Multi-drug resistant bacteria (MDRB) and --- Sludge— By:  Dr. Edward McGowan Those most affected are the immunocompromised, elderly, and those with barrier disruptions to the skin or mucosal membranes. In the last case it may be merely from beach sand scratching the skin at the waistline of bathing suits or under wet suites, or swallowing contaminated water. This was produced somewhere between 2002 and 2003 To: Scottish Executive Environment Group Multi-drug resistant bacteria (MDRB) and --- Sludge—The Scottish Executive consulting paper on proposals to amend the statutory controls and the UK Code of Practice for the agricultural use of sewage sludge I am a sub-group member of a US counterpart to your task group studying the agricultural land application of sewer biosolids—, i.e., treated sewer sludge. I am appointed to this group because of my background: primary degree in agricultural and environmental sciences from UC Davis, Ph.D. in water quality and a degree in medicine. Accordingly, I wish to comment for the record. Although, I will be speaking from the American experience, my main concern is more universal. The subject deals with pathogens and, within that macrocosm of inquiry, the more important issue is one of multi-drug resistant bacteria (MDRB). There is a distinct lack of recognition for issues relating to multi-drug resistant bacteria in land applied sewer sludge as well as biosolids. The former term relates to essentially untreated sludge, the latter, i.e., biosolids—is a  term developed by EPA under the guidance of a PR firm. Sewage sludge (biosolids) is defined by two separate levels of treatment— Class B and Class A, with the second to contain lower levels of detectable pathogens. The recent report by the US National Academy of Sciences has not only called into question the U.S. EPA Part 503 guidelines for land application of sewage sludge (biosolids) but also more specifically indicated a need for the U.S. EPA to consider MDRB. In a recent meeting of our task-group, one of the members, a well respected wastewater engineer, raised the question relating to land application of composted biosolids. Composting raises the level of manipulation of biosolids to that of a manufactured product, often incorporating green-waste, i.e., trimmings from various forms of vegetation. The essence of the question related to the survival of pathogens, hence the underlying issue of surviving MDRB. The question went something like this---"If Staphyloccus aureus were found dead, did that mean that the problem was solved?" The corollary--- was it dead or merely in the viable but non-culturable (VNC) state? Was it a classic persister? (see below). Further, this says nothing for uptake of released naked DNA. Additionally, during the above noted meeting, I had mentioned that there is now strong medical evidence that about ½ of the non-hospital but community acquired skin infections in the Greater Los Angeles area are now MRSA. MRSA stands for methicillin resistant Staphylococcus aureus. Multi antibiotic-resistance happens because mobile genetic elements can be shifted between microbes. These microbes can be in different genera and even in different kingdoms and still make these shifts. Small lengths of DNA, called transposons can carry a variety of antibiotic resistance or virulence genes, are able to move from the bacterial chromosome to plasmid or to a virus DNA (think phage) which can then be freely exchanged between bacteria. Harmless gut and soil bacteria have become reservoirs for multi resistance plasmids which may be gained from pathogens or where there are other commensals that contained the shared genetic information. This means that the normal flora can contain this information and thus later pass it to pathogens. 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, and flies. He notes a Dutch study that followed bacteria from animals to the human food chain and entered the consumer’s kitchen. In other cited examples, he noted the distinct relationship between MDRB in animals and thence to humans attending them, even though the humans used no antibiotics or ate the animals. Levy’s work is not new. (Levy SB, MD. The Antibiotic Paradox. New York, Plenum Press 1997). Rusin and Gerba have written on the transfer of pathogens from common household surfaces via finger to mouth. Gerba in unpublished work has noted the ease in which contamination can be spread within a home. Others have discussed dust as a carrier of viable pathogens. Gerba has written extensively on the movement of pathogens in sediment, their protection for long periods within sediments and their re-transport as viable pathogens. The NRC in its 2002 report admonished EPA to look at off-site movement and resistance. There is no evidence that this re-analysis has taken place, yet the World Health Organization has raised the subject of resistance to a Global crisis. Thus the current U.S. EPA Class B biosolids with its allowed fecal coliform counts of 2 X10/6 per gram may actually constitute a large aliquot when containing MDRB and applied to areas with soil and water movement and animal or vector access. These bacteria are thus able to colonize animals, including humans, through