Pathogenesis of Bacterial Infections in Animals (Gyles/Pathogenesis of Bacterial Infections in Animals) || Mycobacterium

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<ul><li><p>113</p><p> 7 Mycobacterium </p><p> I. Olsen , R. G. Barletta , and C. O. Thoen </p><p> INTRODUCTION Mycobacteria belong to the Order Actinomycetales , Family Mycobacteriaceae . The genus Mycobac-terium includes the Mycobacterium tuberculosis and Mycobacterium avium complexes, other patho-genic mycobacteria, and numerous species of sap-rophytic microorganisms present in soil and water. The Mycobacterium tuberculosis complex includes M. tuberculosis , M. africanum , M. canettii , M. bovis , M. pinnipedii , M. caprae , and M. microti (fi g. 7.1 A ). The M. avium complex includes M. avium subsp. avium , M. avium subsp. hominissuis , M. avium subsp. paratuberculosis , and M. intracellu-lare (fi g. 7.1 B). Some other mycobacteria of clinical signifi cance are Mycobacterium chelonae , Myco-bacterium fortuitum , Mycobacterium kansasii , Myco bacterium leprae , Mycobacterium marinum , Mycobacterium ulcerans , and Mycobacterium scrofulaceum . </p><p> CHARACTERISTICS AND SOURCES OF THE ORGANISMS Mycobacteria are obligate aerobes, nonspore forming and nonmotile bacilli, and are 0.6 1.0 1.0 10 m in size. Their high cell wall lipid content excludes standard aniline dyes, so that once stained with special staining procedures, mycobac-teria are resistant to decolorization even by acid alcohol. This property is termed acid fastness, so that mycobacteria are commonly referred to as acid - fast bacilli. In contrast, these microorganisms are not readily stained with the Gram method and are considered weakly gram - positive. Growth rates </p><p>for mycobacteria are slow, with generation times ranging from 2 to more than 20 h. Based on different generation times, mycobacteria can be divided into slow and rapid growers. Slow growers require more than 7 days to form visible colonies on solid medium, whereas rapid growers form colonies within 7 days (Holt et al. 1994 ). </p><p> Tuberculosis remains the leading cause of death in humans caused by a single infectious agent, being responsible for nearly 2 million deaths annu-ally. It is caused primarily by M. tuberculosis ; M. africanum , M. bovis , and M. canettii account for less than 1% of tuberculosis in humans (Thoen and LoBue 2007; Thoen et al. 2009 ). In addition to the host adaptation of M. bovis largely to cattle, other host - adapted variants of M. bovis have been desig-nated (Hewinson et al. 2006 ), such as M. pinnipedii (seal - adapted) and M. caprae (goat - adapted). Interestingly, M. bovis bacille Calmette - G erin (BCG), attenuated by in vitro passages on potato slices, is used to vaccinate humans throughout the world. M. africanum and M. canettii are human pathogens. Mycobacterium microti has been iso-lated from humans, voles, and some other animals (Thoen et al. 2009 ). Pathogenic mycobacteria all produce granulomatous lesions in tissues of humans and a wide range of domestic and wild animals. Although the tubercle bacillus was discovered more than 120 years ago, defi nitive information on its pathogenesis is not yet available, although understanding is developing at a remarkable rate. Unex plained differences in susceptibility of differ-ent animals to various acid - fast bacilli occur (Thoen and Barletta 2004 ). M. tuberculosis , the human </p><p>Pathogenesis of Bacterial Infections in Animals, Fourth EditionEdited by C. L. Gyles, J. F. Prescott, J. G. Songer, and C. O. Thoen 2010 Blackwell Publishing. ISBN: 978-0-813-81237-3</p></li><li><p>114 Chapter 7</p><p> Figure 7.1. Proposed evolution of the M. tuberculosis and M. avium complexes The tree diagrams for the proposed evolution of members of the M. tuberculosis (A) and M. avium (B) complexes are shown. See text for further details and citations. </p><p>M. tuberculosiscomplex</p><p>M. canettii(humans)</p><p>M. tuberculosis(humans)</p><p>M. africanum(humans)</p><p>M. microti(rodents)</p><p>M. bovis(humans,ruminants)</p><p>M. pinnipedii(fin-footedmammals)</p><p>M. caprae(goats)</p><p>(A)</p><p>M. aviumcomplex</p><p>M. avium subsp.hominissuis</p><p>(humans, porcine,ruminants)</p><p>M. avium subsp.paratuberculosis</p><p>(ruminants)</p><p>Cattle strains Sheep strains</p><p>M. aviumsubsp.avium</p><p>(birds)</p><p>M. intracellulare(birds, humans)</p><p>(B)</p><p>tubercle bacillus, produces progressive generalized disease in nonhuman primates, dogs, swine, and guinea pigs, although cattle and cats are quite resis-tant (Thoen 2010 ). M. tuberculosis may induce tuberculin skin sensitivity in cattle and other ani-mals. M. bovis , the agent of bovine tuberculosis, is a slow - growing nonphotochromogenic organism that also causes disease in other domestic and wild animals, and has been reported in humans in several countries (Thoen et al. 2006 ; Thoen et al. 2009 ). Biochemical tests are available for differentiating bacteria of the M. tuberculosis complex, but molec-ular techniques are now widely