UNIVERSITY OF CINCINNATI Date:___________________ I, _________________________________________________________, hereby submit this work as part of the requirements for the degree of:
in:
It is entitled:
This work and its defense approved by:
Chair: _______________________________ _______________________________ _______________________________ _______________________________ _______________________________
Detecting Mycobacterium spp. in Hospital Water
A thesis submitted to the Division of Research and Advanced Studies of the University of Cincinnati in partial fulfillment of the requirements for the degree of Master of Science in the Department of Biological Sciences of the College of Arts & Sciences 2007 by Kristin Lake Mack B.S. Vanderbilt University, 2003 Committee Chair: Dr. Brian Kinkle
Abstract
The occurrence of Mycobacterium was determined using traditional culturing methods and a novel device called the Biochip. Mycobacterium spp. were determined to be present in the water collected from every hospital and health care facility that participated (N= 11 sources). In total, fifty-seven species of Mycobacterium were isolated from the hospital water. Of the thirty-four that have been putatively identified by 16S rRNA sequencing, twenty-three (68%) are strains from species that have been implicated as disease-causing agents. The clinically significant species identified include M. avium, M. chelonae, and M. mucogenicum. The Biochip device was able to detect and identify Mycobacterium in seven of eighteen sites sources (39%) tested. The standard methods agreed that there was Mycobacterium present at these seven sources. The device detected no Mycobacterium in five sources where traditional methods also did not. The total agreement of the device to the traditional methods was twelve in eighteen (67%).
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Without Whom I would like to dedicate my master’s thesis to my committee members: Drs. Daniel B. Oerther, Brian Kinkle, Richard Karp, and Stacy Pfaller. I am grateful to all of them for their unending support. I received an exceptional education from them all through their advice and teaching. Not only was I among these top-rate individuals as a student, but each of them became my friend, and I will always be thankful for those new friendships. I appreciate the other project members involved with the Biochip innovation and work. I know that the Biochip can play an important role for helping people in the near future with continued, diligent research on its capabilities. Dr. Terry Covert is an amazingly patient man and an inspiring researcher. I appreciate all of his advice and support throughout my studies. My research required contact with health care professionals, particularly Infection Control Practitioners at many different facilities around the state of Ohio. I asked permission to do my study at many facilities and was turned away. I am so appreciative of those professionals from the participating healthcare facilities who cared enough about the welfare of their patients and the information this research could provide to jump through the necessary hoops to make it all possible. The Oerther lab is certainly a unique grouping of people, and I am glad to have been a part of it along with Gina, Kai, Rob, Ting, Mau-Yi, Ben, Mike, Ian, Carrie, Chris, and Sarah. Also as a part of that lab, I had undergraduates who helped me with my research: Anne Ryan, Neil Grabowski, and Shintaro Chiba. Each of them did a great job, and were fun to work with. Julie Stacey and Cathy Hayward are incredible women who helped me when I taught laboratory sections for them. Our labs were right across from each other, and they were great to talk to and have fun with, and they always let me talk through whatever was going on with my research or just in general. I had a great time doing our Sam Adams project together. Also in the Department of Biological Sciences, I would be remiss if I didn’t thank Roger Ruff and Charlie Zimmer who brought back to life all the machines I thought I broke, and who lent a hand in whatever way necessary. Also, the administrative staff in the office who processed all of my paperwork and always had a nice thing to say. Last, but certainly not least, is my family. I am a lucky individual to have the unconditional love and support from Dad, Jeremy, Cody, Dave, Pawpaw, Eddie, Danielle, and the rest of my family, who encourage me with every step I take. My husband, Evan, was with me throughout this degree program. He is an incredible man, and I know, unequivocally, that I would not be where I am, or going where I am without him.
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Table of Contents Abstract.................................................................................................................................i Acknowledgments...............................................................................................................ii Table of Contents................................................................................................................iii List of Tables and Figures..................................................................................................iv List of Abbreviations...........................................................................................................v Introduction..........................................................................................................................1 Chapter 1: Mycobacterium: A Literature Review History……………………………………………………………………………..4 General Biology…………………………………………………………………...5 Species Classifications…………………………………………………………….6 MAC Nomenclature Virulence…………………………………………………………………………..7 Infections and Disease…………………………………………………………….9 Treatment………………………………………………………………………….9 Detection…………………………………………………………………………10 Occurrence……………………………………………………………………….12 Geographical Distribution……………………………………………………….15 Chapter 2: Parameters of Biochip & Comparison with Standard Methods Abstract..................................................................................................................16 Introduction.....................................................................................................…...17 Materials & Methods......................................................................................…...21 Results.............................................................................................................…...25 Discussion.......................................................................................................…...30 Chapter 3: Mycobacterium Occurrence in Hospital Water Abstract..................................................................................................................32 Introduction......................................................................................................…..33 Materials & Methods.....................................................................................……35 Results...........................................................................................................…….40 Discussion..............................................................................................……........50 Conclusions........................................................................................................................54 References..........................................................................................................................57 Appendix I: Sequences of Isolates………………………………………………………I Appendix II: Archive of Environmental Mycobacterium…………………………………XIV
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List of Tables and Figures Tables Table 1 – Summary of Occurrence Studies (Chapter 1, pg. 13) Table 2.1 – Removing Organisms from Biochip (Chapter 2, pg. 26) Table 2.2 – Comparison of methods (Chapter 2, pg. 28) Table 3.1 – Detection of Mycobacterium per site (Chapter 3, pg. 41) Table 3.2 – Mycobacterium identification per site (Chapter 2, pg. 48) Table 3.3 – Agreement between methods (Chapter 3, pg. 50)
Figures Figure 2.1 – Biochip Introduction into Water System (Chapter 2, pg. 24) Figure 2.2 – Biochip Culture (Chapter 2, pg. 25) Figure 2.3 – Staining on Biochip (Chapter 2, pg. 26) Figure 2.4 – Multiplex PCR of Biochip (Chapter 2, pg. 27) Figure 2.5 – Agreement Between Methods (Chapter 2, pg. 29) Figure 3.1 – Sample Distribution (Chapter 3, pg. 36) Figure 3.2 – Diagram of Water System (Chapter 3, pg. 39) Figure 3.3 – Multiplex PCR of Isolates (Chapter 3, pg. 42) Figure 3.4A&B – Species Prevalence of Isolates (Chapter 3, pgs. 43-44) Figure 3.5A-C – Phylogenetic analyses of isolate sequences (Chapter 3, pgs. 45-47) Figure 3.6 – Multiplex PCR of Biochips (Chapter 3, pg. 49)
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List of Abbreviations
spp.
species
MAC
Mycobacterium avium Complex
PCR
polymerase chain reaction
EPA
Environmental Protection Agency (US government agency)
NTM
Nontuberculosis Mycobacterium
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Introduction Mycobacterium avium complex (MAC) was placed on ‘the watch list’ (i.e., Contaminant Candidate List, CCL) by the United States Environmental Protection Agency (USEPA) because it is an opportunistic pathogen of humans - especially dangerous for those with diminished immune competency. In the US, it was estimated that more than one million people were infected with HIV at the end of 2003 (Glynn, 2005). Thus, a significant portion of the US population is susceptible to opportunistic pathogens. MAC is one set of strains that are pathogenic, but many other Mycobacterium spp., such as M. fortuitum, M. chelonae, and M. kansasii have pathogenic strains. Strains of Mycobacterium often are responsible for particularly deadly infections because of their ability to thrive within a host’s passive immune system rather than be destroyed by it. According to Nightingale, as many as forty percent of people that have been diagnosed with HIV/AIDS for at least two years will be infected with MAC (1992). Many Mycobacterium infections take six to eight weeks to be definitively diagnosed because there is not an efficient way to isolate and culture Mycobacterium spp. from a patient’s sample. Unfortunately, this limitation for screening human samples is also an impediment for screening environmental water samples for Mycobacterium, as the traditional methods for detection in water or diagnosis in patients require lengthy cultivation. Recent studies utilizing molecular tools to detect and identify Mycobacterium in aqueous environments have found Mycobacterium in more sources than previously described. They have been found in drinking water, source water, pools, and even dental water lines (Glover, 1994; von Reyn, 1993; Iivanainen, 19992; Barbeau, 1998). Because
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Mycobacterium spp. are able to form biofilms in water distribution systems (Falkinham, 2001), it may be difficult to ever regulate municipal water treatment in a way that will limit their occurrence. The traditional methods for isolating Mycobacterium from environmental water sources are based on the use of toxic chemicals to reduce less resistant bacterial growth and culturing for approximately eight weeks due to the interest in species that are slow growers and may cause disease. This process for detecting Mycobacterium is destructive to the viability of the organisms themselves due to the use of toxic chemicals, meaning the accuracy of the traditional methods may be questionable. Also, the latency in detecting the presence of Mycobacterium in the environment makes it difficult to alert people that may be at risk for infection. Likewise, the latency in detecting the presence of Mycobacterium in patient samples makes it difficult to properly treat someone suspected of having a Mycobacterium infection. Although the traditional methods are capable of detecting Mycobacterium in the environment and it is useful to have cultures of isolates from the environment, a more accurate, sensitive, specific, and efficient process is needed. The first goal of my study was to provide information on the occurrence of Mycobacterium in drinking water from hospitals and healthcare facilities, places where at-risk patients will likely come into contact with it. The second goal was to provide information needed to advance the development of the Biochip, which is a culture-based paraffin chip that can bait Mycobacterium due to its lipophilic outer membrane and allows organisms to culture directly on it. The Biochip could become a powerful tool for detection of Mycobacterium in both environmental and clinical samples.
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Based on previous studies, I hypothesized that Mycobacterium would be present in a minority of the water samples, and the isolates would be strains representative of mostly saprophytic species, but Mycobacterium pathogens would be present in a very small number of sources. Concerning the Biochip, my hypothesis was that the device would be sensitive enough to detect Mycobacterium from environmental sources. The design of the experiment was based on getting the most accurate determination of whether or not Mycobacterium was present in the water samples collected. My thesis is organized into three chapters. The first chapter is a literature review to provide detailed information on three important areas: 1) why Mycobacterium is both important and difficult to study, 2) where does Mycobacterium occur according to recent studies, and 3) what are the parameters (advantages and limitations) on the current methods for detecting and identifying Mycobacterium. The second chapter describes the physical and chemical parameters of the Biochip in a series of experiments that provide more basic information on the current first generation device, and begin to elucidate what future steps could make the Biochip even more useful in a variety of settings. The third chapter is an occurrence study – the results of detecting and identifying Mycobacterium spp. in drinking water at hospitals and healthcare facilities in Ohio will be presented for both the traditional methods and the Biochip device.
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Chapter One Mycobacterium: A Literature Review
History Reports by Hansen in 1874 and Robert Koch in 1882, described bacilli of interest as causative agents of leprosy and tuberculosis, respectively. In 1896, the two organisms were named as members of a new genus, Mycobacterium. The conditions caused by the pathogens, however, were not new in the late 19th century. In contrast, tuberculosis and leprosy have ancient histories. Daniel suggests that although the Mycobacterium genus probably predates primates, M. tuberculosis could have differentiated from its predecessor between 20,400 and 15,300 years ago (Daniel, 2000). Reports of tuberculosis were communicated in artistic depictions of malformations as early as 5000 B.C. (ICS, 2001) and the disease was referred to under many different names including pthisis, King’s Evil, Pott’s Disease, Consumption, White Plague, Scrofula, and Wasting Disease throughout history. Similarly, the first written records of cases of leprosy were gathered from as long ago as 600 B.C. and the disease is still being battled across the globe today (Novartis, 2006). There is a rich history in literature describing detriment brought to the sufferers through physical and social issues. In fact, until 1969, even the American government had forcibly displaced sufferers of leprosy onto an island of exile (Tayman, 2006). The Mycobacterium genus, when officially named in 1896, referred to only two organisms. Although many species from 1896 to the 1950's shared the acid-fast staining characteristic with the genus Mycobacterium, it was not until 1948 that another
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pathogenic species was officially identified as Mycobacterium. As some of the generic 'atypical Mycobacterium' showed the ability to cause disease, interest in starting to identify them heightened (Balows, 1992). The slow expansion of the genus is no longer; thirty novel species have been identified as part of the genus since 2003, making the total species in the genus equal to 120 and growing continuously. Even as the numbers of Mycobacterium species are increasing, so is the interest in the pathogenicity of previously described and novel species. The Environmental Protection Agency (EPA) has even identified M. avium and M. intracellulare, otherwise known as the Mycobacterium avium Complex, on the Contaminant Candidate List as a biological organism of concern. Being placed on this list means that more information is needed on whether the organisms are present in drinking waters and about their ability to infect humans from an environmental source. Based on this information, the EPA will decide whether to make regulations on the organisms and how is best to do that.
