JOURNAL OF CLINICAL MICROBIOLOGY,0095-1137/98/$04.0010

Dec. 1998, p. 3694–3697 Vol. 36, No. 12

Copyright © 1998, American Society for Microbiology. All Rights Reserved.

Typing of Human Mycobacterium avium Isolates in Italy byIS1245-Based Restriction Fragment Length

Polymorphism AnalysisNICOLETTA LARI,1 MICHELA CAVALLINI,1 LAURA RINDI,1 ELISABETTA IONA,2

LANFRANCO FATTORINI,2 AND CARLO GARZELLI1*

Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia,Universita di Pisa, I-56127 Pisa,1 and Laboratory of Bacteriology and Medical Micology,

Istituto Superiore di Sanita, I-00161 Rome,2 Italy

Received 10 June 1998/Returned for modification 2 August 1998/Accepted 18 August 1998

All but 2 of 63 Mycobacterium avium isolates from distinct geographic areas of Italy exhibited markedly poly-morphic, multibanded IS1245 restriction fragment length polymorphism (RFLP) patterns; 2 isolates showedthe low-number banding pattern typical of bird isolates. By computer analysis, 41 distinct IS1245 patterns and10 clusters of essentially identical strains were detected; 40% of the 63 isolates showed genetic relatedness,suggesting the existence of a predominant AIDS-associated IS1245 RFLP pattern.

Mycobacterium avium, long recognized as a primary patho-gen of birds, behaves as an opportunistic human pathogen. Inimmunocompetent patients, the organism causes pulmonaryinfections and cervical lymphadenitis and, occasionally, soft-tissue infections. However, in a high proportion (25 to 50%) ofpatients with AIDS, M. avium causes severe disseminated in-fections (reviewed in reference 3).

M. avium can be isolated from environmental, animal, andhuman sources. Tap water is regarded as the main reservoir ofthe organism, at least for human infections (13), but the epi-demiology of M. avium infections has not been completelydefined. Analysis of restriction fragment length polymorphism(RFLP), based on the insertion sequence (IS) IS1245, a 1,414-bp element belonging to the IS256 IS family, has been pro-posed as a suitable technique for typing of M. avium isolatesfor epidemiological studies (1, 5, 6, 8–10) and also as a way toprovide more insight into the taxonomy and evolutionary di-vergence of the M. avium complex (1, 6).

M. avium strains isolated from humans and typed by theIS1245-based RFLP technique almost invariably show highlypolymorphic, multibanded IS1245 RFLP patterns that share ahigh degree of similarity with a significant proportion of iso-lates from pigs (1, 6, 9). In contrast, the IS1245 banding pat-terns of isolates from a wide variety of bird species are char-acterized by a very low IS number (1, 6, 9); the “bird pattern”M. avium strains are rarely found among human or pig isolates(1, 9).

The purpose of this study was to characterize, by the IS1245-based RFLP technique, 63 M. avium strains isolated fromhuman infections in distinct geographic areas of Italy. Theisolates were from 52 human immunodeficiency virus (HIV)-positive patients, of whom 47 had monoclonal infections and 5had polyclonal infections, and from 2 HIV-negative patients.Thirty-nine isolates were from the Pisa area, and 24 isolateswere from four other geographic areas (Rome, Milan, Flo-rence, and Ancona) of Italy. Forty-one isolates were fromblood, 11 were from respiratory specimens, 3 were from urine,

2 were from stool, and 1 was from cerebrospinal fluid. Fiveisolates were from unknown types of specimens. All isolateswere passed onto Middlebrook medium plates (Becton Dick-inson), and at least two colonies from each isolate, selected onthe basis of different colony morphology (if any), were grownin liquid Middlebrook medium and typed by a previously de-scribed IS1245-based RFLP assay (5) that also takes into con-sideration the 85% homologous insertion element IS1311 (10).The assay generates fingerprints with a number of IS1245-and IS1311-specific bands that is the sum of the copy num-bers of the two insertion elements (5). Briefly, 4.5 mg ofgenomic DNA, prepared from approximately 1.5-ml bacterialcultures, was digested overnight at 37°C with 10 U of therestriction endonuclease NruI (Amersham) in a final volume of20 ml. The DNA fragments generated were electrophoreticallyseparated on a 0.8% agarose gel and blotted onto a nylon filter(Hybond N-plus membrane; Amersham). A mixture of aPvuII-digested supercoiled DNA ladder (Gibco BRL) andHaeIII-digested FX174 DNA (Boehringer Mannheim) wasrun in two lanes of each gel and served as molecular sizemarkers ranging from 16.2 to 0.603 kb. Filters were hybridizedby addition of approximately 200 ng of a peroxidase-labeledIS1245 probe, prepared from DNA of an M. avium isolate byPCR using oligonucleotides P1 59GCCGCCGAAACGATCTAC and P2 59AGGTGGCGTCGAGGAAGAC as primers,as previously described (5, 6), and 25 to 40 ng of each molec-ular size marker probe. Hybridization was then detected onautoradiographic films by the enhanced-chemiluminescencegene detection system (Hyperfilm-ECL; Amersham). The M.avium RFLP patterns were scanned with an Epson GT 8000scanner at 200 dots/in., and the fingerprints were compared by3.1 GelCompar software (Applied Maths). The Dice coeffi-cients of similarity of all pairwise comparisons of patterns werecalculated, and a dendrogram of pattern relatedness amongthe strains was constructed by using UPGMA clustering inaccordance with a previously described algorithm (12). In gen-eral, the IS1245-based RFLP patterns were polymorphic andcomplex, as reported in other studies (1, 6, 9, 10). Assumingthat those isolates yielding banding patterns with similaritycoefficients of greater than 85% were essentially identical orhighly related, we detected 41 distinct IS1245 fingerprints (Fig.1, left side). Ten banding patterns were shared by more than

* Corresponding author. Mailing address: Dipartimento di Patolo-gia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemio-logia, Via San Zeno, 35/39, I-56127 Pisa, Italy. Phone: 39 050 559427.Fax: 39 050 559462. E-mail: garzelli@biomed.unipi.it.

