertilizer is necessary since many of the US surface water supply has been compromised due to the presence of fecal bacteria like Escherichia coli (E. coli) or anoxic, a condition in which the presence of nutrient runoff can lead to conditions which kill fish. The purpose of this study was to determine a suitable fecal indicator for poultry litter runoff following large-scale rain events. A previous greenhouse study determined that there are other poultry-litter fecal indicators more suitable for runoff than traditional indicators such as E. coli; this study confirms those results and found that of all treatments receiving poultry litter, two indicators proved to be useful. Over a two year period, field plots were land applied with various combinations of poultry litter or inorganic fertilizers and rained upon with an artificial rain maker. The results suggested that Clostridium perfringens and staphylococci were more useful as an indicator of poultry litter horizontal movement than all others. This persisted for up to 30 days following land application of poultry litter. The presence of these two indicators may signal a change for regulators and environmental monitors when investigating poultry litter fecal pollution.

Risk assessment of pathogens in manure

Brooks, J.P., McLaughlin, M.R., Gerba, C.P., Pepper, I.L. 2012. Land application of manure and class B biosolids: an occupational and public quantitative microbial risk assessment. Journal of Environmental Quality. 41:2009-2023.


The land application of wastes, such as wastewater treatment plant biosolids and manures, has been practiced for years and serves as a method to dispose and treat these wastes. The presence of bacterial, viral, and parasitic pathogens in these wastes can further complicate issues and reduce public confidence in their disposal. Typically the concern and hence the regulations governing their land application and use have revolved around controlling nutrient runoff and public contact with these microbial pathogens. The recent foodborne outbreaks involving bacterial and viral pathogens has brought into question the use of these wastes and potential for public exposures. To properly assess the use of these wastes a quantitative microbial risk assessment was conducted comparing the use of manure and municipal biosolids using current pathogen data and simulating potential public exposures following a variety of exposures ranging from fresh food crop consumption to aerosol exposures. A comparison of the risks demonstrated that public health risks are relatively low during non-intentional exposures such as fresh food crop or aerosol exposures and fall below the USEPA recommended annual 1 per 10,000 risk of infection. Only exposures in which intentional consumption of soil contaminated with fecal matter yielded risks which were greater. Risks were far below occupational exposures for the most part and demonstrated that risks between the two types of wastes were similar. Infectious risks from biosolids were greater from viral pathogens, while bacterial pathogens presented the greatest risks from manure. This study demonstrated that given typical conditions, the risk of infection from land application and use of either of these types of wastes are acceptable given time between application and exposures.

Microorganisms in dairy wastewater

Dungan, R.S., Leytem, A.B. 2013. The characterization of microorganisms in dairy wastewater storage ponds. Journal of Environmental Quality. 42:1583-1588.


Idaho is the third largest dairy state in the United States. With over 500,000 milk cows, a vast quantity of solid and liquid manure is generated, much of which is land applied. Given the fact that cattle feces contain a variety of microorganisms, we undertook a study using a culture-independent approach (i.e. no cultivation) to characterize microbial communities in wastewaters from south-central Idaho. After the microbial DNA was extracted from the wastewaters, it was amplified and then a sequence library was created for identification of putative matches. In general, the dairy wastewaters contained a variety of microorganisms affiliated with the domains Archaea and Bacteria. However, a statistical analysis of the data revealed that an insufficient number of sequences were obtained to sufficiently characterize microbial diversity at the species level. Despite this limitation, the results from our study enhanced our understanding of microbial species and communities in dairy wastewaters more so then if culture-dependent techniques were utilized.

Bioaerosols and open-freestall dairy operations

Dungan, R.S. 2012. Use of a culture-independent approach to characterize aerosolized bacteria near an open-freestall dairy operation. Environment International. 41(2012):8-14.


Animal manures are known to harbor a variety of pathogens that can potentially be transmitted to humans in the form of airborne particles. In this study, air samples were collected downwind from a 10,000 cow open-freestall dairy and nearby fields being sprinkler irrigated with wastewater. Nucleic acids (DNA) were extracted from the air samples, then a highly conserved gene was sequenced for bacterial identification. Of the 191 gene sequences, none were affiliated with bacteria known to be pathogenic to healthy humans. Our results suggest that there is a low incidence of airborne bacterial pathogens immediately downwind from the dairy and wastewater irrigation sites.

Antibiotic Resistant Bacteria

Antibiotic resistant bacteria

               A major area of concern with the general public has focused on the potential for antibiotic resistant bacteria that reside in both animal manures and biosolids, due to the potential for subsequent transfer of the resistance to pathogens. Bacteria are prokaryotic organisms with the ability to metabolize and replicate very quickly.  They are also very adaptable genetically.  When confronted with an antibiotic, there need only be one bacterial cell with a genetic or mutational change that confers resistance to that antibiotic that subsequently allows for the proliferation of antibiotic resistant bacteria.  Thus the more that antibiotics are used, the greater the likelihood of antibiotic resistant strains developing.  The greatest concern with antibiotic resistance is the potential for human pathogenic strains to become resistant to overused antibiotics, which subsequently cannot contain the infectious agent.  As is typical in most niches, commensal bacteria tend to dominate the pathogenic bacteria at levels which are orders of magnitude greater than the pathogens.  This creates a haven for antibiotic resistance genes, which all have the potential to transfer to true or opportunistic pathogens.  The widespread, sometimes indiscriminant, use of antibiotics has raised the questions: i) “Can antibiotic resistant genes be transferred from nonpathogenic bacteria to human pathogenic strains in the environment?” ii) “Can antibiotic resistance in the environment, via residual land application, be transferred to the public?”

