ANTIBIOTIC RESISTANT PATHOGENS     A PRIMER FOR PLANNERS AND POLICY MAKERS SEWAGE SLUDGE (A.K.A. BIOSOLIDS) AND RECLAIMED  SEWAGE WATER By Dr. Edward McGowan Based on wastewater industry dogma and standards, released effluent, its use in irrigation salad crops, and the land application of sewage sludge are benign and beneficial activities. If however, one reviews the current medical and scientific literature, a different picture emerges, one that raises serious questions about the benevolence of this activity and efficacy of the underlying standards. Thus, the issue takes on aspects of a political and not a scientific argument. It is generally believed that the overuse of antibiotics has driven antibiotic resistance. Serious antibiotic resistant pathogens are no longer confined to the very ill within the confines of hospitals; they are now commonly found within the community. Some have gained sufficient resistance to make them invincible to all currently available antibiotics DRAFT FOR REVIEW PURPOSES ONLY Author contact: Dr. Edo McGowan <edo_mcgowan@hotmail.com> ABSTRACT This brief is designed as a primer for planners and policy makers not familiar with sewage and disease. The paper presents an argument that accelerating risks from both antimicrobial resistance and pandemic, especially as now found emerging in the world community, may be related to the disposal of inadequately treated sewage. That sewage may be in different forms, as solids now termed by EPA as biosolids, or as irrigation water used on vegetable crops. On most continents, the practice of land applied sewer sludge has gained unprecedented acceptance based on the need to rapidly rid ourselves of our waste. Recent papers have noted the mixing of genetic material between various organisms provides for new types of pathogens and our immune systems may thus be faced with an unknown foe. Interestingly, much of this mixing goes on every day in sewage treatment plants in almost every city. Millions of gallons of effluent and tons of poorly treated sewage wastes are discharged to the environment and the solids, now termed biosolids are spread across thousands of square miles of farmland. There, this material is open to background organisms, thus allowing the intermixing with numerous species at the micro and macro biological levels. It is generally believed that the overuse of antibiotics has driven antibiotic resistance. Serious antibiotic resistant pathogens are no longer confined to the very ill within the confines of hospitals; they are now commonly found within the community. Some have gained sufficient resistance to make them invincible to all currently available antibiotics. While over-use of antibiotics may play an important role in the advancement of resistance, other causes of resistance to antibiotics are overlooked. A critical but less well understood mechanism for the transfer resistance, thus amplification of multi-drug resistant pathogens, is found at the local sewer-treatment plant [1]. As bacteria, other pathogens, and common background-organisms wind their way through municipal sewage treatment processes there is the intermixing of vast quantities of organisms that might otherwise never come together. In the process of sewage treatment, the selective pressures against these organisms is increased. In consequence, there is a greater effort by these organisms to up-regulate or acquire numerous other survival mechanisms to assure that they and their genetic material persist to pass on genetic information. Part of this defense strategy is to go dormant---shut down metabolic processes, and enter a viable but non-culturable (VBNC) state. In this VBNC state these organisms are essentially invisible to standard laboratory tests. Additionally, as the environmental crowding and stresses increase, these organisms can acquire and then pass on to other non-related organisms the acquired antibiotic resistance, virulence, as well as resistance to heavy metals and chlorine. In many cases, these changes to the cellular and metabolic machinery afford the ability to deal with numerous insults, hence development of cross- resistance mechanisms. Many antimicrobials or their metabolites pass through the body essentially unchanged. Thus feces and urine do contain some impressive levels. As later noted, Kümmerer and others (1999, 2000, 2003, 2004) [2], have followed this and have noted levels of antibiotics in sewage that are able to induce or maintain resistance. Added to this are the other materials dumped into the toilet or down the drain that confer resistance. This includes discarded antibiotics and disinfectants such as Triclosan [3] a ubiquitous biocide has been suspected of inducing resistance. Based on wastewater industry dogma and standards, released effluent, its use in irrigation salad crops, and the land application of sewage sludge are benign and beneficial activities. If however, one reviews the current medical and scientific literature, a different picture emerges, one that raises serious questions about the benevolence of this activity and efficacy of the underlying standards. Thus, the issue takes on aspects of a political and not a scientific argument. In the interim, most regulatory agencies have backed off []. Recent court documents also tend to implicate that that high-level officials within EPA may have conspired with sludge industry scientists to falsify data and thus to deliberately obscure the adverse health impacts from land applied sludge  (see: Qui Tam Complaint via http://www.sludgefacts.org/). This leaves the citizens and patient base standing naked. In