Vol. 30, No. 5JOURNAL OF CLINICAL MICROBIOLOGY, May 1992, p. 1250-12550095-1137/92/051250-06$02.00/0Copyright ©) 1992, American Society for Microbiology

Large Restriction Fragment Patterns of Genomic Mycobacteriumfortuitum DNA as Strain-Specific Markers and Their Use inEpidemiologic Investigation of Four Nosocomial Outbreaks

JANEL S. R. HECTOR,1 YIJUN PANG,2 GERALD H. MAZUREK,3 YANSHENG ZHANG,2BARBARA A. BROWN,2 AND RICHARD J. WALLACE, JR.2*Departments of Biochemistry,1 Microbiology, 2 and Medicine, 3The University of Texas Health Center, Tyler, Texas 75710

Received 12 November 1991/Accepted 10 February 1992

Pulsed-field gel electrophoresis and restriction endonucleases with rare recognition sites were used togenerate large restriction fragment (LRF) patterns of genomic DNA from 48 isolates of Mycobacteriumfortuitum biovariantfortuitum. Epidemiologically unrelated isolates gave highly diverse patterns when AsnI,HpaI, AfllI, DraI, NdeI, XbaI, Spel, or SspI was used. Epidemiologically related isolates produced identical orminimally different LRF patterns. Minor variations in LRF patterns were seen in two epidemic isolates digestedwith XbaI, suggesting that genetic alteration had occurred. LRF patterns were used to study three cardiacsurgery wound infection outbreaks and one respiratory disease nosocomial outbreak. In two outbreaks, LRFpatterns confirmed the reported clustering of isolates on the basis of multiple phenotyping methods. In theremaining two outbreaks, isolates which could not be separated by prior typing methods were easilydistinguished by LRF pattern analysis. Environmental water isolates from two outbreaks had LRF patternsidentical to those of the disease-producing strains, confirming that the local environment was the source ofinfection. Pulsed-field gel electrophoresis of LRFs ofgenomic DNA offers great promise as an epidemiologic toolfor the study of M. fortuitum.

During the past decade, rapidly growing mycobacteriahave gained notoriety as nosocomial pathogens. Epidemicand sporadic nosocomial diseases have been reported fol-lowing cardiac bypass surgery (11, 15, 25, 31), hemodialysis(4, 18), peritoneal dialysis (3), middle ear cleaning andinstrumentation (19), consumption of contaminated ice (16),augmentation mammaplasty (27, 32), and several other typesof surgical procedures. Contamination by local environmen-tal organisms is suspected as the primary mechanism ofinfection, but limitations in the available typing methodshave prevented confirmation of this suspicion. Previouslyavailable epidemiologic methods have included antibioticand heavy metal susceptibility, plasmid profile analyses, andmultilocus electrophoretic typing of multiple cellular meta-bolic enzymes (22, 31, 32). These methods are limited sincesusceptibility patterns frequently do not distinguish betweendifferent strains; only about 50% of strains contain plasmids;the stability of plasmids, including possible changes in sizebecause of mobile genetic units in these mycobacteria, isunknown; and electrophoretic typing is time-consuming andnot readily available.A new approach involves the use of large DNA restriction

fragment patterns as epidemiologic markers. Large restric-tion fragments (LRFs) are created by digesting an organ-ism's DNA with rarely cutting restriction endonucleases(i.e., enzymes with less than one recognition site per 100,000bp). These large fragments are then separated by pulsed-fieldgel electrophoresis (PFGE), stained with ethidium bromide,and visualized by UV illumination. The LRF patterns gen-erated in this way have been used to study the epidemiologyof several bacterial species, including enterococci (23),Escherichia coli (2), Pseudomonas aeruginosa (1, 8), coag-

* Corresponding author.

ulase-negative staphylococci (7), and human ureaplasmas(26). Potentially any bacterial strain or species can bestudied by this methodology. We used the LRF patternsfrom sporadic and epidemic isolates of Mycobacterilumfortuituim to study the epidemiology of nosocomial disease.

(An abstract of this work was presented at the 91stGeneral Meeting of the American Society for Microbiology,Dallas, Texas, 8 May 1991.)