General Biology In order for a novel species to be identified as a member of the Mycobacterium genus, it must have the unique characteristics that identify the genus. Mycobacterium is the only genus in the Mycobacteriaceae family, which belongs to the Actinomycetales order and the Actinobacteria phylum. Actinobacteria stain Gram-positive indicating they have a thick cell wall that allows the microorganisms to retain crystal violet during the Gram staining procedure. Nocardia, Corynebacterium, and Mycobacterium stain acidfast positive due to the high lipid content of the microorganisms’ cell wall, but they are unlike the typical gram-positive cell wall. Mycolata contain alpha- and beta-hydroxy
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mycolic acids with carbon chain lengths between sixty and ninety carbons. Mycobacteirum alone have mycolic acids that have a wide variety of functional groups, while other mycolata have simpler forms with double bonds. The functional groups on the mycolic acids of Mycobacterium are distinct for separate species, and their compositions are a known source of resistance against chemicals and therefore play a role in virulence in a human host. Because these already resistant organisms can form biofilms, they are extremely resistant to drugs, other environmental stresses, and phagocytosis, (Costerton, 1987).
Species Categorization In the second edition of The Prokaryotes, published in 1991, Robert Good reports that there are “fifty four species that make up the genus Mycobacterium,” and of those ten are human pathogens not found in the environment, nine others are opportunistic pathogens, and six others are animal pathogens. As of 2006, the number of species has increased to one hundred twenty, and it is hard to estimate the number considered opportunistic pathogens, because that number continues to increase. Today, Mycobacterium are generally not classified in terms of pathogenicity because as more information becomes available on novel species it is difficult to determine virulence capabilities; general research is finding that more and more predetermined saprophytic species are able to cause infections, especially in immunecompromised hosts. For example, the same chapter in Prokaryotes in 1991 reported M.chelonae as a saprophytic species, yet M.chelonae has been implicated as a water contaminant and is able to infect wounds as an opportunistic pathogen.
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Usually species of Mycobacterium are categorized as rapid-growers or slowgrowers. Within the genus, the variability in time for division is extremely varied taking as long as twenty days for one division, as is seen in the case of M. leprae. The outer layer of lipids and mycolic acids is the reason for the variance in growth rate; the slower growers have a very thick outer layer that takes longer to generate. Since the outer layer builds the organism’s resilience to harsh environmental conditions (such as seen in the immune system), it would follow that slow-growers, with their thicker outer layers and heightened resilience, are more likely to be pathogenic, as they can survive in the midst of immune defenses. However, some rapid-growers, such as M.chelonae have been definitively indicated in some pathologies. MAC Nomenclature Among the 120 species of Mycobacterium, MAC is a complex made up of two distinct species – Mycobacterium avium and Mycobacterium intracellulare. Recently Mycobacterium chimaera (Tortoli et al., 2004) and Mycobacterium colombiense (Murcia et al., 2006) have been suggested to belong to the M. avium complex. And in the past, different species have been named as part of a complex, for instance the MAIS complex consists of M. avium, M. intracellulare, and M. scrofulceum. However, the consensus today is that a reference to MAC is a reference to M. avium, its subspecies, and M. intracellulare.
Virulence The virulence of Mycobacterium is not completely understood, but it is known that the composition of the distinctive outer layer makes the organisms resistant to harsh
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environmental conditions. Resiliency is a major factor in surviving the host’s defenses and therefore not only being able to infect, but able to thrive among the host’s defenses, thus becoming very difficult to treat. The outer layer of Mycobacterium is much thicker than the average Gram-positive organism. Although the outer layer does not share a likeness with the typical Gram-positive bacteria, Mycobacterium does stain Grampositive due to its thick membrane’s ability to retain crystal violet and resist being decolorized. Most of what is known about the virulence of Mycobacterium is based on the numerous studies of M. tuberculosis. The thick outer layer is seen in most pathogenic Mycobacterium, but the mycolic acid composition is different. One reason Mycobacterium is virulent to its hosts is its ability to avoid the immune defenses. Mycobacterium like all foreign invaders is engulfed by the host’s macrophages, but unlike most invaders, Mycobacterium is able to withstand the consequences of being within the macrophage. A Mycobacterium strain expresses the mig gene once it is within a macrophage in order to produce a product that can prevent cross-linking of the lysozymal membrane. Therefore, the lysosomes cannot rupture and spill out toxic chemicals to digest the organism (Plum, 1997). Mycobacterium spp. have the ability to thrive in macrophages because they can prevent the normal processing that occurs in a macrophage and therefore are protected by the host’s cell and can proliferate inside the macrophage. This ability to not only survive the immune defenses of its host, but actually thrive within cells of the immune system makes Mycobacterium infections extremely difficult to combat.
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Infections and Disease Mycobacterium avium Complex (MAC) infections are the most commonly seen Mycobacterium infections. They usually occur in adults with a T-cell count below 50 cells per cubic millimeter of blood, but they may occur in children before a T-cell count is reported as low as 50 cells/mm3 blood (CDC, 1999). MAC causes infections in humans, which manifest as disease of the lungs and lymph nodes. Lymphadenitis, or a swelling of lymph nodes due to direct infection, and chronic tuberculosis-like disease are the clinical signs of MAC infections (AAM, 2004).
Treatment Once a person is infected with non-tuberculosis Mycobacterium, the regime for combating the infection is a cocktail of antibiotics over a long period of time. For an infection of Mycobacterium avium Complex (MAC) in a person without AIDS or HIV, the specific plan of treatment usually consists of a daily regimen of two or three antibiotics for at least eighteen months or until twelve consecutive months of culturenegative tests have been completed (ATS, 1997). The medications most commonly used for all Mycobacterium infections are isoniazid, a prodrug that actively inhibits the formation of mycolic acids in the cell wall of Mycobacterium; rifampin and rifabutin, antibiotics that prevent mRNA transcription by binding to RNA polymerase; ethambutol, a drug that prevents synthesis of sugar residues in Mycobacterium cell walls that attach to mycolic acids, making the cell wall more permeable if able to form; clarithromycin and azithromycin, antibiotics that inhibit translation; streptomycin, an injectable-only antibiotic that prevents protein production by binding to the bacterial ribosome and preventing release of newly synthesized proteins; and amikacin, an injectable-only
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antibiotic that binds to another part of the bacterial ribosome to prevent protein synthesis due to misreading during translation from mRNA. These drugs are used in pairs or groups of three that provide optimal activity against the Mycobacterium, but of them, only rifampin and rifabutin are bacteriocidal, while isoniazid is bacteriocidal only for rapidly growing species of Mycobacterium. For AIDS patients or HIV positive individuals, a low-dose weekly prophylaxis treatment of certain combination of these drugs is recommended. Once a HIV-positive or AIDS patient is infected with a Mycobacterium pathogen, a much higher treatment of multiple drugs must be given daily and must be maintained for life. If cervical lymphadenitis is part of the infection, the only treatment is still complete surgical excision of the affected tissue (ATS, 1997). The results of proper treatment of Mycobacterium infections in AIDS and HIV-positive patients is still not promising, it requires daily attention and is painful due to the blood draws and drug injections.
Detection Unfortunately, the current methods for collecting water samples and then detecting Mycobacterium lack quickness, accuracy, specificity, and require the use of toxic chemicals. It usually takes approximately eight weeks to culture isolated Mycobacterium from a water sample. Even as the exposure to cetylpyridinium chloride and cycloheximide are closely controlled in the filtration and culturing process, some Mycobacterium are certainly lost. The specificity of isolating out Mycobacterium is based on using toxic chemicals to limit the background organisms and on providing media and nutrients that Mycobacterium require to grow. Unfortunately, other
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organisms, especially some species of Bacillus are able to withstand the chemicals also, and grow on the media with the given nutrients much more quickly than Mycobacterium. There are certainly many ways to collect a water sample and process it for culturing or molecular techniques. After obtaining genetic material to use for identifying the resulting bacteria, researchers use a wide variety of molecular techniques to do the actual identifications. The way water samples are collected largely depends on the source of the water. Environmental samples are almost always collected by dipping a vessel into the source water because that is the most logical way. When water is collected from a tap, shower, or some other pipe-derived source, researchers typically choose whether or not to collect starting immediately or flushing the line for several minutes before beginning their collections. No significant differences in results have been described for these two methods of collection. The ultimate goal for most researchers in processing their water sample is to prepare an appropriate template for their intended molecular method that they will use to identify their populations. In some cases, a large scope of discovery can be determined by cloning an entire water collection (Humrighouse, 2006). If there is a desire to obtain strains from water samples, however, a step to culture the bacteria is necessary. Identifying pure cultures of Mycobacterium to the species level requires either a biochemical or a genetic approach. Researchers commonly use Gas-Liquid Chromatography (Iivanainen, 19991,2; Kusnetsov, 2003; Torvinen, 2004) or High Performance Liquid Chromatography of the mycolic acids (Hilborn, 2006). Genetic approaches include DNA probes (Glover, 1994; von Reyn, 1994), PCR (Pfaller, 2003), or
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sequencing of the 16S rRNA or heat shock protein 65 genes (Covert, 1999; Prommasith, 2003). Since infections of M. avium Complex are common as nosocomial infections (Barbeau, 1998), the use of molecular tools in detecting and identifying Mycobacterium in clinical environments was a logical progression, especially considering their long culture times. In early 1990 (Boddinghaus), a report of successful amplification and sequencing of 16S rRNA sequences from isolates helped to stir interest in the possibility of rapid detection of the slow-growing Mycobacterium. A decade later, a review of molecular techniques used to identify Mycobacterium elucidated even more specific genetic targets in the organisms. In order to distinguish M. avium from M. intracellulare, two closely related organisms, PCR amplification of the hsp65 gene proved more accurate than use of a 16S gene probe, Gen-Probe. Still, sequencing of the 16S region and even perhaps a new method of amplifying the mig gene were other molecular tools deemed appropriate (Beggs, 2000). In the future, evaluating the virulence of Mycobacterium strains may be of the utmost concern. Based on the complexities and high phenotypic and genomic diversity, developing an assay for distinguishing virulent strains from avirulent strains may be difficult, but there is already work started with that purpose (Cangelosi, 2003).
Occurrence Several ecological surveys have been performed in order to determine how much and which Mycobacterium are present in the environment. In terms of which species are most prevalent in drinking water and environmental water, the results vary. In general,
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Mycobacterium, even opportunistic pathogens such as Mycobacterium avium, thrive in aquatic environments (AAM, 2004). MAC was found in an average of 26% of environmental water and drinking water sources studied in two separate studies (von Reyn). From 1994 to 2006, Nontuberculosis Mycobacterium (NTMs) were isolated from 48% of environmental and drinking water sources (average based on occurrence studies from Table 1). In 1998, Vantarakis studied the occurrence of NTMs in hospital water systems and found that of the 64 sources surveyed in Greece, only ten had NTMs present. In 2003 and 2006, two Finnish studies found NTMs present in all but one of their hospital water sources. Table 1 further summarizes several recent and important studies that aimed to determine what types and how much Mycobacterium are in different sources of water. Table 1 – Summary of Occurrence Studies 1990-2006.