one of several major studies looking at sewage treatment plants, the scientists followed bacteria through a sewer treatment works using fecal coliforms as the test organism [4]. Coliform bacteria were isolated at various locations in the plant, specifically a) the inlet, b) the primary sedimentation tank, c) the activated sludge digestion tank, d) the final settling tank, e) the outlet and f) the return activated sludge drain. They were then examined the presence of drug resistant plasmids. Using this approach, resistant bacteria and those that were still sensitive to antibiotics were detected [5]. Several drugs were tested and included tetracycline, kanamycin, chloramphenicol and streptomycin, ampicillin, nalidixic acid, rifampicin, and sulfisoxazole. A total of 900 separate tests were conducted, of which more than half contained multi-drug resistant plasmids. While this is interesting, there was a new finding that raised considerable concern. The further along that the wastewater had progressed through the treatment process, the greater the tendency was to encounter strains that had developed multiresistance to antibiotics. Additionally, the study demonstrated that these multi- resistant bacteria also simultaneously carried, and then passed around their multiple transferable drug-resistance plasmids. Thus, the development of drug resistance and the transfer of multi-drug resistance are enhanced in sewage wastewater treatment plants [5]. These findings have been documented for more than a decade. They were a harbinger, yet little impact from such studies has been noted. Under the current practices, sewage treatment practices allow the survival of up to 2- million viable coliform per gram of sludge at the point of land application to farmlands. The new WERF paper by Higgins and Murthy (2006) now raises some serious questions about the efficacy of current standards. These authors noted that in the centrifuge dewatering of sewer sludge the indicators were in a VBNC state and thus centrifuging resuscitated them. The numbers were several magnitudes greater that standard plate count would have indicated. [6]. This finding by Higgins and Murthy should raise some logical questions. For example, if the dewatering by centrifuge brought out the essence of VBNC, then were those sludge lots not subjected to the centrifuge also in the VBNC state and thus would revive in the field. That may explain why we see large and rapid regrowth of pathogens following land application. Further logical questions might be---what of the more robust pathogens—pathogens not easily killed by the low-level disinfection attainable within sewer currently operated plants. The use of low-level indicator bacteria, along with the apparent lack in understanding of antibiotic resistance within EPA (see FOIA search results at bottom of this file) should alert anyone that the issue is anything but closed. By its refusal to adequately present necessary analyses in this area, EPA has not only manufactured uncertainty, but also potentially increased the risk of human disease, disease from some serious pathogens that may not respond to current antibiotics. All Class-B sewage sludge technologies that are normally used in the U.S. such as anaerobic digestion and aerobic digestion and heating at these levels as well as composting and land stabilization do not effectively destroy critical pathogens [7]. These practices also do not destroy the genetic material and this and its lack of acknowledgement is a critical shortcoming within EPA. Thus if there is antibiotic resistance within sewer sludge, it may be passed through these processes to background organisms including man [8]. Actually several studies have documented the horizontal transfer of genetic information to background environmental systems and such systems can act as lending libraries for this genetic information. Man and animals are exposed daily to such backgrounds [9]. What are the chances for inadvertent acquisition of resistance from environmental contamination such as through sewage sludge? Gerba and Rusin [10] conducted research about the passage from finger to mouth of pathogens found on typical household objects. Others have documented dust as a mechanical vector for pathogens. Thus what of the dwellings and towns down wind from land application of sewer sludge or from a sewage sludge composting facility? There are now several workmen’s comp cases filed by staff of the Chino Women’s Prison for complaints accruing to dust from the adjacent and up-wind sewer sludge composting facility in San Bernardino County, California. Further, there are concerns about wash-off from rains and irrigation return flows. Gerba and others have written extensively about the survival of pathogens and their viable infectivity once they are adsorbed onto sediments [11]. Anyone who lives in an agricultural area knows that tillage and wind cause large movements of soil and dust that are equal to that found for water erosion. The USGS has written extensively on the movement of dust from Africa, across the Atlantic and carrying with it viable pathogens thus causing respiratory disease in the Caribbean [12]. The indicator organisms used for Class B biosolids commonly include Escherichia coli and sometimes Salmonella. These are the organisms that are normally killed by low-level disinfection. They are vegetative bacteria that are highly susceptible to both chemical disinfection and heat disinfection. However, sewage sludge contains a large range of organisms besides E. coli, Salmonella, and Staphylococcus. Also highly susceptible and easily