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Year : 2018  |  Volume : 7  |  Issue : 1  |  Page : 48-52

Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model

Department of Translational Research and New Technologies in Medicine and Surgery, University of Pisa, Pisa, Italy

Date of Web Publication7-Mar-2018

Correspondence Address:
prof Laura Rindi
Dipartimento di Ricerca Traslazionale e Delle Nuove Tecnologie in Medicina e Chirurgia, Via San Zeno 35/39, 56127 Pisa
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ijmy.ijmy_11_18

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Background: Mycobacterium avium subsp. hominissuis (MAH) is an environmental opportunistic pathogen for humans and swine worldwide; in humans, the vast majority of MAH infections is due to strains belonging to specific genotypes, such as the internal transcribed spacer (ITS)-sequevars Mav-A and Mav-B that mostly cause pulmonary infections in elderly patients and severe disseminated infections in acquired immunodeficiency syndrome patients, respectively. To test whether the different types of infections in distinct patients' populations might reflect a different virulence of the infecting genotypes, MAH human isolates, genotyped by ITS sequencing and MIRU-VNTR minisatellite analysis, were studied for the capacity to infect and replicate in human macrophages in vitro. Methods: Cultures of human peripheral blood mononuclear cells and phagocytic human leukemic cell line THP-1 cells were infected with each MAH isolate and intracellular colony-forming units (CFU) were determined. Results: At 2 h after infection, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types. At 5 days, Mav-A organisms, sharing highly related VNTR-MIRU genotypes, yielded numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types. MIRU-VNTR-based minimum spanning tree analysis of the MAH isolates showed a divergent phylogenetic pathway of Mav-A and Mav-B organisms. Conclusion: Mav-A and Mav-B sequevars might have evolved different pathogenetic properties that might account for their association with different human infections.

Keywords: Genotyping, mycobacterial interspersed repetitive unit-variable-number tandem repeat typing, macrophage infection, Mycobacterium avium subsp. hominissuis, virulence

How to cite this article:
Rindi L, Lari N, Garzelli C. Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model. Int J Mycobacteriol 2018;7:48-52

How to cite this URL:
Rindi L, Lari N, Garzelli C. Virulence of Mycobacterium avium Subsp. hominissuis Human Isolates in an in vitro Macrophage Infection Model. Int J Mycobacteriol [serial online] 2018 [cited 2022 Jul 4];7:48-52. Available from: https://www.ijmyco.org/text.asp?2018/7/1/48/226771

  Introduction Top

Mycobacterium avium subsp. hominissuis (MAH), one of the four subspecies of M. avium, is an opportunistic facultative intracellular pathogen for humans and swine worldwide and occasionally for other animals.[1],[2] Soil and water are the natural reservoirs of the organism,[3],[4] so that human infections are usually triggered by exposure to environmental clones[5],[6] that seem to be endowed with a remarkable heterogeneity in virulence.[7],[8]

Among the methods developed to define the molecular profiles of pathogenic clones for humans, genotyping based on minisatellite sequences of repetitive elements dispersed in the genome mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR) turned out to be highly discriminative for studies on epidemiology, pathophysiology, and biodiversity of MAH infections.[9],[10],[11],[12] Another molecular approach, based on the sequence of the 16S-23S ribosomal (rDNA) internal transcribed spacer (ITS),[13] that classifies M. avium into sequevars named Mav-A to Mav-H, is of practical epidemiological interest as the vast majority of MAH infections in humans are due to strains belonging to Mav-A and Mav-B sequevars.[14],[15],[16] In particular, Mav-A organisms mostly cause pulmonary infections in elderly patients and cervical lymphadenitis in children, while Mav-B organisms are almost exclusively restricted to severe disseminated infections occurring in a high proportion of severely immunocompromised acquired immunodeficiency syndrome patients.[17],[18],[19] The reason why Mav-A and Mav-B MAH organisms targets distinct patients' populations causing different types of infections are unknown, but it might reflect a different virulence of the infecting clones. To investigate the possible differential pathogenicity of Mav-A and Mav-B organisms, based on the assumption that macrophage infection represents a key event in MAH immunity,[20],[21],[22] we studied a set of MAH human isolates of different sequevars and MIRU-VNTR genotypes for their capacity to infect and replicate in vitro in human macrophages, such as blood monocytes and cells of the THP-1 cell line.