ingestion. There are indications within the literature of E. coli O157:H7 being to travel up the vascular system in lettuce and celery thus obviating attempts to wash surfaces. Since these vegetables are eaten raw, the risk should be clear to most readers. Once ingested, the shiga containing plasmids may be transferable to normal flora, thence later to pathogenic bacteria found in humans or animals, making later treatment with particular antibiotics ineffective. Additionally, one finds that there is a remultiplication of bacterial numbers within standing sludge, biosolids or compost (see Hassen below). Thus, the current Part 503 limits on biosolid marker organisms may have little bearing on the ultimate numbers. During composting, the mesophiles (these function at normal body temperatures) can transfer genetic information to thermophiles (these operate above the lethal fever temperatures). The archaea, which are extreme thermophiles (these can take temperatures above the boiling point of water), are recognized as a separate third domain of life together with the bacteria and eucarya. Transfer of plasmids to bacteria from archaea, has been demonstrated. Thus, in theory, it may be possible to develop a MDRB that can survive temperatures found within composting. Furthermore, there is experimental evidence that even when disrupted by radiation, these ancient organisms can reassemble. This, from a theoretical perspective, then raises questions of the eventual failure of pasteurization. Hassen, et al found that , gram-positive bacteria, especially micrococcus, spores of bacilli, and fungal propagules survived, and reached high concentrations in compost. Not only that, "the appearance of gram- negative rods (opportunistic pathogens) during the cooling phase may represent a serious risk for the sanitary quality of the finished product intended for agronomic reuse." (Bioresour Technol 2001 Dec;80(3):217-25) Methicillin was an anantibiotic developed in the late 1950s which was effective in killing Staphylococcus aureus. The organism by that time had already become resistant to penicillin G and to almost every other available antibiotic. In the case of strain MRSA 16, only one effective antibiotic, vancomycin, remains; it has serious side effects on humans and its armor has been recently pierced. There is now vancomycin resistant S. a. This resistance was acquired from genetic exchanges between enterococci and Staph. Staphylococcus aureus is easily transferred from the skin of one patient to another and it can be fatal if it enters the bloodstream, or the site of a surgical operation. Resistant strains are controlled with great difficulty. Because of this, hospitals maintain stocks of vancomycin which had, until lately, been considered as the 'drug of last resort'. The effective treatment may now be amputation. Enterococci are ubiquitous in sludge and wastewater. One strain of Enterococcus is totally resistant to all antibiotics—including vancomycin. Enterococci are also not easily killed by sewer processing (see below). Although enterococci have historically had a relatively low virulence, the situation has changed. Transfer of resistance genes to Staphylococcus aureus from Enterococcus had been demonstrated in the laboratory and so it was only a matter of time before this was to be seen in nature. In 1997, a strain of MRSA resistant even to vancomycin was reported from Japan. This was shortly followed in Belgium. Two months ago, it was found on the East Coast of the U.S. Three vancomycin-intermediate Staphylococcus aureus (VISA) and four hetero-VISA strains were detected. They emerged from strains that belonged to locally endemic methicillin-resistant S. aureus (MRSA) genotypes. This is a very worrisome development. It was once thought that over-prescribing of antibiotics was the main route for development of multi-drug resistant bacteria (MDRB). It is important to remember that only about one half of all antibiotic use is through the health care system. The remainder of the use is found in industrial or agricultural settings---settings with considerably fewer controls. The disposal of waste streams, whether from agriculture, industry, or urban environments may ultimately flow through sewer treatment works. This paper argues that wastewater treatment plants may now constitute the principal and expanding source of MDRB. Contrary to popular myth, many pathogens survive their passage through a sewer treatment plant thus, remaining to constitute an increased public health risk. That this situation has continued for some time may be attributed, in part, to economics and the antiquated water quality standards. Nonetheless, readily available scientific and medical literature are, and have been for some time, replete with data demonstrating and confirming this fact. Studies reported in the scientific and medical literature date back to at least 1970 showing failure of treatment. The reader is encouraged to review the results of the recent project entitled Occurrence and Fate of Antibiotic Resistant Bacteria in Sewage. The project was conducted at the Department of Veterinary Microbiology of the Royal Veterinary and Agricultural University, which