Citation
Source (Environmental)
Nightingale
Region New Environmental & Hampshire & 1990-1992 Municipal Water Boston Parkland Memorial Hospital 1992 HIV Patients (Dallas, TX)
von Reyn
1993
von Reyn
Glover
Year
1994
Drinking Water Environmental Water
Drinking Water
US, Finland, Zaire, Kenya US, Finland, Zaire, Kenya
Los Angeles
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Overall Organisms Identified Occurrence
M. avium
10/33 (30%)
MAC
21% (1yr) & 43% (2yrs)
MAC
9/44 (20%)
MAC
13/47 (28%) 54/58 (95%) homes; 10/10 hospitals (100%); MAC 26/76 NTMs
MAC & other NTMs
Vantarakis
1998
Hospital Water
Covert
1999
Drinking Water
NTMs
10/64 (15.6%); 64 samples representing 5 Hospitals Overall 33/139 (46%); 67/105 (35%)
NTMs
0/5 (0%)
NTMs
6/6 (100%)
NTMs
0/11 (0%)
Patras, Greece (state of Peloponnese) NTMs
21 States in USA 21 States in Ice - Commercial USA Ice - Hospital 21 States in Machines USA Bottled Water
21 States in USA
1999
Soil
Central Finland NTMs
47/47 (100%) 53/53 (100%)
Iivanainen
1999
Water Central Finland NTMs Indoor Swimming Pools Finland NTMs
Iivanainen
1999
Runoff Water
Finland
NTMs
Kusnetsov
2003
Hospital Water
Finland
Pfaller
2003
Not Given
NTMs MAC & other NTMs
Not Given
MAC
19/60 (31.7%) Overall 172/861 (20%)
Not Given
MAC
0% - 40%
Iivanainen
Drinking Water Drinking Water Prommasith 2003 (NP) municipal Drinking Water residential Torvinen
2004
Hilborn
2006
Perola
2006
Drinking Water
5/7 (71%) (100%) 15/16 (93.8%) showers; ND up to 1400CFU/L over time
Finland NTMs Metropolitan Unfiltered Surface area in Western Drinking Water United States NTMs Showers & Hospital Water Taps NTMs
14/15 (93%) 27% (38/139) sites sampled over time
Hospital Water
Cold Water
NTMs
100%
Hospital Water
Hot Water
NTMs
0%
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23/24 (96%)
Geographical Distribution Although there is no determinant pattern of national distribution, von Reyn, et al. saw a greater occurrence of MAC in the waters of the United States and Finland than in Zaire (1993). In terms of geographical distribution, M. avium Complex and M. scrofulaceum were found to be in higher numbers in soil that is nearer to water sources and in soil that is acidic (Brooks, 1984). In the same study, there was wide variability in the number of organisms found between adjacent sites and even at the same site over time (1984). Mycobacterium appear to occur throughout soil and water without bias to geography, and researchers are not currently able to accurately predict where Mycobacterium spp. occur in higher numbers. The interest in Mycobacterium occurring in the environment and, in particular, in drinking water systems is based on the knowledge that many species cause human health concerns. If Mycobacterium spp. are consistently found in drinking water, it may be possible to trace source of infections back to the water supply. Opportunistic pathogens, Deleted:
such as M. avium, have been determined to have caused an infection via the drinking water of an immune-compromised individual (von Reyn, 1994). Therefore, it would be advantageous to be able to detect Mycobacterium in drinking water to limit contact with those opportunistic pathogens.
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Chapter Two A Novel Bio-MEMS Device: A Tool for Rapidly Detecting Mycobacterium in Water
Abstract The genus Mycobacterium contains several opportunistic pathogen species that detrimentally affect members of the population who are immune compromised. Currently, culture-based approaches to detect Mycobacterium in drinking water require weeks before results are available. A new approach using the Biochip to detect Mycobacterium exploits this organism’s lipid-rich outer layer to “bait” it from the water for detection purposes. The Biochip allows Mycobacterium spp. to attach to it and to grow on its surface, providing a viable culture for any downstream testing. Mycobacterium spp. can be Gram-, Kinyoun-, or nucleic acid-stained directly on the device or the device can be easily processed to lyse cells on its surface and provide a template for genetic methods such as the polymerase chain reaction. Our multidisciplinary group recently developed this biochip for detecting Mycobacterium spp. and used it in an occurrence study, examining thirty-eight drinking water samples from ten hospitals and one healthcare facility in the state of Ohio. The Biochip recovered Mycobacterium spp. in seven of nineteen sites (37%) and yielded consistent results with the standard methods by either identifying the presence or absence of Mycobacterium in a 12 of 38 (32%) samples. Opportunistic strains of Mycobacterium spp. often cause difficult to treat infections in immuno-compromised individuals around the world, so the significance of being able to assay environmental samples and patient samples for
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Mycobacterium related pathogens in a more rapid manner will positively impact epidemiology, patient diagnosis, and environmental source tracking in the future.
Introduction
Current Methods of Detecting Mycobacterium Unfortunately, the current methods for detecting Mycobacterium lack quickness, accuracy, sensitivity, and specificity, and require the use of toxic chemicals. Researchers use a wide variety of molecular techniques to identify the Mycobacterium spp. present in the environment, due to their being present in potentially small numbers. In the first occurrence study done with the Biochip (see chapter three), the alternative technique used to detect Mycobacterium was a commonly used standardized method for culturing Mycobacterium from water samples. This standard method required mixing the sample with cetylpyridinium chloride, a toxic chemical, to eliminate the less resistant, fastergrowing organisms that could mask the presence of Mycobacterium. Once the sample is vacuum filtered, the membrane is placed onto plates made from agar and Mycobacterium-specific nutritional content with added cycloheximide, another toxic chemical for limiting fungus growth on the plate. Different species of Mycobacterium have a wide range of time to culture, anywhere from seven days (rapid-growers) to sixty days (slow-growers). During the occurrence study described in chapter three, cultures had to be observed and colonies isolated continuously from day seven through day sixty. Once the emergent colonies were isolated, the pure cultures could be removed and identified with molecular methods. The Biochip can be processed and prepared in the
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same way for molecular methods after a much shorter culture time, and without the use of any toxic chemicals.
Significance of Rapid Detection The opportunistic pathogens in the Mycobacterium genus cause difficult to treat infections in immuno-compromised individuals around the world. Nontuberculosis Mycobacterium spp. (NTMs) include many opportunistic pathogens that cause a wide range of serious infections in humans. Mycobacterium avium Complex (MAC), M. kansasii, M. bovis M. fortuitum, M. ulcerans, M. chelonae are common pathogens that cause skin disorders and tuberculosis-like respiratory infections. NTMs have been found in drinking water distribution systems all across the United States (Vantarakis, 1998; Covert, 1999; Iivanainen, 1999; Kusnetsov, 2003; Torvinen, 2004; Hilborn, 2006), and drinking water has been determined to be a source of human infections (von Reyn, 1994). Also, Mycobacterium are able to form biofilms, which can make them significantly more resistant to high temperatures, drugs, and other environmental stresses (Colsterton, 1987). The Mycobacterium avium complex (MAC) was placed on the Contaminant Candidate List (CCL) by the United States Environmental Protection Agency (EPA) because it is an opportunistic pathogen of humans, especially for individuals with diminished immune competency. Mycobacterium has been identified as a cause of nosocomial infections, resulting from patients’ contact with water (Barbeau, 1998). In the US, more than one million people were infected with HIV at the end of 2003 (Glynn, 2005). Thus, a significant portion of the US population is susceptible to opportunistic pathogens. Among opportunistic pathogens, MAC strains are responsible for particularly
18
deadly infections because of their ability to thrive within a host’s immune system cells rather than be destroyed. According to Nightingale (1992), as many as forty percent of people that have been diagnosed with HIV/AIDS for at least two years will be infected with strains of MAC. MAC infections are difficult to diagnose and treat – and thus are often are fatal – due to their slow growth, unusual cell envelope, and virulence factors (CDC, 1999). In addition, Mycobacterium infections can take six to eight weeks to be diagnosed definitively because there is no rapid way to isolate and culture MAC strains, and other slow-growing NTMs, from a patient’s sample. Unfortunately, this limitation for screening human samples is also an impediment for screening environmental water samples for Mycobacterium. Therefore there is ecological, epidemiological, and pathological significance in detecting the presence of Mycobacterium in drinking water systems. The use of molecular tools in detecting and identifying Mycobacterium spp. in the environment is a logical step in research efforts because the culture time of the organisms can be astronomical, especially when clinical efforts are based on the timely reception of such information. The Biochip is easily processed and prepared for use with molecular tools, but it has advantages by being a culture-based device as well. Molecular methods without high sensitivity have a limit of detection that may be well below the amount of Mycobacterium in an environmental sample. Since Mycobacterium can be cultured on the Biochip, the number of organisms present can be increased before detection with a molecular method.
19
First Generation Biochip Attachment The Biochip is designed to "bait" Mycobacterium based on their hydrophobic outer membrane. The paraffinophilic nature of the outer membrane of Mycobacterium has been well reported (Ollar, 1990). Paraffin wax is coated onto a glass wafer (as described in Jing, 2005), and the lipid-rich outer layer of the Mycobacterium cells attach to the lipophilic outer layer of wax on the device. The lipid-rich outer layer of Mycobacterium is fairly unique, so only three other genera of organisms have the ability to attach to the surface of the Biochip. Based on the extreme hydrophobicity of their outer membrane, only members of the Mycobacterium genus, and closely related Tsukamurella, Rhodococcus, and Gordonae are able to attach to the paraffin surface of the Biochip (Jing, 2005). Mycobacterium spp. are able to attach to the device, and the Biochip can quantitatively represent the number of organisms present up to a certain threshold (Jing, 2005). The use of paraffin wax in the design of the chip gives it inherent specificity to Mycobacterium, and its close relatives, and the ability to culture the organisms directly on the device. In the occurrence study described in chapter three, polymerase chain reaction was performed to assure that Mycobacterium spp. were present.
Culture Basis The Biochip is a culture-based method, which means that Mycobacterium can be grown directly on the device. After the Biochip is exposed to the environmental sample,
20
it can be washed and incubated with Czapek Broth (Difco), an organic carbon-free medium. Since many species of Mycobacterium can use the paraffin as their carbon source, colonies can grow directly on the Biochip. The ability of the device to culture Mycobacterium means that the device serves as a sort of live-dead assay because nontarget organisms will not be able to survive. Also, since the organisms are living, all techniques requiring live cultures can still be performed. Biochip Parameters The method for developing the Biochip has been previously described by Jing, et al. (2003 & 2005). Controlled experiments with known amounts of Mycobacterium and non-target organisms have determined that the device is specific to Mycobacterium (Jing, 2003 & 2005). The purpose of the following experiments was to elucidate more clearly the capabilities of the Biochip and to determine its ability to detect Mycobacterium spp. in hospital water obtained from around Ohio.
Materials & Methods Culturing on the Biochip Two Biochips were incubated in an identical solution containing known amounts of Mycobacterium avium spiked into sterile water (total volume 2ml). After incubating for at least two hours, both Biochips were rinsed with sterile water, and one was submerged in DAPI (Qiagen), a nucleic acid stain, for 10 minutes, then rinsed with sterile water. The first Biochip was viewed under ultraviolet light by fluorescence microscopy to determine if microorganisms were present. The second, identical Biochip was incubated in Czapek Broth for three to five days, then rinsed again with sterile water and stained
21
with DAPI for 10 minutes, rinsed with sterile water, and viewed by fluorescence microscopy. Forty random images from each Biochip were compiled and the number of cells was totaled for the first Biochip and compared to the second Biochip analyzed after culturing.
Processing the Biochip for Use With Chemical and Molecular Methods Chemical Methods Biochips were incubated with one of three media cultures. The first contained only M. fortuitum; the second contained 50% M. fortuitum, 25% Gram-positive and 25% Gramnegative organisms; and the third contained only a Gram-negative organism. Biochips were allowed to incubate in one of the three solutions for two hours, and then were rinsed with sterile water. Glass slides were prepared with approximately 10ul of each of the original media cultures. Biochips and glass slides were stained according to Gram staining, Ziehl-Nielsen staining, and Kinyoun staining protocols. The Biochips and the glass slides were examined using light microscopy.
Preparation for Molecular Methods Biochips were exposed to different methods of isolating cells from the surface of the chip:
22
A. Biochips were scraped with a razor blade to remove top layer of wax from glass wafer. B. Biochips were exposed to -80oC for 30 minutes then allowed to thaw either on ice or at room temperature; the freeze-thaw cycle was repeated three times for each chip. C. Biochips were heated by either flame or steaming distilled water and tilted over collection tube. D. Biochips were placed directly into a tube with 20ul of 0.2um diameter glass beads and approximately 50ul sterile water, and processed using a beadbeater (Eppendorf) for three minutes at the highest intensity labeled “Homogenize”. The tubes were then centrifuged at 13,000 rpm for ten minutes.
Occurrence Study Using the Biochip Setup. Water was collected from Ohio hospitals and healthcare facilities. Two four gallon aliquots from each source were put into separate, but identical water flow systems and were run simultaneously. Each laboratory water flow system is three feet of six-inch diameter PVC pipe that stands vertically, and water is pumped from the bottom of the pipe and allowed to passively flow out of the overflow, back into the sample reservoir, allowing circulation of the sample (Figure 2.1). The pump is adjusted to maintain laminar flow within the system. The Biochip is introduced into the water system, and circulation of the sample lasts at least two hours.