inactivated are the enveloped viruses such as Hepatitis B., HIV, and influenza. While these organisms are fairly easily destroyed, Class-B allows 2 million viable coliform per gram of land applied sewer sludge. This raises the logical question of survival for the more robust organisms. These bacteria are thus able to colonize environmental niches, and animals, including humans, through ingestion. Once ingested, the plasmids may be transferred to normal flora, and subsequently to pathogenic bacteria found in humans or animals, making later treatment with particular antibiotics ineffective. Also one must consider transfer of genetic information from these organisms to more robust organisms as highlighted by Sjolund et al. (2005) [13] indicating that resistance in the normal flora, which may last up to four-years, might contribute to increased resistance in higher-grade pathogens through interspecies transfer. These authors go on to note that since populations of the normal biota are large, this affords the chance for multiple and different resistant variants to develop. This thus enhances the risk for spread to populations of pathogens. Furthermore, there is crossed resistance. For example, vancomycin resistance may be maintained by using macrolides [14]. Walsh (2003) [15] notes that resistance to antibiotics is not a matter of IF but one of WHEN. Schentag, et al. (2003), in Walsh, followed surgical patients with the subsequent results. Pre-op nasal cultures found Staphylococcus aureus 100% antibiotic susceptible. Pre-op prophylactic antibiotics were administered. Following surgery, cephalosporin was administered. Ninety percent of the patients went home at post-op day 2 without infectious complications. Nasal bacteria counts on these patients had dropped from 105 to 103, but were now a mix of sensitive, borderline, and resistant Staphylococcus sp. By comparison, prior to surgery, all of the patients’ Staphylococcus samples had been susceptible to antibiotics. For the patients remaining in the hospital and who were switched on post-op day 5 to a second generation cephalosporin (ceftazidine), showed bacterial counts up 1000-fold when assayed on post-op day 7 and most of these were methicillin resistant Staphylococcus aureus (MRSA).  These patients were switched to a 2-week course of vancomycin. Cultures from those remaining in the hospital on day 21, revealed vancomycin resistant enterococcus (VRE) and candida. Vancomycin resistant enterococci infections can produce mortality rates of between 42 and 81%. Note in the above, that these patients harbored NO resistant bacteria in their nasal cavities upon entry to the hospital. But what would be the result if there had been inadvertent acquisition of resistance from environmental contamination such as through sewage sludge? Gerba and Rusin [9] conducted research about the passage from finger to mouth of pathogens found on typical household objects. Others have documented dust as a mechanical vector for pathogens. Thus what of the dwelling down wind from land application of sewer sludge or from a sewage sludge composting facility? Gerba and others have written extensively about the survival of pathogens and their viable infectivity once they are absorbed onto sediments [10]. Anyone who lives in an agricultural area knows that tillage and wind cause large movements of soil and dust. The USGS has written extensively on the movement of dust from Africa, across the Atlantic and carrying with it viable pathogens thus causing respiratory disease in the Caribbean [11]. This then brings into question the current paradigm on infection and its dose response to a certain load of a particular pathogen, i.e., ID and LD 50s. Lateral transfer of mobile genetic elements conferring resistance is not considered in this old paradigm. With the prodigious capacity for the gut bacteria to multiply, once the lateral transfer has taken place, very small original numbers---well below the old paradigms can be multiplied into impressive numbers. Since viruses and phages are also involved, their capacity to multiply, which dwarfs that of bacteria, must also be included. Thus there is a need for a new paradigm; unfortunately, the regulatory community seems not to recognize this. When one considers the multiplication within sewer plants and also within their byproducts, disbursement into the environment, the transfer to background organisms, hence to man and his animals, then the remultiplication within commensals, the emerging picture is worrisome. Further, there are opportunities and interrelationships between microbes that can degrade antibiotics, eg. antibiotic resistant bacteria, and those that can degrade metals as well as pesticides and farm chemicals that are already found in agricultural soils. In many cases, the involved cellular machinery is the same or similar, i.e., a duality (see Schlüter and abstracts of others below). This duality may have some interesting synergistic survival advantages for the microbes, but bad- for-human-health effects when considering sewer sludge as applied to heavily farmed lands. The current standards controlling sewer plant operations, the land application of sewer sludge or the composting of sewer sludge for making compost and potting soils consider none of these issues. This paper therefore contends that this unconsidered avenue for the spread of antibiotic resistance and amplification of risk for a pandemic needs greater awareness within the medical and health care community. Perhaps this is an area worthy of further review by policy committies. Without the perspective of a broader