used in reference </p><p>laboratories around the globe (Harris 2006 ; Thoen et al. 2009 ). </p><p> Mycobacterium leprae , the cause of leprosy (also known as Hansen s disease), is a chronic granulo-matous disease. Leprosy is still considered a public health problem in countries in Africa and Southeast Asia, with a prevalence rate of more than 1 case per 10,000 individuals (Meima et al. 2004 ). M. leprae and leprosy has been identifi ed in armadillos in the United States. Mycobacterium lepraemurium has been isolated from leprosy - like lesions in cats, rats, and mice. Mycobacterium chelonei, M. intracellulare , M. marinum , M. nonchromo-</p></li><li><p> Mycobacterium 115</p><p>genicum , and certain other mycobacteria have been isolated from granulomatous lesions in cold - blooded animals. </p><p> Microorganisms of the M. avium complex have the widest host range among all mycobacteria. M. avium subsp. avium (serovars 1, 2, and 3) are iso-lated from tuberculous lesions in humans, birds, domestic, and wild animals (Thoen et al. 1981 ). In birds, disease is usually progressive, with lesions in the liver and spleen, whereas lesions in other animals are usually confi ned to lymph nodes asso-ciated with the intestinal tract. M. avium subsp. hominissuis is the subspecies most frequently cau-sing lesions in humans and swine, and M . intra-cellulare is widely distributed in the environment, causing granulomatous lesions mainly in cold - blooded animals (Thoen 2010 ). Rabbits are highly susceptible to experimental infection with M. avium subsp. avium , but relatively resistant to M. intracel-lulare. Interestingly, birds are susceptible to M. avium subsp. avium but resistant to infection by members of the M. tuberculosis complex (Thoen and Barletta 2004 ). </p><p> Mycobacterium avium subsp. paratuberculosis causes a transmissible intestinal disorder of rumi-nants commonly known as Johne s disease that has a signifi cant economic impact on the livestock industry (Harris and Barletta 2001 ). Cattle, sheep, goats, and certain wild ruminants are susceptible. In addition, it has been suggested that this microorgan-ism may be the etiologic agent of Crohn s disease, an infl ammatory bowel disease in humans (Chacon et al. 2004 ). However, this issue still remains controversial. A characteristic that is useful in differentiating this organism is its dependency on mycobactin, an iron - chelating agent, for in vitro growth. Mycobactin was initially extracted from Mycobacterium phlei , but later mycobactin J and certain extracellular iron - binding compounds were isolated from M. avium . Molecular techniques such as PCR and restriction endonuclease analysis have been developed for identifying M. avium subsp. paratuberculosis (Harris and Barletta 2001 ). </p><p> Mycobacterium ulcerans causes chronic skin ulcers in humans, termed Buruli ulcer or Bairnsdale ulcer. These lesions are caused by the effects of mycolactone, a polyketide - derived macrolide iso-lated from M. ulcerans (George et al. 1999 ). Studies have pointed to water insects from the family Naucoridae as a possible vector for the transmission of M. ulcerans (Marsollier et al. 2002 ). M. marinum </p><p>causes tuberculosis in fi sh and amphibians as well as cutaneous granulomatous disease in humans, known as swimming pool granuloma. </p><p> Other species of mycobacteria have been iso-lated from various animals (Thoen et al. 1981 ). Mycobacterium fortuitum , a rapid - growing, non-chromogenic organism has been isolated from humans and dogs with lung lesions, cattle with mastitis, and lymph nodes of slaughter cattle and swine. Mycobacterium chelonae, also a rapid grower, has been isolated from swine and humans. Granulomatous lesions in swine and cattle, which closely resemble lesions caused by M. bovis , have been reportedly caused by M. kansasii , a slow - growing, photochromogenic organism. </p><p> BACTERIAL VIRULENCE FACTORS In recent years, remarkable progress has been made in understanding the basis of virulence and the pathogenesis of mycobacterial infections particu-larly through the application of whole genome sequencing and comparative genomic analysis. </p><p> Genomics The science of genomics has made possible the elu-cidation of the complete genetic blueprint of several mycobacterial species of importance in human and veterinary medicine, as well as environmental species. Complete genome sequences are now available for Mycobacterium abscessus , M. avium subsp. hominissuis, M. avium subsp. paratuberculo-sis , M. bovis, M. bovis BCG, Mycobacterium gilvum , M. leprae , M. marinum, Mycobacterium smegmatis , three strains of M. tuberculosis , M. ulcerans , Mycobacterium vanbaalenii , and four mycobacteria unclassifi ed at the species level These genomes possess high GC content ( 65%). Major fi ndings for two mycobacterial species of veterinary importance are reported below. </p><p> Sequencing and annotation of the M. avium subsp. paratuberculosis genome from strain K - 10, isolated from a cow with Johne s disease, have been