  Methods Top

Study strains and molecular typing

A set of 23 MAH strains, identified by AccuProbe and/or InnoLipa probes and by a multiplex polymerase chain reaction (PCR) designed to discriminate MAC organisms,[23] isolated in the Laboratory of Clinical Mycobacteriology of the Santa Chiara University Hospital of Pisa, Italy, from the same number of patients, were studied. Ethics approval was not needed for in vitro experimental research not involving humans or animals in our Institution; written informed consent was obtained from volunteer blood cell donors.

Mav subgrouping was determined by sequencing the 16S-23S rDNA ITS region as described by Frothingham and Wilson,[13] with slight modifications.[16] MIRU-VNTR typing was performed by PCR using specific primers for the eight loci identified as polymorphic for M. avium subsp. paratuberculosis K10 and coded 32, 292, X3, 25, 3, 7, 10, and 47, as described previously.[24]

Human monocytes and THP-1 cells

Peripheral blood mononuclear cells (PBMC) from healthy individuals were isolated by a density gradient according to standard procedures and then plated in 24-well plates at 1 × 106 cells/well in Roswell Park Memorial Institute (RPMI) medium. After 3 h, nonadherent cells were removed with two washes of RPMI, and the cells were reincubated in RPMI with 10% human serum, 2 mM L-glutamine, 100 U/ml penicillin, 100 g/ml streptomycin, and 0.25 g/ml fungizone (Sigma). Monocytes were then incubated at 37°C without change of medium for 3 days. Before infection, cells were washed twice with phosphate-buffered saline (PBS) and 1 ml of fresh culture medium without antibiotics was added.

The human phagocytic monocyte cell line THP-1[25] was maintained in culture medium consisting of RPMI supplemented with 5% fetal bovine serum, 2 mM L-glutamine, and antibiotic/antimycotic solution. For infection, 1 × 105 cells per well were plated in 2 ml of culture medium in the presence of 40 nM phorbol 12-myristate 13-acetate (PMA, Sigma) in 24-well plates. Two days later, cells were washed twice to remove PMA, and 1 ml of fresh culture medium without antibiotics was added.

Inoculum preparations and cell infection

MAH strains were grown at 37°C in Middlebrook 7H9 medium, washed twice in PBS, passed through an insulin-syringe, and resuspended in PBS to an optical density corresponding approximately to 100 × 106 colony-forming units (CFU) per ml. Cell cultures were infected at a multiplicity of infection (MOI) of approximately 50 bacteria per macrophage resuspended in 1 ml of antibiotic-free culture medium. After 2 h, cultures were washed three times to remove nonphagocytosed bacteria and further incubated at 37°C in antibiotic-free culture medium. Intracellular CFUs were counted at 2 h and 5 days after cell infection by lysing cultured cells for 15 min with 1 ml of 0.1% Tween 80 and plating appropriate aliquots onto Middlebrook 7H10 agar medium.

  Results and Discussion Top

[Table 1] reports the characteristics of MAH clinical isolates used in this study. Mav-A organisms included a total of 10 isolates, 5 from HIV-positive, and 5 HIV-negative individuals; 4 isolates were from sputum, 2 from urine, 1 from blood, CFS, pleural fluid and stools, respectively; Mav-B organisms included a total of 13 isolates, all from HIV-positive individuals (11 from blood, 2 from respiratory specimens).
Table 1: Characteristics of Mycobacterium avium subspp. hominissuis clinical isolatesa