MATERLALS AND METHODS

M. fortuitum isolates. Clinical and environmental isolatesof M. fortluitum biovariant fortuitium were selected fromthree cardiac surgery wound infection outbreaks that oc-curred in Colorado (5 clinical and 1 environmental isolate)(11), Texas (2 clinical and 5 environmental isolates) (15), andNebraska (6 clinical isolates) (25) and one nosocomial respi-ratory disease outbreak in Washington, D.C. (20 clinical and3 environmental isolates) (5). The cardiac isolates werekindly provided by V. Silcox of the Mycobacterial ReferenceSection of the Centers for Disease Control, Atlanta, Ga. Therespiratory isolates were kindly provided by D. Burns and F.Gordin of Washington, D.C. The clinical features (5, 11, 15,25) and results of detailed phenotypic evaluation of theseisolates by biovariant identification; antibiotic susceptibility;plasmid profile; heavy metal resistance analyses; lactosefermentation; and multilocus enzyme electrophoresis of 15metabolic enzymes with determination of electrophoreticpatterns (designated as clectrophoretic types) have beenpublished previously (31). Eleven epidemiologically unre-lated clinical isolates submitted to the Mycobacteria/Nocar-dia Research ILaboratory of the University of Texas forsusceptibility testing and the type strain of M. fortuitum(ATCC 6841), which was obtained from the American TypeCulture Collection, Rockville, Md., were selected as con-

1250

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from

LRF PATTERNS OF GENOMIC M. FORTUITUM DNA 1251

trols. Isolates were identified to the species and subspecieslevels by standard methods (28). Most identifications wereperformed by the Mycobacterial Reference Section of theCenters for Disease Control.

Preparation ofgenomic DNA. Genomic DNA was preparedas described by Smith and Cantor (29) and Levy-Frebault etal. (17) with modifications. Mycobacterial cells were grownto an optical density of 1 to 1.5 in 10 ml of Mueller-Hintonbroth. Cycloserine (1 mg/ml) and ampicillin (0.1 mg/ml) wereadded, and incubation was continued for 12 to 16 h. Cellswere collected by centrifugation and then resuspended in 2ml of 1% low-melting-point agarose and cast into plugs. Theplugs were shaken overnight at 37°C in 5 ml of Tris-EDTA(TE) buffer containing 1 M NaCl and 1 mg of lysozyme perml to lyse the cells. The plugs were then incubated for 48 hat 50°C in 5 ml of TE buffer with 1% sodium dodecyl sulfateand 1 mg of proteinase K per ml. Plugs were washed with TEbuffer, incubated overnight with 10 mg of phenylmethylsul-fonyl fluoride per ml in TE buffer, washed four times for 12h with TE buffer, and stored at 4°C until use.

Restriction enzyme digestions. Restriction endonucleaseswith rare recognition sites were selected on the basis of aG+C content of 62 to 70% reported for mycobacterial DNA(33). Enzymes with four to six A and T base pairs or thesequence CTAG in the recognition site (20) were chosen andincluded AsnI (AT/TAAT), DraI (TTlT/AAA), SspI (AAT/ATT), Aflll (C/TTAAG), NdeI (CAJTATG), HpaI (GTT/AAC), SpeI (A/CTAGT), and XbaI (T/CTAGA). The plugscontaining the embedded DNA were equilibrated in theappropriate restriction buffer for 1 h and were then incubatedovernight in 60 ,ul of restriction buffer with 10 U of enzyme.Sterile conditions were maintained to avoid contaminationwith DNase.PFGE. PFGE was performed by using the GeneLine

transverse alternating-field electrophoresis (TAFE) system(Beckman) or a contour-clamped homogeneous electric field(CHEF) system (CHEF-DR II; Bio-Rad). For the GeneLinesystem, gels were made of 1% low-endosmosis agarose andwere run at 12°C for 18 to 20 h at 150 mA of constant currentin Tris-acetate-EDTA buffer (0.01 M Tris, 0.0001 M EDTA,0.00435 M acetic acid) with pulse times of from 20 to 60 s.For the CHEF system, 1% low-endosmosis agarose gelswere run in Tris-borate-EDTA buffer (0.025 M Tris, 0.5 mMEDTA, 0.025 M boric acid) at 14WC with ramped pulse timesof from 5 to 20 s for 20 h at 200 V. The gels were stained withethidium bromide and photographed by using a UV transil-luminator (302 nm; Spectroline). Saccharomyces cerevisiaewhole chromosomes and 48.5-kb bacteriophage lambda con-catemers (Beckman or Bio-Rad) were used as DNA stan-dards. Plasmids were separated from genomic DNA byPFGE of undigested DNA.