analysis of this issue, future policy may be no more that the post hoc rationalization for a series of missed opportunities. It would seem reckless to proceed without a broader picture. Unfortunately, the principal regulatory body, U.S. EPA seems to be essentially oblivious to these concepts, yet it has been promoting the land application of sewer sludge. As seen below, based on a FOIA request, EPA seems less than knowledgeable in the area of antimicrobial resistance. +++++++++++++++++++++++++++ Citations and notes [1] Ribeiro-Dias JC, Vicente AC, Hofer E. Fecal coliforms in sewage waters. I. Resistance to antibiotics, heavy metals and colicinogeny. Appl Environ Microbiol 1983 Jul;46(1):227-32. Others, have found similar results. Mach PA, et al. R-plasmid transfer in a wastewater treatment plant. AEM 1982 Dec;44(6):1395-403. Fontaine TD et al. Transferable drug resistance associated with coliforms from hospital and domentic sewage. Health Lab Sci. 1976 Oct; 13(4): 238-45. Walter, M. V., and J. W. Vennes. 1985. Occurrence of multiple-antibiotic-resistant enteric bacteria in domestic sewage and oxidation lagoons. Appl. Environ. Microbiol. 50:930-933. Rhodes G, Huys G, Swings J, McGann P, Hiney M, Smith P, Pickup RW. Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A. Appl Environ Microbiol 2000 Sep;66 (9):3883-90. Grol A, Szymanska B, Wejner H, Kazanowski A, Wlodarczyk K. The role of mechanically purified city sewers in the spread of antibiotic-resistant bacteria of the Enterobacteriaceae family] Med Dosw Mikrobiol 1989;41(2):100-5. Lewis GD, et al. Enteroviruses of human origin and faecal coliforms in river water and sediments down stream from a sewage outfall in the Taieri River, Otago. New Zealand Journal of Marine and Freshwater Research, 1986, Vol.20: 101-105. Marcinek H, Wirth R, Muscholl-Silberhorn A, Gauer M. Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants. Appl Environ Microbiol 1998 Feb;64(2):626-32. [2] Kummerer K. Resistance in the environment. J Antimicrob Chemother. 2004 Aug;54(2):311-20. Epub 2004 Jun 23. Kummerer K. Promoting resistance by the emission of antibiotics from hospitals and households into effluent. Clin Microbiol Infect. 2003 Dec;9(12):1203-14. Kummerer K. Standardized tests fail to assess the effects of antibiotics on environmental bacteria. Water Res. 2004 Apr;38(8):2111-6. Kummerer K. Biodegradability of some antibiotics, elimination of the genotoxicity and affection of wastewater bacteria in a simple test. Chemosphere. 2000 Apr;40(7): 701-10. Kummerer K. Drugs, diagnostic agents and disinfectants in wastewater and water--a review. Schriftenr Ver Wasser Boden Lufthyg. 2000;105:59-71. Al-Ahmad A, Daschner FD, Kummerer K.   Biodegradability of cefotiam, ciprofloxacin, meropenem, penicillin G, and sulfamethoxazole and inhibition of waste water bacteria. Arch Environ Contam Toxicol. 1999 Aug;37(2):158-63. Rooklidge SJ. Environmental antimicrobial contamination from terraccumulation and diffuse pollution pathways. Sci Total Environ. 2004 Jun 5;325(1-3):1-13. Review. [3] Aiello AE, Marshall B, Levy SB, Della-Latta P, Lin SX, Larson E. Antibacterial cleaning products and drug resistance. Emerg Infect Dis. 2005 Oct;11(10):1565-70). [4] Ribeiro-Dias JC, Vicente AC, Hofer E. Fecal coliforms in sewage waters. I. Resistance to antibiotics, heavy metals and colicinogeny. Appl Environ Microbiol 1983 Jul;46(1):227-32. Others, have found similar results. Mach PA, et al. R-plasmid transfer in a wastewater treatment plant. AEM 1982 Dec;44(6):1395-403. Fontaine TD et al. Transferable drug resistance associated with coliforms from hospital and domentic sewage. Health Lab Sci. 1976 Oct; 13(4): 238-45. [5] Nakamura S, Shirota H. Behavior of drug resistant fecal coliforms and R plasmids in a wastewater treatment plant] Nippon Koshu Eisei Zasshi 1990 Feb;37(2):83-90. [6]  Reference--National Research Council of the National Academy of Sciences (NAS) Biosolids Applied to Land: Advancing Standards and Practices. Washington, DC: National Academy Press, 2002. See also: Examination of Reactivation and Regrowth of Fecal Coliforms in Anaerobically Digested Sludge WERF Report: Biosolids and Residuals (03-CTS-13T) Author(s): MJ Higgins, S Murthy. [7] This report incorporates portions of a personal conversation with Dr. David Lewis of the EPA on the processes used for preparation of sewer sludge. Lance JC et al. Virus movement in soil columns flooded with secondary sewage effluent. AEM Oct 1976 p. 520-26. Gerba CO. Poliovirus removal from primary and secondary sewage effluent by soil filtration. AEM Aug 1978 p. 247-51. Schaub SA et al, Virus and bacteria removed from wastewater by rapid infiltration through soil. Bacteriophage movement in ground water at distances of 600 feet from site of application. AEM 33:609-18. Ward RL et al. Inactivation of poliovirus in digested sludge. AEM 31:921-930. Digested sludge also protects poliovirus during heat treatment. Polio virus nucleic acid from heat ruptured capsuls will maintain infectivity. Breindl M. The structure of heated poliovirus particles. J. Jen Vir 11:147-156. Ward RL, et al. Minimum infective dose of animal viruses. Curt Rev Environ Control 14:278-310. Abbaszadegan M et al. Detection of of enteroviruses in groundwater with PCR. AEM May 1993 1318-24. [8] Rooklidge SJ. Environmental antimicrobal contamination from terraaccumulation and difuse pollution pathways. Sci Toatl Environ 2004 Jun 5;325(1-3):1-13. Golet EM et al. Determination of fluoroquinolone antimicrobial agents in sewage sludge and sludge treated soils using accelerated solvent extraction followed by solid phase extraction. Anal Chem. 2002 Nov 1;74(21):5455-62. Overall recovery ranged from 82 to 94% from sludge and 75 to 92% for soils. Golet EM, et al. Environmental exposure assessment of fluoroquinolone antibacterial agents from sewage to soil. Environ Sci Technol. 