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MIRU-VNTR typing of isolates detected 8 distinct patterns, 2 of which in single isolates and 6 shared by 2 or more isolates (clusters). The relationships between the MIRU-VNTR patterns were visualized by constructing the minimum spanning tree (MST) illustrated in [Figure 1]. This analysis shows that all but one MIRU-VNTR patterns, occurring either as clustered or unique isolates, differed from the nearest one for one allelic variation thus indicating the high relatedness of strains, as previously reported;[12] isolates of sequevars Mav-A and Mav-B segregated at the sides of the tree and were separated by a cluster including both Mav-A and Mav-B organisms.
Figure 1: Minimum spanning tree based on variable-number tandem repeat patterns of a set of 8 mycobacterial interspersed repetitive unit loci of 23 Mycobacterium avium subsp. hominissuis clinical isolates. Circles include isolates sharing a given mycobacterial interspersed repetitive unit-variable-number tandem repeat pattern (the number is given in parenthesis); circle callouts report the mycobacterial interspersed repetitive unit-variable-number tandem repeat pattern expressed as a string of 8 numbers, each representing the number of tandem repeats at the variable-number tandem repeat loci 32, 292, X3, 25, 3, 7, 10 and 47. Light grey and dark grey circles include isolates exclusively of genotype Mav-A and Mav-B, respectively; white circle includes isolates of Mav-A and Mav-B. Numbers next to the branches connecting circles indicate the level of changes induced by loss or gain of variable-number tandem repeat copies at a given locus, yielding a change from one allele to another

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To test the capacity of MAH isolates to infect and replicate in human phagocytes, cultures of PBMC and THP-1 cells were infected at a MOI of 50:1 with each isolate and intracellular CFUs were determined at 2 h and 5 days postinfection. At 2 h, i.e., immediately after cell entry, the numbers of intracellular bacteria did not differ between Mav-A and Mav-B organisms in both phagocytic cell types (data not shown). At 5 days, Mav-A organisms yielded mean numbers of intracellular CFUs significantly higher than Mav-B organisms in both phagocytic cell types (P = 0.003 for PBMC; P = 0.015 for THP-1 cells, by Student's t-test) [Figure 2]. Notably, in preliminary experiments M. avium Mav-C isolates of environmental (stream water) source, not identified at the subspecies level, tested in the macrophage infection assay, turned out to be much less efficient in macrophage infection as they were rapidly phagocytosed and killed by PBMC and THP-1 cells (data not shown). It is, therefore, reasonable to assume that Mav-A and Mav-B ITS sequevars represent distinct MAH subgroups that have evolved different capabilities to infect and replicates in human macrophages. Actually, divergent phylogenetic pathways of sequevar Mav-A and Mav-B isolates are also suggested by the MIRU-VNTR-based MST analysis [Figure 1]; indeed, clustered isolates with MIRU-VNTR profiles 80531222 and 80531223 (all of Mav-A) yielded numbers of intracellular CFUs >10-fold higher than 82421222 isolates (Mav-B) in both phagocytic cell types, while clustered isolates with MIRU-VNTR profile 82521222, including both Mav-A and Mav-B isolates, yielded intermediate numbers of intracellular CFUs.
Figure 2: Phagocytosis of Mycobacterium avium subsp. hominissuis human isolates of Mav-A, Mav-B genotype by peripheral blood mononuclear cells (left panel) and THP-1 cells (right panel). Cultures of Peripheral blood mononuclear cells and THP-1 cells were infected at a multiplicity of infection of 50:1 and intracellular colony forming units were counted in cell lysates at 5 days after infection. Each circle represents the mean Log10of intracellular colony forming units determined in triplicate cultures for each isolate. Horizontal continuous and dashed lines represent mean ± standard deviation of intracellular colony-forming units of the tested isolates for each Mav genotype

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Although our results indicate that different MAH genotypes differ in the capacity of infection and replication in human macrophages, the demonstration of a straightforward link with virulence for humans of the MAH subgroups will require the comparative investigation of specific virulence factors and immunogenicity of the distinct genotypes. Some studies have described that M. avium isolates of different subspecies possess genetically distinct features that may be associated with pathogenetic characteristics[26] and show different behaviors during human macrophage infection in terms of cytokine responses and ability to replicate intracellularly.[27],[28] However, only a few studies have reported the association of specific MAH genotypes to certain virulence factors, for example, we showed that M. avium Mav-A and Mav-B isolates of human origin were strong producers of a magnesium-dependent, cell-wall associated haemolysin, a potential virulence factor, as compared to environmental Mav-C organisms;[29] more recently, specific virulence factors of the mammalian cell entry family have been reported in highly virulent MAH isolates sharing the MST22 genotype which also displayed suppression of the interferon-γ and interleukin (IL)-17 responses, and increased IL-10 production.[8] Whole-genome sequencing-based analysis of representative human, animal, and environmental MAH isolates will likely clarify at the genome level the possible link of virulence with the isolate genotype and the association of MAH genotypes with different human infections.