RESULTS

The optimal enzymes for generating LRF patterns wereassessed by digesting the DNA of two randomly chosen M.fortuitum strains with eight restriction endonucleases. AsnIproduced the fewest number of restriction fragments andfragments of the greatest size. AsnI yielded 11 and 12 DNAfragments of over 200 kb (some as large as 800 kb), suggest-ing genomic sizes of over 4,240 and 5,030 kb, respectively,for each of these two isolates. The eight restriction enzymesevaluated included AsnI, HpaI, Aflll, DraI, NdeI, XbaI,SpeI, and SspI (listed in order of increasing numbers ofrestriction fragments that are produced). The clearest pat-terns followed digestion with XbaI. Fragments were less

L. a;Ose t < -3 41 5 6 7 8 9 10 11 12 13

0-

45.5-

8 5-

FIG. 1. PFGE, using the CHEF-DR II system, of epidemiologi-cally unrelated isolates of M. fortuitum that were digested with AbaIand run for 20 h with 5- to 20-s pulses at 200 V. Lane 1, lambdaconcatemers (48.5 kb); lanes 2 to 7 and 9 to 13, randomly selectedstrains of M. fortuitum; lane 8, ATCC 6841.

well separated and bands were more distorted in the TAFEsystem than they were in the CHEF system.Two or more enzymes were used to generate LRF pat-

terns for all M. fortuitum isolates. Bach isolate gave a readilydiscernible pattern, with 5 to 20 individual fragments avail-able for strain characterization. To assess the potential ofLRF patterns as epidemiologic markers, we did three things.First, we compared the patterns of randomly isolatedstrains. Bleven epidemiologically unrelated isolates and atype strain of M. fortuitum (ATCC 6841) each gave unique,highly diverse LRF patterns with all of the enzymes tested.Figure 1 demonstrates the variability in LRF patterns seenfollowing digestion with AbaIL Second, we studied multiplepreparations of the same isolates. A pair of clinical isolatesthat had been passaged multiple times and that had been keptin different laboratories gave identical LRF patterns. Third,four pairs of epidemiologically related strains with identicalDNA patterns following digestion with AsnI or Xbal werecompared after digestion with two or more additional en-zymes. They were found to have identical patterns afterdigestion with these additional enzymes as well.The six isolates from the sternal wound outbreak in

Nebraska (25) were classified into two groups of two andfour identical strains after PFGB of genomic DNA restric-tion fragments. An identical grouping has previously beenobtained on the basis of phenotyping, electrophoretic typing,and plasmid profiles (31).The two clinical and five environmental isolates from the

sternal wound outbreak in Texas (15) were divided into twogroups on the basis of their LRF patterns. One isolate, whichwas recovered from a hospital ice machine, gave a uniquepattern and made up the first group. The second groupconsisted of isolates from a laparotomy incision, a sternalwound, municipal water coming into the hospital, waterfrom the cold-water faucet in the operating room, anotherhospital ice machine, and water used to cool the cardioplegiasolution in the cardiovascular operating room (a total of sixisolates). These six isolates gave LRF patterns which wereidentical when DraI or SspI was used. However, when theywere digested with Xbal, one isolate from an ice machinewas found to be minimally different. The LRF pattern ofDNA from this isolate had two bands which were not seen inthe other epidemic isolates. The same two groups wereidentified previously by using phenotypes and plasmid pro-

VOL. 30, 1992

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from

1252 HECTOR ET AL.

A Std1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 (Kbp)

sr'flW

'.)ts,101 - :.) -.r.=

-6 /,

---291 .0

-242.5

194.0

-145.5

-97.0

FIG. 2. PFGE, using the CHEF-DR II system, of isolates from a

Washington, D.C., respiratory disease epidemic. The isolates were

digested with Xbal. Lanes 1, 2, and 16, epidemiologically unrelatedstrains; lane 18, lambda concatemer (48.5 kb); lanes 3 to 15 and 17,clinical and environmental epidemic isolates. A Std., bacteriophagelambda size standard.