2003 Aug 1;37(15):3243-9. These results suggest sewage sludge as the main reservoir of FQ residues. [9] Ray JL, Nielsen KM. Experimental methods for assaying natural transformation and inferring horizontal gene transfer. Methods Enzymol. 2005;395:491-520. Occurrence and reservoirs of antibiotic resistance genes in the environment. Seveno, Nadine A. et al. Reviews in Medical Microbiology. Jan 2002, 13(1): 15-27. Hassen A., et al. Microbial characterization during composting of municipal solid waste. Bioresour Technol 2001 Dec;80(3):217-25. Ray JL, et al. Experimental methods for assaying natural transformation and inferring horizontal gene transfer. Methods Enzymol. 2005;395:491-520. Fontaine, T. D., III, and A. W. Hoadley. 1976. Transferrable drug resistance associated with coliforms isolated from hospital and domestic sewage. Health Lab. Sci. 4: 238-245.  Grabow, W. O. K., and O. W. Prozesky. 1973. Drug resistance of coliform bacteria in hospital and city sewage. Antimicrob. Agents Chemother. 3:175-180. Linton, K. B., M. H. Richmond, R. Bevan, and W. A. Gillespie. 1974. Antibiotic resistance and R factors in coliform bacilli isolated from hospital and domestic sewage. J. Med. Microbiol. 7:91-103. Walter, M. V., and J. W. Vennes. 1985. Occurrence of multiple-antibiotic-resistant enteric bacteria in domestic sewage and oxidation lagoons. Appl. Environ. Microbiol. 50:930-933.  Rhodes G, Huys G, Swings J, McGann P, Hiney M, Smith P, Pickup RW. Distribution of oxytetracycline resistance plasmids between aeromonads in hospital and aquaculture environments: implication of Tn1721 in dissemination of the tetracycline resistance determinant tet A. Appl Environ Microbiol 2000 Sep;66 (9):3883-90. Seveno NA. Occurrence and reservoirs of antibiotic resistance genes in the environment. Reviews in Medical Microbiology. 13(1):15-27, January 2002. Cooley MB. Colonization of Arabidopsis thaliana with Salmonella enterica and Enterohemorrhagic Escherichia coli O157:H7 and Competition by Enterobacter asburiae.  Applied and Environmental Microbiology, August 2003, p. 4915-4926, Vol. 69, No. 8. Marcinek H, Wirth R, Muscholl-Silberhorn A, Gauer M. Enterococcus faecalis gene transfer under natural conditions in municipal sewage water treatment plants. Appl Environ Microbiol 1998 Feb;64(2):626-32. Iversen A, Kuhn I, Franklin A, Mollby R. High prevalence of vancomycin-resistant enterococci in Swedish sewage. Appl Environ Microbiol 2002 Jun;68(6):2838-42.  Reinthaler FF, Posch J, Feierl G, Wust G, Haas D, Ruckenbauer G, Mascher F, Marth E. Antibiotic resistance of E. coli in sewage and sludge. Water Res 2003 Apr;37(8):1685-90. Cenci G, Morozzi G, Daniele R, Scazzocchio F. Antibiotic and metal resistance in "Escherichia coli" strains isolated from the environment and from patients. Ann Sclavo 1980 Mar-Apr;22(2):212-26.  Heberer T, Reddersen K, Mechlinski A. From municipal sewage to drinking water: fate and removal of pharmaceutical residues in the aquatic environment in urban areas. . Water Sci Technol 2002;46(3):81-8. [10]  Rusin P, et al. Comparative surface-to-hand and fingertip-to-mouth transfer efficiency of gram- positive bacteria, gram-negative bacteria, and phage. J Appl Microbiol. 2002;93(4):585-92; See also:  Shivi Selvaratnam and J. David Kunberger. Increased frequency of drug-resistant bacteria and fecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge. Can. J. Microbiol./Rev. can. microbiol. 50(8): 653-656 (2004). [11] Gerba CP et al. Effect of sediments on the survival of Ericherichia coli in marine waters. AEM July 1976 114-20. LaBelle RL, et al. Influence of pH, salinity and organic matter on the absorption of enterovirus to estuarine sediments. AEM July 1979 93-101---sediment can act as a reservoir for enterovirus. [12] Griffin DW. African desert dust in the Caribbean atmosphere: Microbiology and public health. Aerobiologia. 2001 Sept : Volume 17, Number 3, pp. 203 - 213 [13] Sjolund et al. (2005) Emerging Infectious Diseases (Vol. 11, # 9, Sept 2005 @ p. 1389 et seq), [14]  Giacometti A, Cirioni O, Kamysz W, Silvestri C, Licci A, D'Amato G, Nadolski P, Riva A, Lukasiak J, Scalise G.  In vitro activity and killing effect of uperin 3.6 against gram- positive cocci isolated from immunocompromised patients. Antimicrob Agents Chemother. 2005 Sep;49(9):3933-6. Robertson GT, Zhao J, Desai BV, Coleman WH, Nicas TI, Gilmour R, Grinius L, Morrison DA, Winkler ME. Vancomycin tolerance induced by erythromycin but not by loss of vncRS, vex3, or pep27 function in Streptococcus pneumoniae. J Bacteriol. 