was supported by the Danish Environmental Protection Agency. Again, it should be mentioned that the European governments, as compared to the U.S., are the insurance carriers for public health. The issue at hand is multi-facetted. There is the continued development of chlorine resistance in bacteria and this is synergistic with the development of multi-drug-resistance in bacteria. Heavy metals often associated with waste streams do augment multi-drug resistance and perhaps bacterial virulence. Additionally, there are current indications of increasing beach closures from high bacteria counts. This last issue raises some questions that must be answered. Further, as noted above, recent findings from the National Academy of Science raise questions about the U.S. EPA’s Part 503 regulation of land applied biosolids. The conclusion appears to be that land disposal of biosolids, may have been carried out under questionable science. The issue is not resolved and considering the worrisome acceleration of MDRB, the situation warrants further review. In a review of the literature, one notes that as far back as 1930s there were credible papers discussing not only the survival of pathogens, but the length of that survival. In one of the more recent studies, it was demonstrated that enteric viral pathogens were able to remain viable in estuarian mud for 13 years. Further, there are numerous reports of disease outbreaks from marine shellfish contaminated with viral pathogens. Yet, those shellfish had passed health tests using current health standards for water quality. Thus after their release pathogenic organisms and viruses do constitute a long standing increased risk to not only man but the environment. Additionally, one example of a more recent scientific curiosity is the resuscitation of the recently found 250 million-year-old bacteria. It is apparently well and comfortable in its modern laboratory environment. With the advent of molecular biology and the subsequent advancements from genetic engineering, we are beginning to understand that these pathogens are "smart" and have numerous survival mechanisms available to them. After all, they have been here for billions of years and in that time, have developed wide based mechanisms for surviving many difficult situations. They not only continue today, but thrive. In the last two or three decades, the topic of drug resistant bacteria has moved into increasing prominence in both scientific and lay media. Antibiotic resistance appeared shortly after the development of antibiotics in the late 1930s and early 1940s. However, the seriousness of this issue, as relates to public health and water quality through multi-drug resistance, is rapidly increasing. This accrues to the escalation of antibiotic use as well as the formation and the transmission of antibiotic-resistant strains. Antibiotics act by inhibiting either protein synthesis, cell wall construction, or DNA replication in bacteria (Pharmaceutical Information Associated, Ltd., 1994). A pathogen becomes resistant to an antibiotic used to treat it either through mutation or through the acquisition of a plasmid for resistance from another strain of bacteria (Neu, 1992). Resistant pathogens render an antibiotic ineffective by either destroying or modifying the antibiotic or preventing the antibiotic from recognizing or accessing its target. We are now talking about multi-drug resistant bacteria, and on top of that, what to do. To highlight the veracity of this, the following abstract from a recent article published in SCIENCE is offered. "Pathogenic enterococci are becoming resistant to currently available antibiotics, including vancomycin, the drug of last resort for Gram-positive infections. Enterococci pose a significant public health threat, not least because of the risk of transferring vancomycin resistance to the ubiquitous Staphylococcus aureus." (this is now history) To accomplish this genetic transfer, bacteria have evolved small transferable genetic information packets called plasmids. To provide some analogy, think of copying a piece of software from a friend’s computer and inserting it into your operating system. These bacterial plasmids facilitate rapid and very broad dissemination of the drug resistant genetic information amongst mixed bacteria. This information sharing and transfer is able to cross species as well as genus barriers (DeFlaun & Levy 1989). Thus, resistant enterococci selected in one environment can pass resistance genes not only to other members of their own genus and species but also to other organisms in other genera. This information can be shared with the cells of plant root hairs. Interestingly, genetically manipulated plants often contain bacterial resistance genetic information. Insects feeding on these plants have been found to pick up this resistance in their gut bacteria. In the case of honey bees, it is then not unreasonable to look for wideral lateral transfer into the human food chain. The consequence may be transfer to human gut flora. Staphylococci share their plasmids with Listeria; E. coli can share genes with other members of the Enterobacteriaceae as well as the pseudomonads and Neisseria, just to mention a