files (31), with one exception. The sternal wound isolate hada plasmid that was slightly larger than those in the remainingepidemic strains.The five clinical and one environmental isolate from the

sternal wound outbreak in Colorado (11) were separated intothree different groups on the basis of their LRF patterns.Four isolates (Mf 294, Mf 295, Mf 296, and Mf 297) recov-

ered from sternal wounds of patients over a 2-week period(21 April to 5 May 1976) had identical patterns. A sternalwound isolate (Mf 293) from a patient who had been oper-ated on 6 months previously (October 1975) and an environ-mental isolate recovered from an operating room settle platefollowing the outbreak (Mf 323) were unique. These sixisolates were previously grouped differently on the basis oftheir plasmid profiles and phenotypes, including their elec-trophoretic types (31). Although the four clustered sternalisolates had identical phenotypic patterns, they exhibitedtwo different plasmid profiles (a large plasmid was present intwo strains but was absent from two others). The environ-mental isolate had a phenotypic pattern identical to those ofthe epidemic strains (for technical reasons, plasmid profilescould not be adequately studied for this strain) and wasassumed to be the same as the epidemic strains. However,on PFGE, this environmental isolate had a unique LRFpattern. The October 1975 sternal wound isolate which had a

unique phenotype also had a unique LRF pattern.The 20 clinical respiratory isolates and the 3 environmen-

tal isolates from the nosocomial respiratory disease outbreakin Washington, D.C., were placed in six groups on the basisof their LRF patterns (examples of several of these are

shown in Fig. 2). Two isolates from showerheads and threeisolates from sputa were unique, producing five markedlydifferent LRF patterns. The 18 remaining isolates, whichincluded 16 isolates recovered from the sputa of patients on

an alcoholic rehabilitation floor, one isolate from a broncho-alveolar lavage sample from a patient elsewhere in the

FIG. 3. PFGE, using the CHEF-DR II system, of respiratorydisease epidemic isolates from Washington, D.C. Lanes 2 and 3,clinical sputum isolates digested with SspI; lane 4, environmentalwater isolate digested with SspI; lanes 5 to 7, the same isolates asthose in lanes 2 to 4 but digested with XbaI to show the minordifferences in band patterns (white arrows) that were not apparentby using SspI. X Std., bacteriophage lambda standard.

hospital (with no evidence of M. fortuitum disease), and oneisolate from a water sample from the water line going to thealcoholic rehabilitation shower, were clustered together in asixth group because of similar LRF patterns. The 18 isolateshad identical LRF patterns when their DNAs were digestedwith DraI and SspI. However, minor differences (variationsof two to four bands) were seen in one of the environmentalisolates following digestion with XbaI (Fig. 3). The same sixgroups were evident by phenotyping and plasmid profileanalysis. Preliminary results of these PFGE studies, as theydefined the nature of this outbreak, are reported elsewhere(5).

All isolates were examined by PFGE for the presence ofplasmids (Fig. 4). Plasmid bands in 38 of 40 epidemic isolateswere detected by PFGE of undigested DNA. The plasmidshave previously been identified by standard agarose gelelectrophoresis in the same 38 isolates (5, 31). Plasmids fromfour epidemic sternal isolates (from the Colorado outbreak)were not seen by using the TAFE system for PFGE and wereonly weakly seen by using the CHEF system. The plasmidswere inconsistent and were difficult to visualize by standardmethods as well. Plasmid size could not be determined byPFGE because of variations in the mobilities of the circularplasmids from one experiment to the next. Minor differencesin electrophoretic mobility by PFGE were noted for severalplasmids in the group of 18 epidemic isolates in the Wash-ington, D.C., outbreak, although all isolates contained twoplasmids with similar sizes (mobilities). The reasons forthese differences remain to be resolved.