com-pleted (Li et al. 2005 ). This strain had a low number of in vitro passages, and a genetic system including transposon mutagenesis is available for the creation of mutant strains (Foley - Thomas et al. 1995 ; Harris et al. 1999 ). The K - 10 genome is a circular chromo-some of about 4.8 Mb encoding 4344 open reading frames (ORFs) with a 69.3% GC content. About 60% of the ORFs have known homologues in </p></li><li><p>116 Chapter 7</p><p>databases while 25% encode putative proteins of unknown functions. About 75% of the M. avium subsp. paratuberculosis genes have counterparts in M. tuberculosis , and although most genes have orthologs in M. avium subsp. hominissuis , there are 39 predicted proteins that are unique to M. avium subsp. paratuberculosis. ORFs are identifi ed by a location number following standard conventions (e.g., M. avium subsp. paratuberculosis 1152 signi-fi es ORF1152 from the ORF0001 DnaA in the clockwise direction; see also Wu et al. 2009 for corrections to the original assembly). </p><p> The genome possesses high redundancy because of gene duplication, especially for genes involved in lipid and redox metabolism. Nonetheless, differ-ences from other mycobacterial genomes are noted in the low abundance of PE and PPE families in M. avium subsp . paratuberculosis . In addition, the salicyl - MP ligase gene ( mbtA ) is truncated, which is likely the basis of its defect in mycobactin bio-synthesis. Analysis of genetic polymorphisms, espe-cially those including large sequences, indicates that M. avium subsp. paratuberculosis originated from M. avium subsp. hominissuis in a biphasic evolu-tionary process (Alexander et al. 2009 ). First, an original pathogenic clone of M. avium subsp. para-tuberculosis arose by acquisition of novel DNA and polymorphisms. Second, sheep and cattle strains arose from this ancestral clone by subsequent lineage - specifi c deletion events. </p><p> Genome sequencing demonstrated that the M. bovis genome (4 345 492 bp for the virulent bovine isolate AF2122/97) is a down - sized version of the genome of M. tuberculosis (4 411 532 bp for the human isolate H37Rv), with more than 99.95% identity and no new genetic material as compared to M. tuberculosis (Garnier et al. 2003 ). Thus, DNA deletions in M. bovis are the major contributors to these differences, which have been found to affect genes involved in transport, cell surface structures, and intermediary metabolism. These deletions may remove genes that are unnecessary for host adapta-tion and lead to a different and sometimes even wider host range. Point mutations also play a role in defi ning the phenotype, as it is the case for M. bovis resistance to pyrazimamide. In addition, sequence variations have been found in genes coding for cell wall and secreted proteins, such as the PE_PGRS and PPE protein families. Another notable change is a mutational event in the M. bovis pyruvate kinase gene that renders M. bovis unable </p><p>to use glycerol as a carbon source. Other sequence changes involve master regulatory genes controlling the expression of multiple gene families. The analy-sis of the M. bovis genome challenged the epidemio-logical hypothesis that M. tuberculosis was a human - adapted variety of M. bovis that was acquired from cattle. The irreversible loss of DNA material uncovered by the M. bovis genome sequencing and the systematic analysis of polymorphisms in a large panel of strains led to the new paradigm that M. canettii is likely the ancestral species of the M. tuberculosis complex. Successive DNA deletions, starting by the loss of region RD9 (RD stands for regions of difference), led to differentiation of M. africanum , M. bovis , and M. microti. Moreover, M. bovis BCG experienced further deletions during in vitro laboratory adaptation and its loss of region RD1 has been implicated as the mechanism of viru-lence attenuation. Thus, similarly to M. avium subsp . paratuberculosis , evolution of mycobacterial genomes involves a dominant process of reductive deletions. Based on comparative genomic studies, fi g. 7.1 depicts an overall tree diagram for the evolution of mycobacterial genomes (Brosch et al. 2002 ; Devulder et al. 2005 ; Mueller et al. 2008 ; Alexander et al. 2009 ). </p><p> Genome sequencing of mycobacteria has been enhanced by parallel developments in the genetic systems used to create defi ned mutants and to elu-cidate the function of each gene in the pathophysiol-ogy of mycobacterial infections (Braunstein et al. 2002 ). Both plasmid and mycobacteriophage vectors have been used extensively to create recombinant or mutant strains using all means of genetic exchange, including transformation, transduction, and con-jugation. A variety of reporter genes have been expressed in mycobacterial species including beta - galactosidase, fi refl y luciferase, and the green fl uorescent protein. In addition, several vectors for conditional or antisense expression in mycobacterial systems are now available. Mycobacteriophage vectors have been...</p></li></ul>