few. In fact, the same tetracycline resistance determinants can be found among Gram-positive and Gram-negative bacteria as well as in the mycobacterium (Roberts 1997). The genetic flexibility and versatility of bacteria have therefore contributed largely to the efficiency by which antibiotic resistance has spread among bacteria and among environments globally. Resistance genes reside not only in disease-causing organisms, but in the common and previously non- pathogenic organisms as well. These formerly harmless bacteria, such as E. coli or enterococcus, can now cause a fatal illness in the young, old and in the immunocompromised. Moreover, these bacteria harbor resistance genes which can spread to the bacterial strains that do cause infection. These latter bacteria then constitute a "lending library". Unfortunately, these reservoirs are not being examined by those often charged with protecting the public health and welfare. Another and less well understood mechanism for the transfer of multi-drug resistant bacteria is found at the local sewer treatment plant. As bacteria wind their way through these treatment processes, the selective pressures against them increase. In consequence, there is a greater effort by bacteria to pass on survival enhancing genetic information. Additionally, as the environmental stresses increase, the bacteria up-regulate numerous other survival mechanisms to assure that they and their genetic material survive. These can include chlorine resistance. In one of the several major studies looking at this, the scientists followed bacteria through a sewer treatment works. Fecal coliforms were the test organism. These bacteria were isolated at various locations in the plant as the sewage was passing through the treatment process. They were isolated from: 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 for multi-drug antibiotic resistance. The study looked for the presence of drug resistant plasmids. The scientists were able to distinguish resistant bacteria from those still sensitive to antibiotics. Several drugs were tested and included tetracycline, kanamycin, chloramphenicol and streptomycin, ampicillin, nalidixic acid, rifampicin, and sulfisoxazole. We have seen above, the big gun— vancomycin is now in trouble. A total of 900 separate tests were conducted. Of these over 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 development of multiresistant strains. Additionally, the study demonstrated that these multi-resistant bacteria also simultaneously carried, and then passed around their multiple transferable drug-resistance plasmids. Thus, the take-home message is that drug resistance and the transfer of multi-drug resistant occurs in wastewater treatment plants. [Nippon Koshu Eisei Zasshi 1990 Feb;37(2):83-90.] This information is now over a decade old. These data were a harbinger, yet little impact from this study has been noted. Above, it was mentioned that the survival mechanisms were up-regulated as the stresses increased. As an example, enterococcus will be discussed. It is a common component of waste water, is highly resistant to treatment, is rapidly gaining notoriety as a new multi-drug resistant pathogen, and a large percentage of these organisms do survive treatment to be ultimately released into the environment. Enterococci, which have been known as a cause of infective endocarditis for close to a century, more recently have been recognized as a cause of nosocomial infection and "superinfection" in patients receiving antimicrobial agents. From an environmental perspective, enterococcus is fascinating. It can withstand severe conditions that will easily kill other enteric bacterial pathogens (note here we are NOT yet discussing viruses). In the lab, one sees this shift starting in about 4 hours. When one flushes the toilet, the saline environment of the normal body fluids is rapidly mixed with the fresh water environment. This abrupt shock and shift of environment in which enterococcus finds itself causes the bacteria to switch on protective survival mechanisms. Keep in mind that, in a town of 60,000, it takes about 2.5 hours for the city’s sewage effluent to enter the treatment plant where it stays exposed to that environment; then is ultimately sent to the ocean or down river to the next city’s intake. In large cities the time is considerably longer. When introduced into a fresh water environment enterococcus shifts to a survival mode and this mode renders it much more resistant to normal sanitary controls. The survival state is called "viable but nonculturable" (VNC), and this renders it essentially invisible to laboratory methods relying on plate count. This leads to false-negative results for purposes of water quality (or biosolid) monitoring by agencies relying on plate counts. Thus in these instances, there is the chance of missing highly resistant strains that then become major pathogens and which can, as we have seen, defy most antibiotics. In its VNC state, for example it can take extremely high saline waters, and in the lab can withstand a sodium chloride (NaCl) solution of 6.5%. It is unaffected by higher chlorine levels, up to 0.05% which is considerably higher than that needed to