DISCUSSION

From the results of this study, we concluded that, byPFGE of large genomic DNA fragments, it is possible tocompare any isolate of M. fortuitum biovariant fortuitum,including some isolates that cannot be differentiated by otheravailable typing methods. Isolates could be reliably distin-guished by using only one restriction enzyme and a singleelectrophoretic run under proper conditions. The restrictionendonuclease chosen was not critical, and DNA digests fromany two isolates that were identical by using one restrictionenzyme were identical by using other restriction enzymes

J. CL-IN. MICROBIOL.

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from

LRF PA`TIERNS OF GENOMIC M. FORTUITUM DNA 1253

8 .) rJ K:b

700 k4)

5604_ b

-440 4b

330k-tb

200 kb

FIG. 4. GeneLine PFGE of M. fortuitum biovariant fortuitumplasmids with unrestricted DNA. Electrophoresis parameters were

18 h, 40-s pulses, 150-mA constant current, 12°C, lx Tris-acetate-EDTA buffer. Lanes A and C, strains from the Corpus Christi, Tex.,outbreak (strains Mf 135 and Mf 111; these strains had different LRFpatterns); lanes B, D, and E, strains from the Omaha, Nebr.,outbreak (strains Mf 305, Mf 287, and Mf 291; strains Mf 287 and Mf291 had identical LRF patterns); lanes F, G, and H, randomlyisolated strains; lane I, S. cerevisiae chromosomal standard.

(with the occasional exception ofXbaI, as discussed below).The use ofAsnl resulted in fewer and larger DNA fragmentsthan resulted from the use of the other enzymes, but AsnI ismuch more expensive. Less expensive enzymes such as

DraI and XbaI gave equally good results.The results of PFGE generally supported the clustering of

isolates and the conclusions of previous epidemiologic stud-ies of disease caused byM. fortuitum; those studies relied onother typing techniques such as antibiograms and subspeciesidentification (31). In several instances, however, it providedconclusive evidence of strain relationships which could notbe established by previous methods. In the Texas sternalwound outbreak (15), PFGE provided unequivocal evidencethat a sternal wound isolate was the same (i.e., belonged tothe same clone) as an M. fortuitum strain that was endemicin the local municipal and hospital water supply. The isolateswere previously assumed to be unrelated because of differ-ences in their plasmid profiles (31). In the Colorado sternalwound outbreak (11), PFGE defined the clonal nature of thefour outbreak isolates which previously appeared to repre-sent two isolate groups because of differences in theirplasmid profiles (31).

This study confirmed that environmental water supplieswere the source of disease caused by M. fortuitum in one

sternal wound outbreak (Texas) and one nosocomial respi-ratory disease outbreak (Washington, D.C.) and excluded an

isolate from an operating room settle plate as the cause ofone other outbreak (Colorado). To date, there have been atleast 12 nosocomial outbreaks involving M. fortuitum or

Mycobacterium chelonae subsp. abscessus in which water,ice, or contaminated solutions have appeared to serve as thereservoir of the infecting organism (3-6, 12, 13, 15, 16, 18,19, 27, 34). In no case has medical equipment, bone wax, or

dust been shown to be the source of disease. However,additional environmental studies are needed for organismsthat cause most diseases, especially surgical wound infec-

tions following augmentation mammaplasty for which noorganism source or reservoir has been identified (except forone outbreak that involved contaminated gentian violet)(27). It is possible that for some outbreaks and some dis-eases, organism reservoirs other than water may be identi-fied.An unexpected finding in the current study was the minor

changes in genomic DNA fragment patterns seen betweentwo strains that were otherwise identical to multiple otherisolates recovered from the respective epidemics. Thesechanges appeared to involve two to four large fragments,with the disappearance of one or more fragments and theappearance of one or more new ones. These changes werenoted only with XbaI. Both isolates that exhibited thesechanges were environmental and were recovered 6 monthsor longer after the initial infection. The two isolates did notappear to be unrelated strains, since there was such amarked heterogeneity of genomic DNA patterns by PFGE ofepidemiologically unrelated strains. The two isolates werematched (clonal) with other epidemiologically related iso-lates because of the identity of multiple other LRF fragmentsobtained with XbaI, the identical LRF patterns produced byother restriction endonucleases, plasmid profiles, and multi-ple phenotypic characteristics. This strongly suggests that,rather than there being a similarity among unrelated strains,some change in the genomic DNA occurred in the strains.One possible explanation for these changes would be achromosomal rearrangement event(s) such as an inversion ordeletion. Another, more likely, possibility would be theacquisition or spread of an insertion sequence, since morethan one change in the DNA appears to have occurred. Theintroduction in multiple sites in the chromosome of aninsertion sequence that carries an XbaI recognition sitewould provide a ready explanation for the observed changes.Insertional events have been suggested as a reason for thechanges that have been seen following multiple laboratorypassages in some strains of P. aeruginosa (10). An insertionsequence is known to be widespread in the Mycobactenumtuberculosis genome (9), and an insertion sequence has beenreported for M. fortuitum (30), but its sequence and preva-lence are unknown.PFGE also provided a ready means of screening strains of