Financial support and sponsorship

This work was financially supported by the University of Pisa, Grant “Fondi di Ateneo, 2014–2015.”

Conflicts of interest

There are no conflicts of interest.

  References Top

Turenne CY, Wallace R Jr., Behr MA. Mycobacterium avium in the postgenomic era. Clin Microbiol Rev 2007;20:205-29.  Back to cited text no. 1
Rindi L, Garzelli C. Genetic diversity and phylogeny of Mycobacterium avium. Infect Genet Evol 2014;21:375-83.  Back to cited text no. 2
Hilborn ED, Covert TC, Yakrus MA, Harris SI, Donnelly SF, Rice EW, et al. Persistence of nontuberculous mycobacteria in a drinking water system after addition of filtration treatment. Appl Environ Microbiol 2006;72:5864-9.  Back to cited text no. 3
Vaerewijck MJ, Huys G, Palomino JC, Swings J, Portaels F. Mycobacteria in drinking water distribution systems: Ecology and significance for human health. FEMS Microbiol Rev 2005;29:911-34.  Back to cited text no. 4
von Reyn CF, Maslow JN, Barber TW, Falkinham JO 3rd, Arbeit RD. Persistent colonisation of potable water as a source of Mycobacterium avium infection in AIDS. Lancet 1994;343:1137-41.  Back to cited text no. 5
Alkinham JO 3rd. Ecology of nontuberculous mycobacteria – Where do human infections come from? Semin Respir Crit Care Med 2013;34:95-102.  Back to cited text no. 6
Bruffaerts N, Vluggen C, Duytschaever L, Mathys V, Saegerman C, Chapeira O, et al. Genome sequences of four strains of Mycobacterium avium subsp. Hominissuis, isolated from swine and humans, differing in virulence in a murine intranasal infection model. Genome Announc 4:e00533-16. doi:10.1128/genomeA.00533-16.  Back to cited text no. 7
Bruffaerts N, Vluggen C, Roupie V, Duytschaever L, Van den Poel C, Denoël J, et al. Virulence and immunogenicity of genetically defined human and porcine isolates of M. avium subsp. Hominissuis in an experimental mouse infection. PLoS One 2017;12:e0171895.  Back to cited text no. 8
Inagaki T, Nishimori K, Yagi T, Ichikawa K, Moriyama M, Nakagawa T, et al. Comparison of a variable-number tandem-repeat (VNTR) method for typing Mycobacterium avium with mycobacterial interspersed repetitive-unit-VNTR and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol 2009;47:2156-64.  Back to cited text no. 9
Radomski N, Thibault VC, Karoui C, de Cruz K, Cochard T, Gutiérrez C, et al. Determination of genotypic diversity of Mycobacterium avium subspecies from human and animal origins by mycobacterial interspersed repetitive-unit-variable-number tandem-repeat and IS1311 restriction fragment length polymorphism typing methods. J Clin Microbiol 2010;48:1026-34.  Back to cited text no. 10
Tirkkonen T, Pakarinen J, Rintala E, Ali-Vehmas T, Marttila H, Peltoniemi OA, et al. Comparison of variable-number tandem-repeat markers typing and IS1245 restriction fragment length polymorphism fingerprinting of Mycobacterium avium subsp. Hominissuis from human and porcine origins. Acta Vet Scand 2010;52:21.  Back to cited text no. 11
Rindi L, Buzzigoli A, Medici C, Garzelli C. High phylogenetic proximity of isolates of Mycobacterium avium subsp. Hominissuis over a two decades-period. Infect Genet Evol 2013;16:99-102.  Back to cited text no. 12
Frothingham R, Wilson KH. Sequence-based differentiation of strains in the Mycobacterium avium complex. J Bacteriol 1993;175:2818-25.  Back to cited text no. 13
De Smet KA, Brown IN, Yates M, Ivanyi J. Ribosomal internal transcribed spacer sequences are identical among Mycobacterium avium-intracellulare complex isolates from AIDS patients, but vary among isolates from elderly pulmonary disease patients. Microbiology 1995;141(Pt 10):2739-47.  Back to cited text no. 14