2002 Dec;184(24):6987-7000. ]. [15] Walsch, C. Antibiotics----, Actions, Origins, Resistance, (March 2003) New York: ASM Press. ++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++++ In Febuary 2005, my group had requested, via Freedom of Information Act, certain data from the U. S. EPA on their progress dealing with biosolids and resistance. In providing us answers to this request, EPA delayed its response for about 6 months and then merely directed us to a section of the NERL’s website, which contained no usable information. This site was (www.epa. gov/nerlesd1/chemistry/pharma/fq.htm#disposal), as evidenced by the following search results. Similar results were found for other EPA web addresses. It is now nearly  two years and EPA has yet to respond. Results of Searching the "Environmental Sciences" Area of EPA's Web Site No matches found for transposon; 1402 files searched No matches found for antibiotic resistance + biosolids; 1402 files searched. No matches found for antimicrobial resistance + biosolids; 1402 files searched No matches found for virulent pathogens + biosolids; 1402 files searched. No matches found for plasmids + biosolids; 1402 files searched. No matches found for mobile genetic elements; 1402 files searched. No matches found for high level disinfection + biosolids; 1402 files searched. Results of Searching EPA's Entire Web Site We have searched the entire EPA site and found the following results. You may also return to searching for the same terms within Environmental Sciences. No matches found for high level disinfection + biosolids; 494732 files searched. No matches found for plasmids + biosolids; 494732 files searched. No matches found for transposons + biosolids; 494732 files searched. No matches found for mobile genetic elements + biosolids; 494732 files searched. No matches found for virulent pathogens + biosolids; 494732 files searched. No matches found for antibiotic resistance + biosolids; 494732 files searched. No matches found for antimicrobial resistance + biosolids; 494732 files searched. Results of Searching the "Exposure Research" Area of EPA's Web Site We have searched the area of EPA's site related to Exposure Research and found the following results. You may also search for the same terms across EPA's entire site. No matches found for prions + biosolids; 3352 files searched. Results of Searching EPA's Entire Web Site We have searched the entire EPA site and found the following results. You may also return to searching for the same terms within Exposure Research. No matches found for prions + biosolids; 530969 files searched. +++++++++++++++++++++++++++= The 64 508 bp IncP-1 antibiotic multiresistance plasmid pB10 isolated from a waste-water treatment plant provides evidence for recombination between members of different branches of the IncP-1 group A. Schlüter, et al The complete 64 508 bp nucleotide sequence of the IncP-1 antibiotic-resistance plasmid pB10, which was isolated from a waste-water treatment plant in Germany and mediates resistance against the antimicrobial agents amoxicillin, streptomycin, sulfonamides and tetracycline and against mercury ions, was determined and analysed. A typical class 1 integron with completely conserved 5' and 3' segments is inserted between the tra and trb regions. The two mobile gene cassettes of this integron encode a -lactamase of the oxacillin-hydrolysing type (Oxa-2) and a gene product of unknown function (OrfE-like), respectively. The pB10-specific gene load present between the replication module (trfA1) and the origin of vegetative replication (oriV) is composed of four class II (Tn3 family) transposable elements: (i) a Tn501-like mercury-resistance (mer) transposon downstream of the trfA1 gene, (ii) a truncated derivative of the widespread streptomycin-resistance transposon Tn5393c, (iii) the insertion sequence element IS1071 and (iv) a Tn1721-like transposon that contains the tetracycline-resistance genes tetA and tetR. A very similar Tn501-like mer transposon is present in the same target site of the IncP-1 degradative plasmid pJP4 and the IncP-1 resistance plasmid R906, suggesting that pB10, R906 and pJP4 are derivatives of a common ancestor. Interestingly, large parts of the predicted pB10 restriction map, except for the tetracycline- resistance determinant, are identical to that of R906. It thus appears that plasmid pB10 acquired as many as five resistance genes via three transposons and one integron, which it may rapidly spread among bacterial populations given its high promiscuity…”. ++++++++++++++++++++++++++++++++++++++++ Risk Assessment Water Intelligence Online © IWA Publishing 2003 A Dynamic Model to Assess Microbial Health Risks Associated with Beneficial Uses of Biosolids - Phase 1 John M. Colford*, Jr, Don M. Eisenberg**, Joseph N.S. Eisenberg*, James Scott* and Jeffrey A. Soller** -------------------------------------------------------------------------------- ABSTRACT Maximum allowable levels for chemical contaminants in biosolids were developed for the Part 503 rule using risk-based methodologies. However, maximum allowable levels of microbiological contaminants in the Part 503 rule were based on specific treatment methodologies rather than risk levels, because it was determined at that time that risk assessment methodologies were not sufficiently developed. Given the current interest in the beneficial uses of biosolids and the projected rapid growth of biosolids reuse, there is increasing interest in the development of a microbial risk assessment methodology for regulatory and operational decision making. This document presents a methodology for assessing risks to human health from pathogens via exposure to biosolids. The methodology integrates two fundamental components: an exposure assessment component and a health risk component. The exposure assessment component is used to quantify pathogen levels in the environment and serves as input to the health effects component. The health effects component is used to quantify health risks using a model that explicitly accounts for properties unique to an infectious disease process, specifically secondary transmission and immunity. To demonstrate the applicability of these risk-based methods developed for biosolids exposure, numerical simulations were carried out for a case study example in which the route of exposure was direct consumption of biosolids-amended soil. [McGowan's comment