M. fortuitum for plasmids, and the CHEF technique was assensitive as agarose gel electrophoresis ofDNA prepared byother methods (14). Problems of sizing of the plasmid bandson the basis of their electrophoretic mobilities in PFGEremain to be resolved, however.

Previously, the technology to distinguish isolates of bothrapidly growing and slowly growing mycobacteria (includingM. tuberculosis) has not been available. A better under-standing of the disease epidemiology as a result of infectionwith mycobacteria, especially of the nontuberculous myco-bacteria, is essential for knowing how the organism istransmitted and for any hope of preventing diseases causedby mycobacteria.The recent use of frequently cutting restriction enzyme

digests of genomic DNA has been described for severalmycobacterial species (21, 24, 35). However, the largenumber of fragments generated by common restriction siteendonucleases makes comparison of strains difficult. Whenthis technique is used in combination with a radiolabeledprobe (especially one that has multiple areas of homologywithin the genome), however, strain comparisons are easier.A recently sequenced insertion sequence in M. tuberculosiswas used as a probe and allowed excellent comparisons ofstrains in this species (9, 30). However, usable probes for

VOL. 30, 1992

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from

1254 HECTOR ET AL.

species other than the M. tuberculosis complex are unknownor have not been adequately studied. PFGE offers greatpotential for the study of the epidemiology of multiplemycobacterial species other than M. fortuitum. Studies inour laboratory have shown that M. tuberculosis strains canbe separated by this same technique (36). Studies of genomicDNA by pulse-field technology have also been reported forMycobacterium paratuberculosis (17). Additional studies ofother species by this new technique should be forthcoming.Results of those studies will greatly enhance our knowledgeof the epidemiology of both nosocomial and community-acquired mycobacterial infections.

ACKNOWLEDGMENT

This work was not funded by sources outside the University ofTexas Health Center.

REFERENCES1. Allardet-Servent, A., N. Bouziges, M.-J. Carles-Nurit, G. Bourg,

A. Gouby, and M. Ramuz. 1989. Use of low-frequency-cleavagerestriction endonucleases for DNA analysis in epidemiologicalinvestigations of nosocomial bacterial infections. J. Clin. Micro-biol. 27:2057-2061.

2. Arbeit, R. D., M. Arthur, R. Dunn, C. Kim, R. K. Selander, andR. Goldstein. 1990. Resolution of recent evolutionary diver-gence among Escherichia coli from related lineages: the appli-cation of pulsed field electrophoresis to molecular epidemiol-ogy. J. Infect. Dis. 161:230-235.

3. Band, J. D., J. I. Ward, D. W. Fraser, N. J. Peterson, V. A.Silcox, R. C. Good, P. R. Ostroy, and J. Kennedy. 1982.Peritonitis due to Mycobacterium chelonei-like organism asso-ciated with intermittent chronic peritoneal dialysis. J. Infect.Dis. 145:9-17.

4. Bolan, G., A. L. Reingold, L. A. Carson, V. A. Silcox, C. L.Woodley, P. S. Hayes, A. W. Hightower, L. McFarland, J. W.Brown III, N. J. Petersen, M. S. Favero, R. C. Good, and C. V.Broome. 1985. Infections with Mycobacterium chelonei in pa-tients receiving dialysis and using processed hemodialyzers. J.Infect. Dis. 152:1013-1019.

5. Burns, D. N., R. J. Wallace, Jr., M. E. Schultz, Y. Zhang, S. Q.Zubairi, Y. Pang, C. L. Gibert, B. A. Brown, and F. M. Gordin.1991. Nosocomial outbreak of respiratory tract colonizationwith Mycobacterinumfortuitum: demonstration of the usefulnessof pulse-field gel electrophoresis in an epidemiologic investiga-tion. Am. Rev. Respir. Dis. 144:1153-1159.