Frothingham R, Wilson KH. Molecular phylogeny of the Mycobacterium avium complex demonstrates clinically meaningful divisions. J Infect Dis 1994;169:305-12.  Back to cited text no. 15
Novi C, Rindi L, Lari N, Garzelli C. Molecular typing of Mycobacterium avium isolates by sequencing of the 16S-23S rDNA internal transcribed spacer and comparison with IS1245-based fingerprinting. J Med Microbiol 2000;49:1091-5.  Back to cited text no. 16
Falkinham JO 3rd. Epidemiology of infection by nontuberculous mycobacteria. Clin Microbiol Rev 1996;9:177-215.  Back to cited text no. 17
Inderlied CB, Kemper CA, Bermudez LE. The Mycobacterium avium complex. Clin Microbiol Rev 1993;6:266-310.  Back to cited text no. 18
Karakousis PC, Moore RD, Chaisson RE. Mycobacterium avium complex in patients with HIV infection in the era of highly active antiretroviral therapy. Lancet Infect Dis 2004;4:557-65.  Back to cited text no. 19
Sturgill-Koszycki S, Schaible UE, Russell DG. Mycobacterium-containing phagosomes are accessible to early endosomes and reflect a transitional state in normal phagosome biogenesis. EMBO J 1996;15:6960-8.  Back to cited text no. 20
Oh YK, Straubinger RM. Intracellular fate of Mycobacterium avium: Use of dual-label spectrofluorometry to investigate the influence of bacterial viability and opsonization on phagosomal pH and phagosome-lysosome interaction. Infect Immun 1996;64:319-25.  Back to cited text no. 21
Jha SS, Danelishvili L, Wagner D, Maser J, Li YJ, Moric I, et al. Virulence-related Mycobacterium avium subsp hominissuis MAV_2928 gene is associated with vacuole remodeling in macrophages. BMC Microbiol 2010;10:100.  Back to cited text no. 22
Shin SJ, Lee BS, Koh WJ, Manning EJ, Anklam K, Sreevatsan S, et al. Efficient differentiation of Mycobacterium avium complex species and subspecies by use of five-target multiplex PCR. J Clin Microbiol 2010;48:4057-62.  Back to cited text no. 23
Thibault VC, Grayon M, Boschiroli ML, Hubbans C, Overduin P, Stevenson K, et al. New variable number tandem-repeat markers for typing Mycobacterium avium subsp. para-tuberculosis and M. avium strains: Comparison with IS900 and IS1245 restriction fragment length polymorphism typing. J Clin Microbiol 2007;45:2404-10.  Back to cited text no. 24
Tsuchiya S, Yamahi M, Yamaguchi Y, Kobayashi Y, Konno T, Tada K. Establishment and characterisation of human acute monocytic leukemia cell line (THP-1). Int J Cancer 1980;26:171-6.  Back to cited text no. 25
Uchiya KI, Tomida S, Nakagawa T, Asahi S, Nikai T, Ogawa K. Comparative genome analyses of Mycobacterium avium reveal genomic features of its subspecies and strains that cause progression of pulmonary disease. Sci Rep 2017;7:39750.  Back to cited text no. 26
Thegerström J, Jönsson B, Brudin L, Olsen B, Wold AE, Ernerudh J, et al. Mycobacterium avium subsp. Avium and subsp. Hominissuis give different cytokine responses after in vitro stimulation of human blood mononuclear cells. PLoS One 2012;7:e34391.  Back to cited text no. 27
Agdestein A, Jones A, Flatberg A, Johansen TB, Heffernan IA, Djønne B, et al. Intracellular growth of Mycobacterium avium subspecies and global transcriptional responses in human macrophages after infection. BMC Genomics 2014;15:58.  Back to cited text no. 28
Rindi L, Bonanni D, Lari N, Garzelli C. Most human isolates of Mycobacterium avium mav-A and mav-B are strong producers of hemolysin, a putative virulence factor. J Clin Microbiol 2003;41:5738-40.  Back to cited text no. 29


  [Figure 1], [Figure 2]

  [Table 1]

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