23
Figure 2.1 – Biochip Introduction into Water System
(A)
(B)
(C)
Pump
Culture The Biochips were removed and placed into sterile containers holding Czapek broth and allowed to culture for one to eighteen days. Template Preparation After the indicated incubation, the chips were removed and washed with sterile water, and then they were broken into a 1.7ml microcentrifuge tube. 100ul of 1% PBS or TE buffer and 50ul of a solution containing either 1% PBS or TE buffer and 0.2mm glass beads are added to each tube, and they were processed using a beadbeater, as described above, at the highest intensity for cell lysis.
PCR for identification Polymerase Chain Reaction, as described in Kulski, et al. (1995) was used to identify Mycobacterium avium complex strains to the species level. 24
Data Analysis Results indicating presence or absence of Mycobacterium from a sample were compared for agreement with the standard method for identifying Mycobacterium spp. in drinking water (as described by Covert, 1999). The full details of the occurrence study are available in chapter three.
Results Culturing on the Biochip When incubated with Czapek Broth, organisms were able to grow directly on the Biochip. Figure 2.2 shows the before and after culture period photos, with fluorescent detection of nucleic acid staining of organisms.
Figure 2.2 – (A) Biochip under fluorescence microscopy after incubation with M. fortuitum, (B) Biochip under fluorescence microscopy after incubation with M. fortuitum and subsequent culturing in Czapek Broth.
(A)
(B)
Processing the Biochip for Use With Chemical and Molecular Methods Gram, DAPI, and Kinyoun staining were possible directly on the Biochip. Ziehl-Nielsen staining was possible, but more difficult due to the step where heat is applied to the
25
slide/Biochip. Stain rolled off of the paraffin surface, or if overheated, the paraffin began to melt. Some visualization was difficult through the wax, as seen in Figure 2.3A, but it was still possible.
Figure 2.3– (A) Ziehl-Nielsen stain of mixed culture on chip. (B) DAPI stain of M.fortuitum on surface of Biochip
(A)
(B)
The easiest and most rapid way to obtain the organisms from the surface of the Biochip, so that they could be lysed and be useful for polymerase chain reaction, was to break the device into a 1.7ml centrifuge tube, and sterile water and beads, and procees the entire tube using a beadbeater for three minutes (Table 2.1 for summary of results). Table 2.1 – Attempts to Remove Organisms or Wax and Organisms from the Biochip.
Method
Wax Recovered
Notes
Scraping
None
Glass wafer beneath wax breaks
Freeze-thaw
Partial
Wax flakes away from glass
Flame heat
None
Wax evaporates immediately
Steam heat
None
Wax pools but pouring off is difficult Glass & wax pellet, leaving intracellular components
Beadbeater
Full
free in supernatant
26
Occurrence Study Using the Biochip The Biochip was successful in detecting Mycobacterium spp. from water samples taken from hospitals in the occurrence study described in chapter three. After incubation with the water sample, and a specified amount of culture time in Czapek Broth, the devices were washed and processed using a beadbeater. The supernatant containing intracellular components, including nucleic acids were used as DNA for the Multiplex PCR. PCR of the templates derived from each source indicated whether or not Mycobacterium spp. were present. This method did result in some positive PCR results, meaning that the Biochip was able to attach Mycobacterium spp. from an environmental source, culture it on the chip, and be processed for molecular methods. Figure 2.4 – Multiplex PCR of Biochips. Inverted gel image of Multiplex PCR of Biochips exposed to hospital water, marked A-K. First lane is a Mycobacterium positive control with Mycobacterium genus, and M. avium bands; Second lane contains a DNA ladder, Lanes 3-13 contain template from processed Biochips, and the last lane contains a negative control of E. coli.
M+ L A B C D E F G H I
27
J K -
The Biochip is faster for detection than standard methods, and it does not require the use of toxic chemicals. Like standard methods, it is culture-based, meaning that living cells are available to use for staining and testing, and it can be processed to use in molecular methods. Table 2.2 – Comparison of Biochip to Standard Techniques Method
Toxic Chemicals Time to Completion
Culture-based
Molecular Methods
Biochip
No
3 days
Yes
Yes
Standard Methods
Yes
60 days
Yes
Yes
The Biochip was successful in the occurrence study, because it allowed quick return on the answer of whether Mycobacterium spp. were present. As it is now, the firstgeneration device is not geared to be specific toward any particular species of Mycobacterium, and it is true that not all species have enough of a hydrophobic outer layer to attach to the Biochip (e.g. some rapid-growers) (Jing, 2005). However, the Biochip can be engineered to a greater specificity and it did agree with the standard methods of detecting Mycobacterium for twelve of nineteen sites (63%) (Table 2.5).
28
Figure 2.5 – Agreement between standard methods and biochip results.
Hospital 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 11
Location Patient Care Area (PCA) 1 Dental Clinic Office Area PCA PCA 1 PCA 2 PCA 1 Office Area Office Area PCA Construction Area Endoscopy - post treated PCA 1 PCA 2 PCA PCA Maternity PCA 1 PCA 2 PCA
Standard Methods First Standard Flush Flow + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + +
Biochip First Standard Flush Flow NA NA + + + + + + + -
* Shaded areas indicate results that agree with the standard method.
Discussion New parameters of the Biochips performance were produced on the ability of the Biochip to culture Mycobacterium spp., which chemical and molecular methods are possible downstream, and the abilities of the Biochip to identify Mycobacterium spp. from an environmental source (hospital drinking water), as compared with standard methods.
29
As seen in Figure 2.2, M. avium is able to grow directly on the surface of the Biochip. The Biochip is extremely versatile. It can be stained with only minor difficulties in visualizing cells, due to the uneven surface of the wax. As the patterning of the wax becomes more standardized, it will be even easier to visualize the organisms stained on the surface. Also, the Biochip can be quickly destroyed using a beadbeater, leaving all the intracellular components of the organisms on the surface to be analyzed by molecular methods. The Biochip was able to detect Mycobacterium spp. in hospital drinking water. Twelve of thirty-eight samples (32%) tested gave identical results using both the standard culture method and the Biochip. When looking at just the 19 sites, not the two collections within each, there is a 58% agreement (11/19) of results that the source did or did not have Mycobacterium present in at least one of the collections. The results of the first-generation Biochip are promising, and knowing how to use the Biochip, how organisms can be visualized on the device, and that it is useful in an environmental occurrence study provides extremely useful information on how to further the development of the Biochip. The Biochip has also been studied using a microfluidic device, and with a better-controlled method of how much water passes over the device (Polacyzk, 2006). Although it is important that enough water come in contact with the Biochip to detect the usually small amounts of Mycobacterium spp. that are present in drinking water, the water flow system was much larger than necessary for the device to be effective. There is already considerable interest in further studies using the Biochip in clinical settings to test patient sputum and blood samples for infection and also in other environmental water systems as real-time device to alert immuno-compromised
30
individuals to the presence of Mycobacterium. There are many possibilities for application of the Biochip into fields in medicine, ecology, and biotechnology because the Biochip is versatile, inexpensive to fabricate, and specific. Making the Biochip even more specific, sensitive, accurate, and rapid are continuous goals that are now more defined with new parameters and the first field-test successfully complete. In the future, detecting Mycobacterium spp. in terms of the virulence of the strains present may be of the utmost concern. Based on high phenotypic and genomic diversity, developing an assay for distinguishing virulent strains from avirulent strains may be difficult, but there is already work started with that purpose (Cangelosi, 2003). The Biochip, especially if specific to pathogenic strains of Mycobacterium spp., shows promise to be useful in environmental occurrence studies, as described above, but also in rapid analysis of patient samples to determine whether infection is present. The Biochip is a basic design, and so it can easily be developed further in terms of becoming more specific, more sensitive, and more accurate. Even as a first generation device it has shown possible uses in the fields of ecology, medical technology, water safety, bioterrorism alerts, and bioremediation.
31
Chapter Three Mycobacterium Occurrence and a Novel Method for Detecting it in Hospital Drinking Water
Abstract The genus Mycobacterium contains pathogens that are a danger to people without competent immune systems. Currently, culture-based approaches to detect Mycobacterium spp. in drinking water require weeks before results are available. A new approach to monitoring presence of Mycobacterium exploits their lipid-rich outer layer to “bait” it from the water for detection purposes. In this study, the occurrence of Mycobacterium avium Complex (MAC) and other Mycobacterium spp. was determined using two methods of detection, the standard method of culturing a water sample, and an innovative approach using a device referred to as the Biochip developed at the University of Cincinnati by a multidisciplinary team. Forty drinking water samples representing eleven sources (ten hospitals and one healthcare facility) in the state of Ohio were tested for Mycobacterium by both methods. At nine of the eleven sources, water was collected at two separate sites on two separate visits. Two water samples were collected at each site for a total of forty water samples. The first sample was collected using the standard method of flushing the line for three minutes before collecting, while the other sample was collected immediately after turning on the taps and was stopped within the first fortyfive seconds of flow. Overall, Mycobacterium spp. were found to occur in 100% of the sources (each site sampled at every hospital and health care facility tested). In total, fiftyseven isolates cultured from the twenty different collection sites have been identified as
32
strains of Mycobacterium. Nineteen of the thirty-four identified isolates were M .chelonae, making it the most prevalent species represented. Mycobacterium spp. were found in 16 of 20 (80%) of the first flush collections and in 17 of 20 (85%) of the standard flow collected samples. There is not yet solid evidence of a major difference between the types or numbers of isolates using different collection methods at the same source. The Biochip agreed with the standard culture methods of detecting Mycobacterium in twelve of thirty-eight collections, and showed a positive presence within as few as 72 hours.
Introduction Nontuberculosis Mycobacterium (NTM) includes many opportunistic pathogens that cause an array of serious infections in humans. Mycobacterium avium Complex (MAC), M. kansasii, M. bovis M. fortuitum, M. ulcerans, M. chelonae are common pathogens that cause skin disorders and tuberculosis-like respiratory infections. NTMs have been found in drinking water distribution systems all across the United States (von Reyn, 1993; Glover, 1994; Covert, 1999; Hilborn, 2006), and drinking water has been determined to be a source of human infections (von Reyn, 1994). Mycobacterium spp. are able to form biofilms, which can make them significantly more resistant to high temperatures, drugs, and other environmental stresses (Costerton, 1987). Dental water lines have been suggested to be a model for determining the effects of microorganisms in biofilms ubiquitous in piping (Barbeau, 1998). An infection caused by a MAC organism is often difficult to diagnose and treat – and thus can be fatal. MAC are difficult to target and destroy by the immune system or using drugs because of their slow growth, unusual
33
cell envelope, and various virulence factors (CDC, 1999). Just as they are difficult to diagnose clinically, MAC strains are difficult to detect and enumerate in drinking water systems using traditional cultivation methods, which require up to eight weeks for confirmation of results. The Mycobacterium avium complex (MAC) was placed on ‘the watch list’ (i.e., Contaminant Candidate List, CCL) by the United States Environmental Protection Agency (USEPA) because it contains strains that are opportunistic pathogens of humans, especially for individuals with diminished immune competency. MAC strains have the opportunity to cause nosocomial infections because patients come in direct contact with them via their drinking water (Barbeau, 1998). In the United States, more than one million people were infected with HIV at the end of 2003 (Glynn, 2005). Thus, a significant portion of the US population is susceptible to opportunistic pathogens. Among opportunistic pathogens, MAC strains are responsible for particularly deadly infections because of their ability to thrive within a host’s immune system cells rather than be destroyed. According to Nightingale (1992), as many as forty percent of people that have been diagnosed with HIV/AIDS for at least two years will be infected with strains of MAC. Mycobacterium infections take six to eight weeks to be diagnosed definitively because there is no rapid way to isolate and culture from a patient’s sample. Unfortunately, this limitation for screening human samples is also an impediment for screening environmental water samples for Mycobacterium. The newly developed miniaturized culture-based device (Jing, 2005), the Biochip, may make it possible to rapidly detect Mycobacterium spp. in environmental samples. The Biochip uses a paraffin surface to selectively isolate Mycobacterium spp. from water
34
samples based on their extremely hydrophobic outer layer. Because of this distinctive cellular property, Mycobacterium can bind to the Biochip and accumulate relative to other microorganisms, which remain in the water sample. The goals of this study are to determine the occurrence of Mycobacterium in the drinking water at hospitals and healthcare facilities around the state of Ohio and identify what strains are present. In addition, the newly developed paraffin device will be used in parallel with traditional methods in order to test accuracy, sensitivity, and quickness of the device.