Brooks (2006) evaluated the incidence of antibiotic resistant bacteria (ARBs) in biosolids and a variety of other environmental samples and foodstuffs.  Table1.docx shows that Class B biosolids did not contain unusually high numbers of ARBs, and that in fact, the relative incidence was less than that found in pristine soil.  Interestingly, ARB concentrations were also lower than those found in common foodstuffs such as lettuce. Therefore food itself could be an important route of exposure to ARBs.  Rates of gene transfer in soil are thought to be a relatively infrequent event without selective pressure (Neilson et al., 1994), which reduces the risk of antibiotic resistant gene transfer to human pathogenic bacteria. Finally, note that soil itself is the original source of human antibiotics.

               Antibiotic use in the livestock and poultry industries has gradually increased over the past three decades in direct relation to the increasing number of CAFOs in operation.  Throughout this gradual cultural shift in livestock production, the need for antibiotics has increased as stocking densities and production cycles have increased.  The Union of Concerned Scientists predicted the number of antibiotics used in the industries at up to 50 million pounds annually (Chee-Sanford et al., 2009), with nearly half being used as a means to increase production.  The Animal Health Institute refutes this number stating that approximately 20.5 million pounds of antibiotic are used annually with approximately 1/10 of these used to increase production (Chee-Sanford et al., 2009).  These discrepancies highlight how little is known regarding this topic, and how contentious these issues truly are, particularly with news-cycles reporting increasing antibiotic resistance in our food supply or higher incidences of nosocomial infections.  Regardless, livestock industries account for a large amount of antibiotic use in the United States.  Antibiotics are used: 1) to treat infections and to prevent diseases; and 2) as a prophylactic, thus increasing production.  It is with the latter, that most concern or blame is placed. 

               In either case, as opposed to human antibiotic use, treating livestock with antibiotics is conducted in a manner that promotes the treatment of non-diseased animals.  Typically, CAFO animals are not individually treated for a disease.  If there is an outbreak of a disease-causing pathogen, farm managers typically react by not treating just the diseased individuals (perhaps only 100 of 20,000), but by treating the entire flock or herd.  This increases the likelihood for antibiotic resistance, as resistance genes can be promoted in healthy as well as diseased members of the host population.

               Brooks and McLaughlin (2009a) and Brooks et al. (2010) described the presence of antibiotic resistant bacteria in swine and poultry CAFOs.  The presence of antibiotic resistant bacteria in swine CAFOs appeared to be influenced by the type of management employed by the producer, specifically; the presence of younger piglets increased the amount of resistance in commensal E. coli.  In general, younger piglets led to resistance to an extra class of antibiotics (Brooks and McLaughlin, 2009a).  In some instances, regulatory and media pressures have forced industries to reduce antibiotic use, as has been noted in the poultry industry.  Brooks et al. (2010) noted the overall lack of antibiotic resistance in poultry CAFO manure, and an overall decrease among staphylococci, enterococci, and E. coli when compared to previous studies (Brooks et al., 2009a).    

                 Ultimately, the concern is for the potential movement of antibiotic resistant bacteria and genes from the “farm to the plate”.  Movement from the farm to the product and ultimately the consumer remains a poorly understood area (Marshall et al., 2011).  Three potential routes exist for the transfer to occur: 1) via consumption of undercooked food; 2) clonal spread from the occupationally exposed; 3) or from indirect manure contamination onto fresh food crops (e.g. environmental spread).  Sufficient evidence exists to support clonal spread from the occupationally exposed (Marshall et al., 2011), while the other two routes are poorly understood.  Contamination of fresh food crops either via runoff, land application of manure/biosolids, or feral animal has been hypothesized as a potential sources of contamination (Brooks et al., 2012a).  Antibiotic resistance phenotypes have been demonstrated to move via aerosols or runoff, though in very small amounts and over small distances from the CAFO (Brooks et al., 2009b, 2012b; Chinivasagam et al., 2009).  Brooks et al. (2009b) demonstrated that runoff from plots receiving litter was more concentrated with antibiotic resistant enterococci, which was characteristic of the litter and thus demonstrated that antibiotic resistant bacteria will transport as readily as any other bacteria.

Modified from Environmental Microbiology, 3rd Ed, Ed. I.L. Pepper, C.P. Gerba, and T.J. Gentry.  Chapter 26 “Land application of organic residuals: municipal biosolids and animal manures” by J.P. Brooks, C.P. Gerba, and I.L. Pepper.  

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