maintain residual fresh water sanitation, and also a pH down to 3.3. It is not readily killed by standard antibiotics and has developed resistant strains that defy vancomycin and other lesser materials. In the VNC mode it is insensitive to chloramphenicol. Additionally, it grows at 45 degrees C (115 F), which is well above the lethal fever temperature for man. It can survive 60degrees C (140 F), which is well above the required sludge cooking temperatures (98 F) used by many sewer plants for sludge digestion. Additionally, there are indications of developing chlorine resistance. In the VNC state, the bacteria when plated may actually die, thus giving a false impression of its status. This death is caused by a lack of internally generated antioxidants to overcome the peroxides and other reactive oxygen species. In animals, we see this as reperfusion injury. Thus in the VNC state enterococcus can withstand severe environmental insults, and survive when in bottom mud at the outfall or estuarian mud. Thus, it is able to survive for extended periods. This survival in estuarian mud may see it later flushed into the ocean during early rains. One would expect that the likelihood of survival for enterococcus to be accompanied similarly mechanisms in enteric viruses, some of which are extremely resistant to current sanitary controls. The viral particles also have very long survival rates in estuarian mud. Thus, it is possible that a "witches brew" flushes from the mud with the first rains. Such survival, then begs the issue of pathogen survival in sludge or biosolids applied to the land. This then warrants further critical analyses. Is there is reason to suspect that survival of pathogenic organisms in sewer effluent is different from survival in sludge? Thus in soil applied sludge or biosolids, if there is much topographic relief, these pathogens may move with the surface water or soil water to streams. Gerba notes in one of his papers, viral viability and survival at slightly more than 13 years---yes years. In America, under the Clean Water Act’s Phase II NPDES permits (U.S. controls and permitting requirements) for storm water and return flows, setbacks and slopes need further consideration. Stream set back limits may require reanalysis when considering MDRB. This may show to be of critical importance when compared to the former rational that did not consider the transfer of MDR genetic information to non-pathogens, thence to the environment. There are sufficient studies in the literature tying viral and bacterial pathogen counts to community rates of infection and then following these through treatment works and streams to the near-shore marine environment. As to its impact on ground water, there are numerous studies demonstrating contamination by pathogens from spread sewage. From this one needs to tease out the studies that discuss biosolids as differentiated from sludge. Again, this begs the question of other pollutants reaching the ground water. Additionally, many of the aging and failing trunk lines leading to sewer plants are, and have been for years, leaking into the groundwater beneath many towns and cities across America. As the mission environmental and health officer for the USAID Mission to Somalia, I had first hand experience with this. The aquifer under the capital became so polluted that a new well field was required some miles from the city. Again, we may use enterococcus as an example. From a clinical and pathophysiological perspective, we have previously demonstrated that enterococcus is important. Approximately 20% of bacterial endocarditis is attributed to this organism. It is, however, not necessarily seen until it reaches an acute state, often when there has been valvular damage in the heart. This may necessitate valve replacement surgery—assuming one is actually a surgical candidate. Some people can not tolerate surgery and thus must attempt to live with the defect. The organism may lie clinically silent producing a smoldering subclinical level of disease. Thus, the 20% figure for bacterial endocarditis may actually be an understatement. Those most affected are the immunocompromised, elderly, and those with barrier disruptions to the skin or mucosal membranes. In the last case it may be merely from beach sand scratching the skin at the waistline of bathing suits or under wet suites, or swallowing contaminated water. 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. To conclude, the following thought is a statement by the WHO’s chief of Communicable Disease, David Heymann, before the US Senate hearing on The Spread of Communicable Disease, in 2001. Some microbes have accumulated resistant genes to virtually all currently available drugs. Thus, these have the potential to cause untreatable infections. Accordingly, such diseases may have no effective cures over the next 10 years unless there is some uncharacteristic breakthrough in drug therapy. Therefore, if current trends continue, many important medical and surgical procedures, including cancer therapy, bone marrow and organ transplant, hip and knee replacement, and coronary bypass surgery could no longer be undertaken without undue risk of unstoppable infection.