Materials & Methods Sample Collection Drinking water samples were collected from ten participating hospitals and one healthcare facility in the state of Ohio. All water was collected from faucets, taps, or drinking fountains into sterile one gallon (U.S.) containers provided by Trauth Dairy (Covington, KY). The containers were marked on the outside with a code that allowed a confidential identification of the source of the water samples. All containers contained 0.46g of sodium thiosulfate, a preservative, to limit the free chlorine destruction of the water samples. All samples were collected by the same person wearing a laboratory jacket and gloves to maintain the integrity of the samples; the faucets, taps, or fountains were not cleaned prior to collections. The water samples taken at each source were collected using two distinct collection methods: “First Flush and “Standard Flow” (described below). Each of those samples was analyzed using two distinct methods: “Standard Culture” and “Biochip” as described below (Figure 2.1).
35
Figure 3.1 – Sample distribution into standard methods and Biochip analysis.
Total First Flush Collection (22.7L or 5 US Gallons)
Standard Culture (1.5L)
Total Standard Flow Collection (22.7L or 5 US Gallons)
Standard Culture (1.5L)
Biochip (21.2L)
Biochip (21.2L)
First Flush The first flush method for collecting drinking water has not been previously reported. At each source, five gallons of water were collected immediately after turning on the tap, faucet, or drinking fountain, and the collection was halted within 45 seconds of the water running. Because water could only be collected for 45 seconds, and five gallons of water were needed, up to four taps were used from the same common area. Standard Flow The standard methods for collecting drinking water were followed as applied in Covert (1999). At each source, five gallons of water were collected after allowing the tap, faucet, or drinking fountain to run for at least three minutes prior to collection into the standard sterile gallon container containing sodium thiosulfate.
Sample Processing Standard Methods 1.5 L of the standard flow collection and 1.5 L of the first flush collection water samples were returned to the lab to be analyzed by the following standard methods (also described 36
in Covert, 1999). 1.5 L samples were further divided into three 500ml portions, and membrane filtered, using 0.45um HABG filters (Millipore) after the water samples were exposed to 0.04% cetylpyridinium chloride for thirty minutes. Culturing Membranes from each of the three repetitions was placed onto a Middlebrook 7H10 agar plate (Difco) with 500mg/L added cycloheximide and incubated at 28oC for eight weeks. Observations were made approximately once per week during the culture period and records were collected describing time of appearance and colony morphology. Staining A portion of each bacterial colony of each distinct morphology type was isolated and acid-fast stained (Kinyoun stain). All acid-fast bacteria were isolated to pure culture and a portion was removed and processed to prepare DNA for molecular analysis. DNA isolation To isolate DNA, a loopful of a colony was placed into a 1.7ml microcentrifuge tube containing 100ul of 1% Phosphate Buffered Saline (PBS) or 10mM Tris-Cl EDTA, pH 7.2 (TE) buffer. 50ul of a solution containing both 1% PBS or TE buffer, and approximately 20ul of 0.2mm glass beads were added to each tube. The tubes were processed in a beadbeater (Eppendorf) set at “Homogenize”, which is the highest intensity for three minutes. The lysate was retained, and the pellet thrown away, after centrifuging each sample at 13,000 rpm for at least ten minutes (Eppendorf, Microcentrifuge 5415D). PCR for identification Multiplex polymerase chain reaction (PCR), as described by Kulski (1995), was used to
37
identify colonies as Mycobacterium (genus-specific) or MAC after isolation from culture. Sequencing of 16S for identification The 16S rRNA genes of all cultures of interest were also sequenced. First, PCR was performed in a 50ul reaction using universal primers 8F (5’AGAGTTTGATCCTGGCTCAG-3’) and 1492R (5’-GGTTACCTTGTTACGACTT-3’). Reagents for 16S rRNA PCR were used at the following concentrations: 10X PCR Buffer, 25mM MgCl2, 2.5mM dNTPs, 10uM each of 8F and 1492R primers, and 0.8ul taq, along with 3ul DNA template. The PCR product (approx. 1550bp) was purified and concentrated using Microcon 50 spin filters (Amicon) and sequenced through an outside service (Cincinnati Children’s Hospital Medical Center).
Biochip Processing Four gallons of both the first flush and the standard flow collections from each source were put into separate, but identical water flow systems and were run simultaneously. Each laboratory water flow system is three feet of six-inch diameter PVC pipe that stands vertically, and water is pumped from the bottom of the pipe and allowed to passively flow out of the overflow, back into the sample reservoir, allowing circulation of the sample (Figure 3.2). The pump is adjusted to maintain laminar flow within the system. The Biochip is introduced into the water system, and circulation of the sample lasts at least two hours.
38
Figure 3.2- (A) Diagram of flow directions of water system, (B) Photo of whole water system, two identical separate systems running on one pump.
(A)
(B)
Pump
Culture The Biochips were removed and placed into sterile containers holding Czapek broth and allowed to culture for one to eighteen days.
Template Preparation After the indicated incubation, the Biochips were removed and washed with sterile water, then broken into a 1.7ml microcentrifuge tube. 100ul of 1% PBS or 10mM Tris-Cl EDTA, pH 7.2 buffer and 50ul of a solution containing both 1% PBS or TE buffer and approximately 20ul of 0.2mm glass beads are added to each tube, and then were processed with a beadbeater (Eppendorf) for cell lysis at “Homogenize” setting for 3
39
minutes. The homogenized device was centrifuged (Eppendorf) and the supernatant containing intracellular components was maintained. PCR for identification As in Standard Methods above, Multiplex PCR, as described in Kulski, et al. (1995), was used to identify Mycobacterium (genus-specific) and MAC.
Results Standard Methods Occurrence Mycobacterium was isolated from each of the 20 sites tested (100%). Mycobacterium was detected in 16 of 20 (80%) of the first flush collection method samples, and 17 of 20 (85%) standard flow collection method samples. (Table 3.1)
40
Table 3.1 – Detection of Mycobacterium per site at each source. The + indicates that Mycobacterium was present as indicated by Multiplex PCR (Kulski), the species present were determined by sequencing of the 16S rRNA gene and phylogenetic analysis. Hospital Location
First Flush Species Present
Standard Flow Species Present +
(1B) M.petroleophilum (99%) (3B) Unidentified
(8) M.avium (96%) (6) Unidentified
+
(6B) M.mucogenicum (100%)
+
(17A) M.anthracenium (99%)
+
(17) M.anthracenium (98%) (17.2) M.anthracenium (99%)
PCA
+
(21) M.mucogenicum (100%)
+
(21B) M.mucogenicum (100%) (20) Unidentified
3
PCA 1
+
(27C) M.chelonae (99%) (27A) Unidentified
+
(26) M.chelonae (99%) (27) M.chelonae (100%)
3
PCA 2
-
+
(33.2B) M.chelonae (97%)
4
PCA 1
+
(36.4A) M.neglectum (99%) (35A & 36.2) Unidentified
+
(35) M.chelonae (97%) (36 & 36.4B) Unidentified
4
Office Area
+
(43) Unidentified
+
(40) Unidentified
5
Office Area
+
(45A) M.anthracenium (99%) (44A & 46A) Unidentified
+
(44) M.anthracenium (99%) (46) Unidentified
5
PCA
+
(55A) M.anthracenium (99%) (57A) Unidentified
+
(55) M.salmoniphilum (100%)
6
Construction Area
+
(48A) Unidentified
+
(50) M.chelonae (100%) (48) Unidentified
6
Endoscopy - post treated
+
(51) Unidentified
-
7
PCA 1
+
(58A & 59A) Unidentified
+
(57 & 58) Unidentified
7
PCA 2
+
(60A) M.chelonae (99%)
+
(60) M.chelonae (100%) (62) Unidentified
8
PCA
+
(66) Unidentified
-
1
Patient Care Area (PCA) 1
-
1
Dental Clinic
+
2
Office Area
2
8 9
PCA Maternity
+ -
9
PCA 1
+
10
PCA 2
-
11
PCA
+
(70) M.chelonae (100%) (69 & 71A) Unidentified (77) M.chelonae (100%) (79) M.chelonae (99%) (78) Unidentified (83A2) M.chelonae (99%)
+ +
(67) M.chelonae (100%) (69B) M.chelonae (100%) (70B) M.chelonae (99%) (76) M.chelonae (99%)
+
(78B) M.lentiflavum (98%)
+
(82) M.petroleophilum (100%)
+
(83B2) M.chelonae (100%)
* Isolate designation is given in parentheses (1B) for reference to Figure 3.4. ** Percentage in parentheses (99%) is percentage of similar nucleotides between isolate sequence and identity given when searched with BLAST.
41
Identification of Mycobacterium by PCR Presence or absence of Mycobacterium was based on a Multiplex PCR (Kulski, 1999), which indicates a Mycobacterium genus-specific band (1030bp), as well as speciesspecific bands for M. intracellulare (850bp), M. avium (180bp), and M. tuberculosis (372bp). (Figure 3.3)
Figure 3.3- Multiplex PCR (Kulski) of isolates. Lane L is a DNA ladder, Lanes marked 1-11 are isolates from standard method, lane marked M+ is a Mycobacterium positive control with Mycobacterium genus, and M. avium bands, and the last lane contains a negative control from an E. coli culture.
L 1 2 3 4 5 6
7 8 9 10 11 M+ -
Sequencing of Isolates The species found at each hospital or health care facility are listed in Table 3.1 as identified by sequencing the 16S rRNA gene. (Full sequences can be found in Appendix 1) Of the thirty-four that have been identified, species names include M. avium, M. chelonae, M. mucogenicum, M. neglectum, M. salmoniphilum, M. petroleophilum, M. lentiflavum, and M. anthracenium. The list of species present is putative data. The identity of the organisms so far is based on their 16S rRNA gene sequencing and the phylogenetic analysis. More definitive results will be provided when the Heat Shock Protein 65 gene is sequenced for all organisms. The percentage following each
42
organism’s species identification is based on Blast searches of their raw sequencing information, which was on average 805 nucleotides. The sequences were cut to the smallest size, and ambiguous areas were removed before the phylogenetic analysis was performed. Figure 3.4 (A&B) represent the species prevalence of the isolates from all sites sampled.
Figure 3.4A – Species prevalence of all isolates (Mycobacterium isolates without definitive species identification included, N=58). Pathogenic species are highlighted.
M.avium (1)
M.chelonae (19)
Mycobacterium, species unknown (24)
M.neglectum (1) M.petroleophilum (2)
M.anthracenium (6) M.mucogenicum (3)
43
M.salmoniphilum (1) M.lentiflavum (1)
Figure 3.4B – Species prevalence of all identified organisms (N=34). Pathogenic species are highlighted.
M.avium (1)
M.anthracenium (6)
M.mucogenicum (3) M.lentiflavum (1)
M.chelonae (19)
M.salmoniphilum (1) M.petroleophilum (2)
M.neglectum (1)
Figure 3.5 (A-C) depicts a phylogenetic relationship of isolates found at each facility along with other known Mycobacterium spp.
44
Figure 3.5A – Phylogenetic tree of isolates based on Neighbor-Joining algorithm.
45
Figure 3.5B - Phylogenetic tree of isolates based on Parsimony algorithm
46
Figure 3.5C - Phylogenetic tree of isolates based on Maximum Likelihood algorithm.