6. Foz, A., C. Roy, J. Jurado, E. Arteaga, J. M. Ruiz, and A.Morgas. 1978. Mycobacterium chelonei iatrogenic infections. J.Clin. Microbiol. 7:319-321.

7. Goering, R. V., and T. D. Duensing. 1990. Rapid field inversiongel electrophoresis in combination with an rRNA gene probe inthe epidemiological evaluation of staphylococci. J. Clin. Micro-biol. 28:426-429.

8. Grothues, D., U. Koopmann, H. von der Hardt, and B. Tummler.1988. Genome fingerprinting of Pseudomonas aeruginosa indi-cates colonization of cystic fibrosis siblings with closely relatedstrains. J. Clin. Microbiol. 26:1973-1977.

9. Hermans, P. W. M., D. van Sooligen, J. W. Dale, A. R. J.Schuitema, R. A. McAdam, D. Catty, and J. D. A. van Embden.1990. Insertion element IS986 from Mycobacterium tuberculo-sis: a useful tool for diagnosis and epidemiology of tuberculosis.J. Clin. Microbiol. 28:2051-2058.

10. Hjelm, L. N., A. A. Branstrom, and R. L. Warren. 1990.Detection of restriction fragment length polymorphisms in clin-ical isolates and serially passaged Pseudomonas aeruginosastrains. J. Clin. Microbiol. 28:2178-2182.

11. Hoffman, P. C., D. W. Fraser, F. Robicsek, P. R. O'Bar, andC. U. Mauney. 1981. Two outbreaks of sternal wound infectionsdue to organisms of the Mycobacterium fortuitum complex. J.Infect. Dis. 143:533-542.

12. Hoy, J., K. Rolston, and R. L. Hopfer. 1987. Pseudoepidemic ofMycobacterium fortuitum in bone marrow cultures. Am. J.

Infect. Control 15:268-271.13. Inman, P. M., A. Beck, A. E. Brown, and J. L. Stanford. 1969.

Outbreak of injection abscesses due to Mycobacterium absces-sus. Arch. Dermatol. 100:141-147.

14. Kado, C. I., and S.-T. Liu. 1981. Rapid procedure for detectionand isolation of large and small plasmids. J. Bacteriol. 145:1365-1373.

15. Kuritsky, J. N., M. G. Bullen, C. V. Broome, V. A. Silcox, R. C.Good, and R. J. Wallace, Jr. 1983. Sternal wound infections andendocarditis due to organisms of the Mycobacterium fortuitumcomplex. Ann. Intern. Med. 98:938-939.

16. Laussucq, S., A. L. Baltch, R. P. Smith, R. W. Smithwick, B. J.Davis, E. K. Desjardin, V. A. Silcox, A. B. Spellacy, R. T.Zeimis, H. M. Gruft, R. C. Good, and M. L. Cohen. 1988.Nosocomial Mycobacterium fortuitum colonization from a con-taminated ice machine. Am. Rev. Respir. Dis. 138:891-894.

17. Levy-Frebault, V. V., M. F. Thorel, A. Varnerot, and B.Gicquel. 1989. DNA polymorphism in Mycobacterium paratu-berculosis, "wood pigeon mycobacteria," and related myco-bacteria analyzed by field inversion gel electrophoresis. J. Clin.Microbiol. 27:2823-2826.

18. Lowry, P. W., C. M. Beck-Sague, L. A. Bland, S. M. Aguero,M. J. Arduino, A. N. Minuth, R. A. Murray, J. M. Swenson, andW. R. Jarvis. 1990. Mycobacterium chelonae infection amongpatients receiving high-flux dialysis in a hemodialysis clinic inCalifornia. J. Infect. Dis. 161:85-90.

19. Lowry, P. W., W. R. Jarvis, A. D. Oberle, L. A. Bland, R.Silberman, J. A. Bocchini, H. D. Dean, J. M. Swenson, and R. J.Wallace, Jr. 1988. Mycobacterium chelonae causing otitis mediain an ear-nose-and-throat practice. N. EngI. J. Med. 319:978-982.

20. McClelland, M., R. Jones, Y. Patel, and M. Nelson. 1987.Restriction endonucleases for pulsed field mapping of bacterialgenomes. Nucleic Acids Res. 15:5985-6005.