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one isolate (i.e., cluster); the 10 clusters, indicated in Fig. 1 asa through j, included 31 (49%) of the 63 isolates. Clusters a andd were the largest, as they included six and seven isolates,respectively. Clusters b, c, e, f, g, h, i, and j each included twoor three isolates. A reference strain of bird origin, i.e., M.avium ATCC 35712, occurred in cluster j together with twohuman isolates sharing the bird-type RFLP pattern; both iso-lates showed the glycopeptidolipid antigen of serotype 3, asassessed by thin-layer chromatography (2, 11). This findingreinforces the view that infections with bird-type M. aviumstrains do occur in humans, although rarely (1, 9). Moreover,as shown in Table 1, where the fingerprints of the isolatesoccurring in clusters are matched with their geographic origins,clusters a, b, c, e, f, g, i, and j each contained isolates from thesame geographic area, suggesting the existence of a commonsource of infection for patients. For example, the isolates incluster i were from three AIDS patients hospitalized in onehospital in Rome during the same period, which suggests thepossibility of nosocomial transmission of M. avium infection.Clusters d and h contained, respectively, seven and three iden-tical or highly related isolates from different geographic areas.Isolates in cluster d, in particular, derived from as many as fourdistinct areas.

To visualize more objectively the genotype relatedness among

all of the isolates, a similarity matrix was generated. This ma-trix shows the degree of relatedness of each IS1245 bandingpattern with any other in the collection. In Fig. 1 (right side),the “families” of related IS1245 banding patterns are shown ingroupings of gray-shaded values. In general, all of the group-ings with related fingerprints contained few strains, with theexception of one large family, including 25 (40%) of the 63isolates with similarity values of greater than 55%. This family,which comprises clusters c, d, e, f, and g, includes isolatesderived from the five different geographic areas. The geneticrelatedness among numerous strains isolated from AIDS pa-tients in different areas may support the possibility of theexistence of an IS1245 RFLP pattern(s) associated with HIV-induced immunodeficiency. Conflicting evidence has been re-ported on the genetic characters of M. avium strains that causedisseminated disease in AIDS patients. It has been suggestedthat M. avium strains that infect AIDS patients form a distinctand genetically conserved group of highly similar isolateswithin the M. avium complex (7). This hypothesis is also rein-forced by the quantitative demonstration of a high degree ofrelatedness of the AIDS-associated isolates, compared withisolates from non-HIV-infected individuals (4). In contrast,several studies have shown that AIDS patients are infected byunrelated, highly variable strains, at least according to RFLP

TABLE 1. Geographic origins of Italian M. avium isolates occurring in clusters

a Clusters of isolates were defined in accordance with the results shown in Fig. 1.b Abbreviations for geographic areas in which the clustered M. avium strains were isolated: PI, Pisa; FI,

Florence; RM, Rome; MI, Milan. The number of isolates from each geographic area is given in parentheses.c One fingerprint occurring in cluster j in Fig. 1 is not included in this table, i.e., that of reference strain

ATCC 35712.

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analysis (1, 6, 9, 10). Our results indicate that these two con-ditions may coexist. AIDS patients may be infected by differ-ent, genetically unrelated strains, but at the same time, thepossibility cannot be ruled out that one or more strains may beselected in or adapted to a particular environmental niche thatfacilitates diffusion to AIDS patients, with the consequentemergence of a predominant strain in these patients.

In conclusion, the results of RFLP typing of Italian M. aviumisolates described in this paper confirm the marked polymor-phism of most isolates from AIDS patients but also raise thequestion of the possible definition of the predominant genomicfingerprints of virulent clones for these patients.

This work was financially supported by MURST (ConfinanziamentoProgetto di Ricerca 1997 “Controllo della patogenicita microbica”)and partly by Istituto Superiore di Sanita, Ministero della Sanita (Pro-gramma Nazionale sull’AIDS), grants 50A.0.17 and 10/A/2.

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12. van Soolingen, D., P. W. M. Hermans, P. E. W. de Haas, D. R. Soll, andJ. D. A. van Embden. 1991. The occurrence and stability of insertion se-quences in Mycobacterium tuberculosis complex strains: evaluation of inser-tion sequence-dependent DNA polymorphism as a tool in the epidemiologyof tuberculosis. J. Clin. Microbiol. 29:2578–2586.

13. Von Reyn, C. F., J. N. Maslow, T. W. Barber, J. O. Falkinham III, and R. D.Arbeit. 1994. Persistent colonisation of potable water as a source of Myco-bacterium avium infection in AIDS. Lancet 343:1137–1141.

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