47
Biochip Occurrence Mycobacterium were detected in 7 of the 19 sites tested (37%) using the Biochip. The presence of Mycobacterium from the first flush collection method was detected from Biochips in 2 of 19 (11%) samples, and in 5 of 19 samples (26%) from the standard flow collection. (Table 3.2) Table 3.2 – Mycobacterium Identification per source by the Biochip
Hospital 1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 10 11
Location Patient Care Area (PCA) 1 Dental Clinic Office Area PCA PCA 1 PCA 2 PCA 1 Office Area Office Area PCA Construction Area Endoscopy - post treated PCA 1 PCA 2 PCA PCA Maternity PCA 1 PCA 2 PCA
Culture First Culture Time Standard Time Flush (Hours) Flow (Hours) NA NA NA NA + 88 & 136 88 & 136 72 & 120 + 72 & 120 72 & 120 + 120 ONLY 72 & 120 + 72 & 120 88 & 136 + 88 & 136 96 & 144 96 & 144 + 120 ONLY 72 & 120 72 & 120 + 120 ONLY - 48, 72, & 168 168 72 & 120 72 & 120 192 72 & 192 72 & 144 72 & 144 96 & 120 96 & 120 24 & 144 24 & 144 72 & 168 72 & 168 48 & 144 48 & 144 432 432 168 & 192 168 & 192 48 & 144 48 & 144
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Culture impact on Biochip detection The Biochip was incubated with Czapek Broth (Difco) to allow any attached Mycobacterium to culture. The incubation times were different for each of the two or three Biochips (repetitions) per collection as indicated in Table 3.1. In 3 of the 7 (43%) positive detections of Mycobacterium, only the Biochip with a longer culture time was successful. PCR detected the presence of Mycobacterium on the Biochip with as few as 72, and up to 136, hours of culture time.
Biochip - Identification of Mycobacterium by PCR Presence or absence of Mycobacterium was based on a Multiplex PCR (Kulski), as above with standard methods. (Figure 3.5)
Figure 3.6 - Inverted gel image of Multiplex PCR of Biochips marked A-K. First lane is a Mycobacterium positive control with Mycobacterium genus, and M. avium bands; Second lane contains a DNA ladder, Lanes A-K contain template from processed Biochips, and the last lane contains a negative control of E. coli.
M+ L A B C D E F G H I
J K -
Comparison of standard methods and the Biochip The standard methods and the Biochip detected the same presence or absence of Mycobacterium in 12 of the 38 samples (19 sites, each with two samples: first flush and standard flow), which represents a 32% agreement. Overall, 11 of the 19 sites (58%) had
49
agreement between the results from the standard culture method and the Biochip. (Table 3.3)
Table 3.3- Agreement between standard methods and biochip results
Hospital Location Patient Care Area (PCA) 1 1 1 Dental Clinic 2 Office Area 2 PCA 3 PCA 1 3 PCA 2 4 PCA 1 4 Office Area 5 Office Area 5 PCA 6 Construction Area 6 Endoscopy - post treated 7 PCA 1 7 PCA 2 8 PCA 8 PCA 9 Maternity 9 PCA 1 10 PCA 2 11 PCA
Standard Methods Biochip First Standard First Standard Flush Flow Flush Flow + + + + + + + + + + + + + + + +
+ + + + + + + + + + + + + + + + + +
NA + + -
NA + + + + + -
* Shaded boxes indicate agreement between standard methods and the Biochip.
Discussion The main objectives of this study were to determine the occurrence of opportunistic pathogens in the drinking water of healthcare facilities and to determine if there is a better way to detect Mycobacterium spp. in drinking water. This study
50
confirms a recent studies that suggest Mycobacterium occurrence is higher than previously reported (100% of sites tested were positive for Mycobacterium). Where it may be that there is more Mycobacterium in drinking water systems now than one hundred years ago, when such systems were not as intricate, there is no reason to believe that the occurrence of Mycobacterium has increased in the last few decades. It is probably not true that Mycobacterium spp. are more prevalent now than in the recent past, but rather that the methods for detecting their presence and the increased knowledge about their ability to form biofilms are more advanced. Thirty-four isolates were identified in the current study, including M. avium, M. chelonae, M. mucogenicum, M. neglectum, M. salmoniphilum, M .petroleophilum, M. lentiflavum, and M. anthracenium. Of which, the first three (M. avium, M. chelonae, and M. mucogenicum) have been implicated in infections (CDC, 1999; Hong, 2003). The threat that Mycobacterium spp. pose to the general population is still being investigated, but there is no question how much damage Mycobacterium infections can cause to those whose immune systems are compromised. It is certainly helpful to monitor water that is regularly consumed or used for drinking, bathing, etc. for these opportunistic pathogens, but the delay caused by culture-based detection of these slow-growing bacteria is a serious problem. Although the Biochip did not identify Mycobacterium at every source, it did agree with the standard methods on presence or absence of Mycobacterium in twelve of thirty-eight (32%) samples collected. The device not only was able to identify Mycobacterium in the collected samples, but the process of identifying took as little as seventy-two hours, while the standard methods took as long as seventy-two days.
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The Biochip provides hope for a more rapid way to detect Mycobacterium spp. that may be present in drinking water, and perhaps in the future, in patient sputum and blood. A quick turn-around time for detecting and identifying for diagnosis will help to prevent infections and allow for proper treatment once infections have occurred. This has been the first field test for the Biochip, and it was successful in detecting the presence of Mycobacterium in a significantly shorter time than is expected with standard methods. The Biochip confirmed the standard culture method’s detection of Mycobacterium spp. in 12 of 38 (32%) collections, and had the advantage of showing a positive presence of Mycobacterium within as few as 72 hours. Further studies on the paraffin device will provide the multidisciplinary team working on it with novel ideas to further the device by making it more specific, more sensitive, more accurate, and even more rapid. One future goal is to be able to make the Biochip a real-time device able to be used as an alert system for pathogenic Mycobacterium spp. in drinking water. The Biochip is extremely versatile because of its simple design and could also be modified for detecting Mycobacterium spp. that are useful for bioremediation. Therefore, there are many possibilities for the development and use of the Biochip in areas as diverse as medicine, medical technology, ecology, and water safety. Monitoring for Mycobacterium may be the next step rather than environmental regulation of the organisms, since the organisms are extremely adept at forming biofilms in environments such as piping systems, hot water returns, and dental lines. The focus of research projects concerning Mycobacterium may be shifting to monitoring and alert systems, and biocidal treatments of piping to prevent biofilm formation in water systems. The conventional methods for culturing and identification for many organisms of interest
52
from a clinical perspective have already yielded to molecular methods for the sake of saving time, and the trend will only continue, especially with Mycobacterium spp., due to their extremely long culture times. Because of the diversity of the genus Mycobacterium occurrence studies should focus on species relevant to the specific research area (i.e. degradative species for bioremediation studies and pathogenic species for epidemiological studies). Although this study was mainly concerned with detecting and identifying pathogenic Mycobacterium spp. in hospital water systems, a large archive of isolates representing a variety of species was produced.
Further studies on the immunology of pathogenic
Mycobacterium spp., especially the differences between virulent and saprophytic strains, may elucidate a way to make monitoring specific for pathogens. It is an important first step to recognize that Mycobacterium spp. are more prevalent in our drinking water than was originally speculated. It is an important step to continue research to define the pathogenic species and learn more about their basic biology in order to elucidate drug targets, since it is unlikely that Mycobacterium can be removed from every pipe and water source. In the meantime, it is a necessary step to monitor our water for these pathogens, and protect our susceptible population from infection by having a rapid warning system to alert us of the threat of Mycobacterium.
53
Conclusions
The Biochip device was successful in detecting Mycobacterium in drinking water. The Biochip detected Mycobacterium in two of the first run collections and five of the standard flow collections for a total occurrence of 7/38 (18%) in terms of collections or 7/19 (37%) in terms of sites sampled. It agreed with the standard methods for detecting presence of Mycobacterium at seven sites and for detecting the absence (negative result) at five sites for a total agreement with the standard methods of 12/19 (63%). The importance of the Biochip certainly cannot be discounted because it can detect Mycobacterium with fairly high sensitivity, as seen in the occurrence study where Mycobacterium is not present in large quantities. And not only is it a viability assay because of its culturing abilities, its detection of Mycobacterium in the present study occurred within approximately seventy-two hours (3 days), whereas the traditional methods took up to seventy-two days. It is possible to use the Biochip for chemical detection using nucleic acid staining or mycolic acid staining and for molecular detection using PCR. The Biochip is versatile and useful, because it can be cultured to allow more growth of the organisms of interest, it provides information only on living Mycobacterium, and it can be used for molecular methods without any complicated procedure to remove the organisms from its surface. The Biochip is relatively inexpensive to fabricate, and has plenty of room for further development to make it more specific, sensitive, accurate, and faster. The current study is the first of its kind, and provides information needed to progress the development of the Biochip to its fullest potential.
54
In the occurrence study, 57 strains of Mycobacterium were isolated from twenty different sources of potable water in ten hospitals and one healthcare facility. So far of 34 that have been putatively identified by 16S rRNA sequencing and phylogenetic analysis, twenty-three (68%) are clinically significant organisms. Since each of the twenty sources sampled had Mycobacterium present in the first flush collection, the standard flow collection, or both, the occurrence of Mycobacterium per site is 100% (20/20). Among the first flush collections, four sites had no detectable Mycobacterium present, so the occurrence for the first flush collections is 14/20 (80%). Two samples collected by standard flow had no detectable Mycobacterium present, making the occurrence of Mycobacterium in that subset 18/20 (90%). There does not seem to be any statistical difference between the two collection methods. However, it is still being determined which strains were identified in each collection, and if one collection method leads to the detection of more clinically-significant organisms. In other words, the two collection methods may have both detected Mycobacterium, but it cannot be discounted that the populations recovered from one method were distinct from the other in an important way. There have been studies that have done either the first flush collection method or the standard flow collection method, but this is the first study to collect samples at each site with both methods. Since neither collection method alone gave the full picture of 100% occurrence without the other, the combination of the two in this study was important. The clinically-significant species found thus far (M.avium, M.chelonae, and M.mucogenicum) can cause diseases ranging from tuberculosis-like respiratory infections to disseminated lymphadenitis and neurological infections (CDC, 1999; ATS, 1997;
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AAM, 2004; Hong, 2003). The present study supports many recent studies (Torvinen, 2004 ; Iivanainen, 1999) that suggest Mycobacterium occurs in water and in water distribution systems at a much higher rate than previous studies indicate. The knowledge that Mycobacterium spp. are more prevalent in drinking water than previously reported is important for preventing human infection. Not only is Mycobacterium present more often than previously described, but a greater percent of those present are being identified as pathogenic species. This study was able to determine that pathogenic Mycobacterium are more prevalent and therefore may present a real threat to human health. This study was able to use the Biochip to determine the presence of Mycobacterium within three days, which may provide earlier warnings to individuals to prevent Mycobacterium infections.