21. McFadden, J. J., P. D. Butcher, R. Chiodini, and J. Hermon-Taylor. 1987. Crohn's disease-isolated mycobacteria are identi-cal to Mycobacterium paratuberculosis as determined by DNAprobes that distinguish between mycobacterial species. J. Clin.Microbiol. 25:796-801.

22. Meissner, P. S., and J. 0. Falkinham III. 1984. Plasmid-encodedmercuric reductase in Mycobacterium scrofulaceum. J. Bacte-riol. 157:669-672.

23. Murray, B. E., K. V. Singh, J. D. Death, B. R. Sharma, andG. M. Weinstock. 1990. Comparison of genomic DNAs ofdifferent enterococcal isolates using restriction endonucleaseswith infrequent recognition sites. J. Clin. Microbiol. 28:2059-2063.

24. Patel, R., J. T. Kvach, and P. Mounts. 1986. Isolation andrestriction endonuclease analysis of mycobacterial DNA. J.Gen. Microbiol. 132:541-551.

25. Preheim, L. C., M. J. Bittner, D. K. Giger, and W. E. Sanders,Jr. 1982. Program Abstr. 21st Intersci. Conf. Antimicrob.Agents Chemother., abstr. 564.

26. Robertson, J. A., L. E. Pyle, G. W. Stemke, and L. R. Finch.1990. Human ureaplasmas show diverse genome sizes bypulsed-field electrophoresis. Nucleic Acids Res. 18:1451-1455.

27. Safranek, T. J., W. R. Jarvis, L. A. Carson, L. B. Cusick, L. A.Bland, J. M. Swenson, and V. A. Silcox. 1987. Mycobacteriumchelonae wound infections after plastic surgery employing con-taminated gentian violet skin-marking solution. N. Engl. J.Med. 317:197-201.

28. Silcox, V. A., R. C. Good, and M. M. Floyd. 1981. Identificationof clinically significant Mycobacterium fortuitum complex iso-lates. J. Clin. Microbiol. 14:686-691.

29. Smith, C. L., and C. R. Cantor. 1987. Purification, specificfragmentation, and separation of large DNA molecules. Meth-ods Enzymol. 155:449-467.

30. Thierry, D., M. D. Caves, K. D. Eisenach, J. T. Crawford, J. H.Bates, B. Gicquel, and J.-L. Guesdon. 1990. IS6110, an IS-likeelement ofM. tuberculosis complex. Nucleic Acids Res. 18:188.

31. Wallace, R. J., J. M. Musser, S. I. Hull, V. A. Silcox, L. C.Steele, G. D. Forrester, A. Labidi, and R. K. Selander. 1989.Diversity and sources of rapidly growing mycobacteria associ-

J. CLIN. MICROBIOL.

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from

LRF PATTERNS OF GENOMIC M. FORTUITUM DNA 1255

ated with infections following cardiac surgery. J. Infect. Dis.159:708-716.

32. Wallace, R. J., Jr., L. C. Steele, A. Labidi, and V. A. Silcox.1989. Heterogeneity among isolates of rapidly growing myco-

bacteria responsible for infections following augmentation mam-maplasty despite case clustering in Texas and other southerncoastal states. J. Infect. Dis. 160:281-288.

33. Wayne, L. G., and W. M. Gross. 1968. Base composition ofdeoxyribonucleic acid isolated from mycobacteria. J. Bacteriol.96:1915-1919.

34. Wenger, J. D., J. Spika, V. Pryor, G. Kubica, R. Smithwick, and

R. C. Good. 1989. Program Abstr. 29th Intersci. Conf. Antimi-crob. Agents Chemother., abstr. 390.

35. Whipple, D. L., R. B. LeFebvre, R. E. Andrews, and A. B.Thiermann. 1987. Isolation and analysis of restriction endonu-clease digestive patterns of chromosomal DNA from Mycobac-terium paratuberculosis and other mycobacterium species. J.Clin. Microbiol. 25:1511-1515.

36. Zhang, Y., G. H. Mazurek, M. D. Cave, K. D. Eisenach, Y.Pang, D. T. Murphy, and R. J. Wallace, Jr. Submitted forpublication.

VOL. 30, 1992

on June 30, 2019 by guesthttp://jcm

.asm.org/

Dow

nloaded from