interjected---what about respiratory or the interaction of contained chemicals on the inflammatory reaction, thus breaking barriers?] The output from the case study yielded a decision tree that differentiates between conditions in which the risk from biosolids exposure is high and those conditions in which the relative risk from biosolids is low. This decision tree illustrates the interaction among the important factors in quantifying risk. For the case study example, those factors include biosolids treatment processes, the pathogen shedding rate of infectious individuals, secondary transmission and immunity. Further work in determining biosolids exposures is required before this methodology can be used in a comprehensive risk assessment. McGowan's comment---EPA which controls he land-spreading of sewage sludge has never done a health risk assessment on pathogens. The interesting thing here is that in relying on technology, that technology has failed to consider the viable but non-culturable (VBNC) aspect as well as persisters and biofilms. Further, as noted by the recent paper by Higgins & Murthy, pathogens in the VBNC state are missed by standard lab analysis, hence potentially vastly understating actual risk. Their paper noted that using centrifuges to dewater sewage sludge rather than belt presses saw bacterial counts rocket within 20 minutes to several magnitudes above what the lab had just noted. Additionally, where is the potential for transfer of antibiotic resistance from a very small number to the gut bacteria and then its (the gut bacteria's) prodigious capacity to multiply that information? Thus their whole study---absent this aspect is badly flawed. Reliance on this study would then vastly underestimate the real risks. ---See: Maria Sjölund's paper below indicating a long-standing ability for these bacteria to remain in the gut . "resistant strain may persist for 4 years, in the absence of further antimicrobial treatment." Also, for example, Levy found that the resistance in gut bacteria of cattle moved to gut bacteria of mice having access to the same area, then from the mice to pigs, chickens, ++++++++++++++++++++++++++++ Risk Analysis Volume 24 Page 221 - February 2004 doi:10.1111/j.0272-4332.2004.00425.x Volume 24 Issue 1 A Dynamic Model to Assess Microbial Health Risks Associated with Beneficial Uses of Biosolids Joseph N. S. Eisenberg1* Jeffrey A. Soller2, James Scott1, Don M. Eisenberg2, and John M. Colford, Jr.1 There is increasing interest in the development of a microbial risk assessment methodology for regulatory and operational decision making. This document presents a methodology for assessing risks to human health from pathogen exposure using a population-based model that explicitly accounts for properties unique to an infectious disease process, specifically secondary transmission and immunity. To demonstrate the applicability of this risk-based method, numerical simulations were carried out for a case study example in which the route of exposure was direct consumption of biosolids-amended soil and the pathogen present in the soil was enterovirus. The output from the case study yielded a decision tree that differentiates between conditions in which the relative risk from biosolids exposure is high and those conditions in which the relative risk from biosolids is low. This decision tree illustrates the interaction among the important factors in quantifying risk. For the case study example, these factors include biosolids treatment processes, the pathogen shedding rate of infectious individuals, secondary transmission, and immunity. Further refinement in methods for determining biosolids exposures under field conditions would certainly increase the utility of these approaches. McGowan's comments on the Risk Analysis paper------ A brief read of this paper produced the following comments. Principal amongst my thoughts is the paper’s limit to pathogens that would not likely multiply outside the host---i.e., viruses. Thus, the model is quite limited from this important perspective. Secondly, there is no consideration of transfer of mobile genetic elements (MGEs) to terrestrial reservoirs, the potential for shifts in genetic information passing through multiple species, and thus the potential for newly emerging diseases. Consequently the issue of transferred antibiotic resistance and similar molecular and cellular machinery is missed. They also do not discuss colonization or later acquiring of resistance, the fecal veneer and thus movement into other organ systems or orifices. +++++++++++++++++++++++++ Hospital effluent: A source of multiple drug-resistant bacteria V. Chitnis, D. Chitnis*,†, S. Patil** and Ravi Kant* The present work was carried out to study the spread of multiple drug-resistant (MDR) bacteria from hospital effluent to the municipal sewage system. The MDR bacteria population in hospital effluents ranged from 0.58 to 40% for ten hospitals studied while it was less than 0.00002 to 0.025% for 11 sewage samples from the residential areas. Further, the MDR bacteria carried simultaneous resistance for most of the commonly used antibiotics and obviously the spread of such MDR bacteria to the community is a matter of grave concern. ++++++++++++++++++++++++++++++= Increased frequency of drug-resistant bacteria and fecal coliforms in an Indiana Creek adjacent to farmland amended with treated sludge Shivi Selvaratnam and J. David Kunberger Can. J. Microbiol./Rev. can. microbiol. 