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Appendix I Sequences of Isolates 1B (782bp) TGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAA CACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTG GGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGT GTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGG AGTACGGCCGCAAGGCTAAAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGG AGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAG GACGCCGGCAGAGATNTCNGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTC ATGTTGCCAGCACGTAATGGTGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGG AAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTA CAATGGCCGGTACAAAGGGCTGCGATGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCG GTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAAT CGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA CGTCATGAAAGTCGGTAACACCCGAAGCCG 6B (805bp) GGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGT GGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAA CTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCC ACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTGCCGTAGCT AACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT TGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGA ACCTTACCTGGGTTTGACATGCACAGGACGCCGGCAGAGATGTCGGTTCCCTTGTGGC CTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTC CCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTTATGCCGGGGACTCGTAG GAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCT TATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGATGCCGTGA GGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACC CCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCC CGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCG 8 (804bp) ACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATAT CAGGAGGAACACCGGTGGCGAAGGCGGGTCTTCTGGGCAGTAACTGACGCTGAGGAG CGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGT GGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTAC CCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGC ACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTT GACATGCACAGGACGCGTCTAGAGATAGGCGTTCCCTTGTGGCCTGTGTGCAGGTGGT GCATGGCTGTTGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCA
I
ACCCCTGTCTNATGCTGCCAGCGGGTAATGCCGGGGACTCGTGAGAGACTGCCGGGGT CAACTCGGAGGAAGGTGGGGATGACTGTCAAGTCATCATGCCCCTTATGTCCAGGGCT TCACACATGCTACAATGGCCGGTACAAAGGGCTGCGATGCCGTAAGGTTAAGCGAATC CTTTTAAAGCNGGTGTCAGTTCGGATCGGGGTAAGCAACTCGACCGCAGNAANTCGNA CGTNNGGTAGTAATCTGCAGATCAGGCAACGCTCGCGCGTGAATACNGTTCCCAAGTC CNNGTACAGCGGCCGCNCNAGCGCTACTTNCNCAAACCGTCCGCACACTCNA 17A (795bp) ACAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCA GATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGA GGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAA CGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAG TACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCC CGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGG TTTGACATGCACAGGACGCTGGTAGAGATATCAGTTCCCTTGTGGCCTGTGTGCAGGT GGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGC GCAACCCCTATCTTATGTTGCCAGCGCGTTATGGCGGGGACTCGTAAGAGACTGCCGG GGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGG CTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGAATCCGCGAGGTGGAGCGA ATCCCTTGAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTT GGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGT ACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCG 17 (804bp) ATACGGGCAGACTAGAGTACTGCAGGGGGAGACTGGAATTCCTGGTGTAGCGGTGGA ATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCNGGTCTCTGGGCAGTAACTG ACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACA CCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTGCCGTAGCTAAC GCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGA CGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACC TTACCTGGGTTTGACATGCACAGGACGCTGGTAGAGATATCAGTTCCCTTGTGGCCTGT GTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG CAACGAGCGCAACCCNTATCTTATGTTGCCAGCGCGTTATGGCGGGGACTCGTAAGAG ACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTAT GTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGAATCCGCGAGGT GGAGCGAATCCCTTNAAAGCCGGTCTCAGTTCGGATCGGNGTCTGCAACTCGACCCCG TGAAGTTGGAGTCGTATAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCG GGCCTTGTACACACCGCCCGTCACGTACATGAAAGTCGTGTACACACCNNAAGG 17.2 (806bp) GCGATACGGGCAGACTAGAGTACTGCAGGGGGAGACTGGAATTCCTGGTGTAGCGGT GGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCNGGGTCTCTGGGCAGTA ACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTC CACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTGCCGTAGCT AACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAAT TGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGA
II
ACCTTACCTGGGTTTGACATGCACAGGACGCTGGTAGAGATATCAGTTCCCTTGTGGCC TGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCC CGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCGCGTTATGGCGGGGACTCGTAAG AGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTT ATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGAATCCGCGAG GTGGAGCGAATCCCTTGAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCC CGTGAAGTTGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCC GGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGGCG 21 (815bp) AGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTG GTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC TGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGT GCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTC AAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAA CGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGCCGGCAGAGATGTCGGTTCC CTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGG GTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTTATGCCGGGG ACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCAT CATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCG ATGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGC AACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCG AAGGCG 21B (813bp) AGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGT GTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTG GGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCT GGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTG CCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCA AAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAAC GCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGCCGGCAGAGATGTCGGTTCCC TTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTTATGCCGGGGA CTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATC ATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGA TGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCA ACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAA TACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGA AGCCG 26 (808bp)
III
AGCGATACGGGGCAGACTAGAGTACTGCAGGGGGAGACTGGAATTCCTGGTGTAGCG GTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGT AACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGT CCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAG CTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGA ATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAA GAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTG GCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAA GTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCG TAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGC CCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCC GCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCG ACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGT TCCCGGGTCCTTGTACACACCGCCCGTCACGTCATGAAAGTCNGTAACACCCGAAGNC C 27 (814bp) AGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTG GTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTC TGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACC CTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCG TGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCA ACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTC CCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTG GGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGG GACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCA TCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGC GAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGC AACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGA ATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCG AAGCC 27C (819bp) AACTGTGGGCGTGCGGNCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGG TATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATG CCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCA AGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGG GCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGG
IV
TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCC 33.2B (821bp) ACTNTTGGGCGTGCGGGCGANACNGGCAGACTAGAGTGCTGCAGGGGAGACTGGAAT TCCTGGTGAAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGTCGG GTCTCTGGGCAGTAACTGACGCTAAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTTCCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGG TATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCNTATGTTGCCAGCGGGTAATG CCGNGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCA AGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGG GCTGCGAAGCCGCAAGGTAGAACGAATCCNNTAAAGCTGGTCTCAGTTCGGATTGGGG TNTGCAACTCGACCCCATGAAGTCGGAGTCGTNTAGTAATCGCAGATCACGCAACGCT GCNGNGAATACGTTCCCGGGCCTNGTANACACCGCCCGTCACNTCATGAAAGTCGGTA ACANCCGAGGCC 35 (820bp) CTNTTGGGCGTGCGGGCGANACNGGCAGACTAGAGTGCTGCAGGGGAGACTGGAATT CCTGGTGAAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGTCGGG TCTCTGGGCAGTAACTGACGCTAAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGA TACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGA TCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAA AACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGA TGCAACGCGAAGAACCTTTCCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGT ATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGAT GTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCNTATGTTGCCAGCGGGTAATGC CGNGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGG CTGCGAAGCCGCAAGGTAGAACGAATCCNNTAAAGCTGGTCTCAGTTCGGATTGGGGT NTGCAACTCGACCCCATGAAGTCGGAGTCGTNTAGTAATCGCAGATCACGCAACGCTG CNGNGAATACGTTCCCGGGCCTNGTANACACCGCCCGTCACNTCATGAAAGTCGGTAA CANCCGAGGCC 35A (822bp) AACTGTGNGCGTGCGGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTA
V
AAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGCCGGCAGAGATGTC GGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGCCAGCACGTTATGG TGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGG CTGCGATGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGG TCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGCGTAA CACCCGAAGCCG 36.4A (821bp) AACTGTGGGCGTGCGGNCGATACGGGCAGACTTGANTACTGCAGGGGAGACTNGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACNCCGCCTGGGGAGTACGGCCGCAAGGCTA AAACTCAAAGAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTGCCTAGAGATAGG TATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCTTATGTTGCCAGCGCGTAATGG CGGGGACTCGTGAGAGACTGCCGGGGTCAANTCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAANGG NTGCGATGCCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGG TCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCCG 45A (820bp) AACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGCTGGTAGAGATATC AGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCGCGTTATGG CGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGG CTGCGAATCCGCGAGGTGGAGCGAATCCCTTGAAAGCCGGTCTCAGTTCGGATCGGGG TCTGCAACTCGACCCCGTGAAGTTGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCCG 44 (820bp)
VI
AACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGCTGGTAGAGATATC AGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCTTATGTTGCCAGCGCGTTATGG CGGGGACTCGTAAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAA GTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGG CTGCGAATCCGCGAGGTGGAGCGAATCCCTTGAAAGCCGGTCTCAGTTCGGATCGGGG TCTGCAACTCGACCCCGTGAAGTTGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCCG 50 (819bp) AACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGG TATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATG CCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCA AGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGG GCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGG TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCC 55 (819bp) AACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCGCAGGACGTATCTAGAGATAGG TATTCCCTTGTGGCCTGCGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATG CCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCA AGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGG GCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGG
VII
TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCC 55A (791bp) ACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATAT CAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGC GAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTG GGTACTAGGTGTGGGTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCC CGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAAGGAATTGACGGGGGCCCGCAC AAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGA CATGCACAGGACGCTGGTAGAGATATCAGTTCCCTTGTGGCCTGTGTGCAGGTGGTGC ATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCCTATCTTATGTTGCCAGCGCGTTATGGCGGGGACTCGTAAGAGACTGCCGGGGTCA ACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCA CACATGCTACAATGGCCGGTACAAAGGGCTGCGAATCCGCGAGGTGGAGCGAATCCCT TGAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTTGGAGTC GCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCCG 60 (773bp) AAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGT GGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCG AACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTT TCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACG GTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATG TGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTA CCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCT CGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCC AGCGGGTAATGCCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGG GGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGC CAGTACAGAGGGCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGT TCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATC AGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGA AAGTCGGTAACACCCGAAGCC 60A (800bp) ATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAA TGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGA CGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGC CGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACG CATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACG GGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTT ACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGT GCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCA
VIII
ACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTNGTAGGAGAC TGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGT CCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTG GAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTNTGCAACTCGACCCCATG AAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACNTCATGAAAGTCGGTAACACCCGAAGCC 67 (819bp) AACTGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAAT TCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGG GTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAG ATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGG ATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTA AAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCG ATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGG TATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGA TGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATG CCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCA AGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGG GCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGG TCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGC GGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAAC ACCCGAAGCC 69 (787bp) AGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCA GGAGGAACACCGGTGGCGAAGGCGGGTCTNTGGGCAGTAACTGACGCTGAGGAGCGA AAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGT ACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCG CCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAA GCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACA TGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGCAGGTGGTGCAT GGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC TTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGACTGCCGGGGTCAAC TCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACA CATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGGAGCGAATCCCTT AAAGCTGGTCTCAGTTCGGATTGGGGTTTGCAACTCGACCCCATGAAGTCGGAGTCGC TAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC 69B (780bp) TGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAA CACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTG GGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGT GTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGG AGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGG
IX
AGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCACAG GACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTC GTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCT ATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAG GAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATGCT ACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCT GGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAA TCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTC ACGTCATGAAAGTCGGTAACACCCGAAGCC 70 (816bp) TGTGGGCGTGCGGGCGATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCC TGGTGTAGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTC TCTGGGCAGTAACTGACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATA CCCTGGTAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATC CGTGCCGTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAA CTCAAAGGAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATG CAACGCGAAGAACCTTACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTAT TCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCG GGGACTCGTAGGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGT CATCATGCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCT GCGAAGCCGCAAGGTGGAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCT GCAACTCGACCCCATGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGT GAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACC CGAAGCC 70B (801bp) GATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGA ATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTNTGGGCAGTAACTG ACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACG CCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAAC GCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGAC GGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCT TACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTG TGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGA CTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATG TCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTG GAGCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATG AAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC 76 (799bp) ACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATG CGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACG
X
CTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCG TAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCA TTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGG GGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTA CCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTG CAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAA CGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTNGTAGGAGACT GCCGGGGTCAANTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTC CAGGGCTTCACACATGNTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGG AGCGAATNCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTTTGCAACTCGACCCCATGA AGTCGGAGTCGCTATGTAATNGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC 77 (788bp) AAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGCGCAGATATC AGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGCTGAGGAGCG AAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGGTGG GTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCATTAAGTACCC CGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCAC AAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGA CATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGCAGGTGGTGC ATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAAC CCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGACTGCCGGGGTCA ACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCA CACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGGAGCGAATCCC TTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAAGTCGGAGTC GCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACAC CGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC 78B (784bp) ACTGCAGNGGGGNAGAANNNNTTCTTGTGTAGCGGTGGAATGCGAGATATCAGGAGG AACACCGGTGGCGAAGGCGGGTCTNTCGGCAGTAACTGACGCTGAGGAGCGAAAGCG TGGGGAGCGAACAGGATTAGATACCCTGGTACTCCACGCCGTAAACGGTGGGTACTAG GTGTGGGTTTCCTTCCTTGGAATCCGTGCTAGTAGATAACGCATTAAGTACCCCGCCTG GGGAGTACGGCCGCAAGACTAAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGG CGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACCTGGGTTTGACATGCA CAGGACGCCGGCAGAGATGTCGGTTCCCTTGTGGCCTGTGTGCAGGTGGTGCATGGCT GTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGT CTCATGTTGCCAGCGGGTAATGCCGGGGACTCGTGAGAGACTGCCGGGGTCAACTCGG AGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCCAGGGCTTCACACATG CTACAATGGCCGGTACAAAGGGCTGCGATGCCGTAAGGTTAAGCGAATCCTTTTAAAG CCGGTCTCAGTTCGGATCGGGGTCTGCAACTCGACCCCGTGAAGTCGGAGTCGCTAGT AATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCG TCACGTCATGAAAGTCGGTAACACCCGAAGCC 79 (801bp)
XI
GATACGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGA ATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTG ACGCTGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACG CCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAAC GCATTAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGAC GGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCT TACCTGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTG TGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGC AACGAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGA CTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATG TCCAGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTG GAGCGAATNCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTTTGCAACTCGACCCCATG AAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGC CTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC
82 (813bp) GCGTGCGGGCGATACGGGCAGACTTGAGTACTGCAGGGGAGACTGGAATTCCTGGTGT AGCGGTGGAATGCGCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGG CAGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGG TAGTCCACGCCGTAAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCC GTAGCTAACGCATTAAGTACCCCGCCTGGGGAGTACGGCCGCAAGGCTAAAACTCAAA GAAATTGACGGGGGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGC GAAGAACCTTACCTGGGTTTGACATGCACAGGACGCCGGCAGAGATGTCGGTTCCCTT GTGGCCTGTGTGCAGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTT AAGTCCCGCAACGAGCGCAACCCTTGTCTCATGTTGCCAGCACGTAATGGTGGGGACT CGTGAGAGACTGCCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCAT GCCCCTTATGTCCAGGGCTTCACACATGCTACAATGGCCGGTACAAAGGGCTGCGATG CCGTGAGGTGGAGCGAATCCTTTCAAAGCCGGTCTCAGTTCGGATCGGGGTCTGCAAC TCGACCCCGTGAAGTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATA CGTTCCCGGGCCTTGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAG CCG 83A2 (798bp) TACGGGCAGACTAGAGTACTGCAGGGGAGACTGAATTCCTGGTGTAGCGGTGGAATGC GCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTNTGGGCAGTAACTGACGC TGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGT AAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCAT TAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACC TGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGC AGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGACTG CCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCC AGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGGA
XII
GCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAA GTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCT TGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC 83B2 (797bp) CGGGCAGACTAGAGTACTGCAGGGGAGACTGGAATTCCTGGTGTAGCGGTGGAATGC GCAGATATCAGGAGGAACACCGGTGGCGAAGGCGGGTCTCTGGGCAGTAACTGACGC TGAGGAGCGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGT AAACGGTGGGTACTAGGTGTGGGTTTCCTTCCTTGGGATCCGTGCCGTAGCTAACGCAT TAAGTACCCCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAAAGGAATTGACGGG GGCCCGCACAAGCGGCGGAGCATGTGGATTAATTCGATGCAACGCGAAGAACCTTACC TGGGTTTGACATGCACAGGACGTACCTAGAGATAGGTATTCCCTTGTGGCCTGTGTGC AGGTGGTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAAC GAGCGCAACCCTTGTCCTATGTTGCCAGCGGGTAATGCCGGGGACTCGTAGGAGACTG CCGGGGTCAACTCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGCCCCTTATGTCC AGGGCTTCACACATGCTACAATGGCCAGTACAGAGGGCTGCGAAGCCGCAAGGTGGA GCGAATCCCTTAAAGCTGGTCTCAGTTCGGATTGGGGTCTGCAACTCGACCCCATGAA GTCGGAGTCGCTAGTAATCGCAGATCAGCAACGCTGCGGTGAATACGTTCCCGGGCCT TGTACACACCGCCCGTCACGTCATGAAAGTCGGTAACACCCGAAGCC *All sequences listed are reverse complement strands, so they are presented in 3'- to -5' format.
XIII
Appendix II Archive of Isolates
Isolate Source
Day Found
Description: color/shape/location colony; microscopic appearance
mPCR
FL-MM203-A-1 I
Pink clustered cocci or bacillus Bright yellow, circular colony
-
FL-MM203-B-1 I
Pink bacillus Bright yellow, circular colony
M+
FL-MM203-A-1 III
Pink bacillus White, circular only interior colony
-
FL-MM203-B-1 I
Pink bacillus White, circular only interior colony
M+
6
FL-MN133-A-2 I
Pink & purple rods and cocci White, circular only interior colony
M+
6B
FL-MN133-B-2 I
Pink & purple rods and cocci White, circular only interior colony
M+
7
FL-MN133-B-2 I
Pink, circular spread out among white interior colonies
1
1B
3
3B
XIV
-
Sequencing
M.petroleophilum
M.mucogenicum
8
FL-MN133-A-2 III
17A AZ-MO276-A-1 II
Isolate Source
17
AZ-MO276-B-1 I
Day Found
LATENT yellow
M+
M.avium
Pink bacillus White, somewhat wet;
M+
M.anthracenium
mPCR
Sequencing
M+
M.anthracenium
M+
M.anthracenium
Description: color/shape/location colony; microscopic appearance Pink & blue cocci (bad stain, think negative), bipolar? White, somewhat wet;
17.2 AZ-MO276-?-1 II
LATENT yellow
18A AZ-MO276-A-1 I
Large pink & blue vacuoles Grey-White, eruptive mold?
-
Pink & blue cocci (bad stain) Grey-White, eruptive mold?
Lost
Blue rods & some pink cocci pink
-
18
AZ-MO276-B-1 I
20A AZ-MO117-A-2 III
20
AZ-MO117-B-2 II
Blue rod & pink cocci - Not alone pink
XV
M+
21
Pink rods bipolar stain? white
AZ-MO117-A-2 II
Pink rods white Pink rods (bipolar stained some) look just like #21 white, eruptive, cloudlike
21B AZ-MO117-B-2 I
26
MH-MO258-B-1 III
Isolate Source
27A MH-MO258-A-1 II
27
MH-MO258-B-1 II
27C MH-MO258-A-1 II
29
MH-MO258-A-1I
33.2A MH-MO113-A-1
Day Found
Description: color/shape/location colony; microscopic appearance Pink rods & some blue cocci very small white Pink rods (bipolar?) like #21 and #26 chaining very small white Too few on stained slide to tell Bright white, singular among little tmtc/smooth together white bkgd. Pink big outlined in blue too loarge for MO/bacteria? cream like, big watery
White
M+
M.mucogenicum
M+
M.mucogenicum
M+
M.chelonae
mPCR
Sequencing
M+
M+
M.chelonae
M+
M.chelonae
-
Lost
XVI
33.2B MH-MO113-B-1
White
M+
Pinkish/purple small rods bright yellow, big, wet
M+
PA-MO150-B-1 II
Pinkish/purple small rods bright yellow, big, wet
M+
M.chelonae
PA-MO150 B-1 III
Pink rods yellow, eruptive
M+
M.petroleophilum
mPCR
Sequencing
35A PA-MO150-A-1 II
35
36
Isolate Source
Day Found
Description: color/shape/location colony; microscopic appearance
36.2 PA-MO150-A-1
Orange sectioned
36.3 PA-MO150-B-1
Pink watery
M.chelonae
M+
-
36.4A PA-M0150-A-1
White
M+
36.4B PA-M0150-B-2
White
M+
XVII
M.neglectum
40
PA-MP194-B-2 II
43
PA-MP194-A-2 III
Chained blue rods, some big & smaller pink rods (some in chains), together w/ #41 below yellow, eruptive Most large, not bacteria & some blue rods in chains, & some pink rods in chains large cream colored
M+
M+
43.2A PA-MP194-A-2
White
-
43.2B PA-MP194-B-3
White
-
Pink rods & some blue orange eruptive, white center
44A SC-MP292-A-1 II
Isolate Source
44
SC-MP292-B-1 I
45A SC-MP292-A-1 II
45
SC-MP292-B-1 I
Day Found
Description: color/shape/location colony; microscopic appearance
M+
mPCR
Sequencing
Large purple rods & small pink rods orange eruptive, white center
M+
M.anthracenium
Pink & blue rods yellow circular, creamy
M+
M.anthracenium
Pink rods (some purple-ish) yellow circular, creamy
XVIII
-
46A SC-MP292-A-1 II
46
SC-MP292-B-1 I
48A MD-MP254-A-1 II
48
MD-MP254-B-1 II
Small pink rods white circular, creamy
M+
Pink rods cream colored eruptive
M+
Large capsule/cylinder; light pink (57,58) cream colored eruptive
M+
Small pink rods/some purple-ish large white, creamy
MD-MP254-B-1 III
Isolate Source
M+
Large white, creamy (difficult to separate from pink #49)
50A MD-MP254-A-1 II
50
Small pink rods white circular, creamy
Day Found
Description: color/shape/location colony; microscopic appearance
51
MD-MP173-A-2 III
Pink rods latent dark yellow, creamy, difficult to isolate
53
SC-MP196-B-2 III
Small purple/pink rods very small bright yellow, hard to isolate
XIX
-
M+
M.chelonae
mPCR
Sequencing
M+
-
55A SC-MP196-A-2 II
Blue rods & cocci large, white & eruptive, hard to isolate
M+
M.anthracenium
55
SC-MP196-B-2 III
Small pink rods large, white & eruptive, hard to isolate
M+
M.salmoniphilum
56
SC-MP196-B-2 II
Pink & purple small rods - aggregates white, circular grouped/sectioned
-
Bacillus lichenformis
57A SC-MP196-A-2 I
57
HI-MP296-B-1 III
HI-MP296-B-1 III
Isolate Source
59A HI-MP296-A-1 II
M+
Small pink rods & large pink cylinder/capsule white, creamy, flat (48,58)
M+
Large pink cylinder/capsules (48,57) star-shaped eruptive cream Large pink cylinder/capsules (48,57), some small pink rods star-shaped eruptive cream
58A HI-MP296-A-1 I
58
Large pink cylinder/capsule white, creamy, flat (48,58)
Day Found
Description: color/shape/location colony; microscopic appearance Large pink vacuoles dark orange, flat, hard to isolate from white
XX
M+
M+
mPCR
M+
Sequencing
59
Small pink rods (some in chains), pink cocci dark orange, flat, hard to isolate from white
HI-MP296-B-1 III
-
Pink rods white
M+
M.chelonae
HI-MP172-B-2 II
Pink rods (some bipolar) white
M+
M.chelonae
62
HI-MP172-B-2 I
Mix of pink rods & big pink vacuoles cream
M+
65
NM-MP181-A-1 II
Large pink vacuoles red (-> pink? Day 14), same as pink
-
65B NM-MP181-B-1 II
Large pink & blue vacuoles red (-> pink? Day 14), same as pink
-
60A HI-MP172-A-2 II
60
66
Blue rods & pink vacuoles- Not Isolated white
NM-MP181-A-1 I
Pink rods white
67A TN-MP241-A-1 II
Isolate Source
Day Found
M+
-
Description: color/shape/location colony; microscopic appearance
XXI
mPCR
Sequencing
67
TN-MP241-B-1 I
Pink rods white
68
TN-MP241-A-1 I
Pink vacuoles pink (outside)
-
Blue rods & vacuoles pink (outside)
-
68B TN-MP241-B-1 III
M+
M.chelonae
Pink rods "ringworm" cream (white before?)
M+
M.chelonae
Pink bipolar rods "ringworm" cream (white before?)
M+
M.chelonae
Pink rods symmetrically sectioned white
M+
M.chelonae
70B TN-MP241-B-1 I
Pink rods symmetrically sectioned white
M+
M.chelonae
71A TN-MP241-A-1 I
Large pink rods (??yellow isolate) yellow
-
Too few on slide to tell yellow
-
69
TN-MP241-A-1 II
69B TN-MP241-B-1 I
70
71
TN-MP241-A-1 III
TN-MP241-B-1 I
XXII
Isolate Source
72
TN-MP278-A-2 III
72B TN-MP278-B-2 I
73
TN-MP278-A-2 III
75A TN-MP278-A-2 III
Day Found
Description: color/shape/location colony; microscopic appearance Pink rods white, became pointed at day 50 (maybe same as 76) Large pink rods white, became pointed at day 50 (maybe same as 76) Pink rods green/yellow coverage Pink rods yellow
mPCR
Sequencing
-
-
-
Lost
TN-MP278-B-2 III
Pink rods yellow Tiny pink rods "ringworm" sectioned cream (maybe same as 72)
M+
M.chelonae
77
CO-MQ150-A
Pink rods white
M+
M.chelonae
78
CO-MQ150-A
Pink rods yellow
M+
75
TN-MP278-B-2 III
76
XXIII
-
78B CO-MQ150-B
Isolate Source
LATENT yellow Day Found
Description: color/shape/location colony; microscopic appearance
CO-MQ150-A
Pink rods cream
81
CO-MR150-A-2-II
Small pink cocci/rods White accumulation of very tiny colonies?
82
CO-MR150-B-I
Pink rods and cocci (or some bipolar staining); sectioned yellow (14)
79
83A OR-MS92-A-I
Pink rods & blue cocci White, wet
83A2 OR-MS92-A-III
Pink rods Bright white, raised circular
83B OR-MS92-A-I
Pink rods & blue cocci or rods White, wet
83B2 OR-MS92-A-II
Pink rods Bright white, raised circular
XXIV
M+
M.lentiflavum
mPCR
Sequencing
M+
M.chelonae
-
M+
M.petroleophilum
-
M+
M.chelonae
-
M+
M.chelonae
84A OR-MS92-A-I
Mostly blue rods & some pink Pink on outside perimeter, difficult to isolate
-
84B OR-MS92-B-I
Blue rods Pink on outside perimeter
-
Isolate Source
85A OR-MS92-A-I
85B OR-MS92-B-I
Day Found
Description: color/shape/location colony; microscopic appearance Mostly blue cocci, some pink rods Yellow/Orange, difficult to isolate Mostly pink rods along with blue cocci Yellow/Orange, very difficult to isolate (smeared in white surrounding colonies by accident)
XXV
mPCR
-
-
Sequencing