50(8): 653-656 (2004) Abstract: Many studies indicate the presence of human pathogens and drug-resistant bacteria in treated sewage sludge. Since one of the main methods of treated sewage disposal is by application to agricultural land, the presence of these organisms is of concern to human health. The goal of this study was to determine whether the frequency of drug-resistant and indicator bacteria in Sugar Creek, which is used for recreational purposes, was influenced by proximity to a farmland routinely amended with treated sludge (site E). Surface water from 3 sites along Sugar Creek (site E, 1 upstream site (site C) and 1 downstream site (site K)) were tested for the presence of ampicillin- resistant (AmpR) bacteria, fecal and total coliforms over a period of 40 d. Site E consistently had higher frequencies of AmpR bacteria and fecal coliforms compared with the other 2 sites. All of the tested AmpR isolates were resistant to at least 1 other antibiotic. However, no isolate was resistant to more than 4 classes of antimicrobials. These results suggest that surface runoff from the farmland is strongly correlated with higher incidence of AmpR and fecal coliforms at site E. Key words: drug-resistant bacteria, indicator bacteria, treated sludge, surface runoff. +++++++++++++++++++ Antibiotic resistance genes (ARGs) should be considered emerging environmental contaminants with more research devoted to the mechanisms by which they spread, scientists say in a report scheduled for the Dec. 1 issue of the semi-monthly ACS journal Environmental Science & Technology. Colorado State University's Amy Pruden and colleagues reached that conclusion after a study that documented occurrence of tetracycline and sulfonamide Antibiotic resistance genes in irrigation ditches, river sediments, and other spots in the environment in northern Colorado. They detected tetracycline resistance genes in treated drinking water, suggesting that it may be a pathway for spread of Antibiotic resistance genes to humans. Antibiotic resistance genes are pieces of DNA that make bacteria resistant to common antibiotics - recognized as an increasingly serious global health problem. The genes can spread in different ways. Bacteria, for instance, exchange Antibiotic resistance genes among themselves. Pruden and colleagues note that even if cells carrying ARGs have been killed, DNA released to the environment can persist and spread to other cells. "ARGs in and of themselves can be considered to be emerging 'contaminants' for which mitigation strategies are needed to prevent their widespread dissemination," they state. ++++++++++++++++ The Importance of Municipal Sewage Treatment in the Spread of Antibiotic Resistance 106th General Meeting of the American Society for Microbiology May 21-25, 2006, Orlando, Florida For more information on any presentation at the 106th General Meeting of the ASM contact Jim Sliwa, ASM Office of Communications at jsliwa@asmusa.org EMBARGOED UNTIL: Monday, May 22, 9:00 a.m. EDT (Session 041/Q, Paper Q-032) Sara Firl University of Minnesota Minneapolis, MN, United States Phone: 612 626 8865 firl0002@umn.edu Our study determined that substantial numbers of antibiotic-resistant bacteria were present in municipal wastewater, and that the existing treatment infrastructure did not adequately prevent release of antibiotic-resistant bacteria into the environment. Many of the bacteria found in the wastewater treatment plant and in the plant effluent were tentatively identified as potential pathogens and were also resistant to multiple antibiotics, raising public health concerns. We believe that wastewater treatment plants could be modified to further prevent the release of resistant bacteria to the environment. Sara Firl and Leslie Onan performed this study under the supervision of principal investigator Dr. Timothy LaPara at the University of Minnesota, Department of Civil Engineering. Funding was provided by the Center for Urban and Regional Affairs at the University of Minnesota and Geomatrix Consultants, Inc. The work is being presented as a poster at the 106th General Meeting of the American Society for Microbiology in Orlando on May 22. The spread of antibiotic-resistant bacteria is a major public health concern. Infections previously treatable are increasingly resistant to antibiotics. Scientists believe that the spread of antibiotic resistance results from both misuse of antibiotics and transfer of resistance between bacteria. A potentially large reservoir for antibiotic-resistant bacteria is municipal wastewater. People release resistant bacteria with fecal matter into the wastewater stream, which is collected and treated at municipal treatment facilities before release to the environment. The objective of this study was to investigate how many resistant bacteria were present at municipal wastewater plants and if the existing infrastructure of waste treatment was adequate to remove resistant bacteria before discharge. In our study, the effect of effluent treatment (clarification and disinfection) and biosolids treatment (sludge digestion) on the removal of antibiotic-resistant bacteria was investigated at three wastewater treatment facilities. We found substantial numbers of resistant bacteria at the wastewater treatment facilities and that, although effluent treatment reduced the numbers of bacteria, large quantities of resistant bacteria were discharged. Numerous bacteria isolated from the effluent stream were resistant to multiple antibiotics and closely related to potentially pathogenic bacteria. Our research suggests that the existing wastewater treatment infrastructure should be modified to better prevent release of these potentially dangerous bacteria to the environment.