Laboratory aspects of "Mycobacterium genavense," a proposed species isolated from AIDS patients

(1)

Laboratory Aspects of

"Mycobacterium

genavense,"

a

Proposed

Species

Isolated from AIDS Patients

MARIE B. COYLE,1,2* LADONNAC.

CARLSON,'

CAROLYN K.

WALLIS,'

REBECCA B.

LEONARD,'

VIDMANTAS A. RAISYS,1JAMES 0. KILBURN,3

MANSUR SAMADPOUR,4ANDERIK C. BOTrGER5

DepartmentofLaboratory Medicine, Harborview MedicalCenter, University of Washington, Seattle,

Washington

981041;

Departmentsof Microbiology2and EnvironmentalHealth,4University ofWashington, Seattle, Washington98195; DivisionofBacterial andMycoticDiseases, CenterforInfectious Diseases,

CentersforDisease Control, Atlanta, Georgia 303333; and Institut fur Medizinische Mikrobiologie, MedizinischeHochschule Hannover, 3000 Hannover 61, Germany5

Received 17July 1992/Accepted14September1992

"Mycobacteriumgenavense"is aproposed new species

recently

reported to cause disseminated infectionsin 18 patients with AIDS in Europe. We have recovered "M. genavense" as

slowly

growing fastidious mycobacteria in blood cultures of seven patients withAIDS. In the original studies of "M. genavense," the fastidiousorganism grew

only

inBACTEC 13A vials. The Seattle, Washington, isolates of "M. genavense" also failedtogrow whensubcultured from 13A vialstoroutine solidmedia, butdysgonic colonieswereproduced on Middlebrook 7H11 agarsupplemented with mycobactinJ. The mycolic acid pattern of patients' isolates

closely

resembledthat of thetype strain ofMycobacteriumsimiae whenanalyzed by one- and two-dimensional thin-layer chromatography and byhigh-performance liquid chromatography. Whole-cell

fatty

acid

analyses

by gas-liquidchromatography distinguished the isolates from M. simiae but misidentified them asMycobacterium fortuitum. Sequence determinations of thehypervariable regions of the 16S rRNA gene indicate that these organisms belongtothe

recently

proposednewspecies "M.genavense." Growth from Middlebrook 7H11 agar supplemented with mycobactin J

consistently

yieldedpositive tests for catalase (semiquantitative and at 68°C), pyrazinamidase, and urease which enable mycobacteriology laboratories to presumptively identify "M. genavense" without nucleic acid

analyses.

The failure of "M.genavense" togrow on conventional mycobac-terialsolid media suggests that mycobacterial blood cultures should includeabroth mediumincubated forat least8 weeks.

Theincidence ofmycobacterialinfections inpatientswith AIDS continues to climb, withMycobacteriumavium

com-plexaccounting for thevastmajorityof thesedisseminated

infections(13, 15). StudiesofpatientswithAIDShave found thatdisseminated M. aviumcomplex infections occur in 15 to24%ofpatients monitoredinclinics (15). Reportsof the incidence of M. tuberculosis infections in

patients

with AIDS range from 4to21%, depending on thegeographical region (7). Other Mycobacterium species that have been

reportedasinfrequentor rare causesof disseminated infec-tions inpatients withAIDSinclude M. kansasii,M. scrofu-laceum,M. fortuitum, M. chelonae,M. gordonae,M. xe-nopi, M. asiaticum, M. flavescens, M. malmoense, M.

szulgai, M. terrae (10-12), M. haemophilum (22, 31), M. simiae(21, 26,32,36),M.marinum (28),and M.leprae (18).

Aproposed species, "M.genavense," hasrecently been reportedto causedisseminated infectionsin 16patients with AIDS inSwitzerland andoneeach inAustria and Germany

(4). Because "M. genavense" failed to grow on all media except the radiometric BACTEC 13A (13A) mycobacterial blood culturebroth, biochemicaltestsforcomparisonswith other

Mycobacterium

species could not be done. Mycolic acidanalysesbythin-layer chromatography (TLC) indicated thatthe fastidiousorganismwassimilar to M. simiae and M.

malmoense(14). Byusing primers complementary to myco-bacterial 16S rRNA, the 16S rRNAgene was amplified by

polymerase chain reaction. Sequence determination

re-*

Corresponding

author.

vealedaunique 16S rRNA sequence, suggestingthatthese fastidious organisms are members of a proposed species,

"M. genavense," which was found to be most closely

related to M. simiae (4). It was suggested that "M.

genavense"may be anunrecognizedcauseofdisseminated infectionsin manypatients with AIDS because its failure to growon solid mediaprecludes identificationand character-ization in routinemycobacteriologylaboratories (4).

Our laboratoryisolated fastidious unidentified

mycobac-teria from 15 blood cultures fromsevenpatientswith AIDS

duringa2-yearperiod.Sequencingof the gene for 16S rRNA

confirmed the identity of these organisms as "M.

genavense." Like the Swiss isolates, they could not be subcultured from the original 13A vials onto routine solid

media; however, subcultures yielded dysgonic colonies on

Middlebrook7H11

(M7H11)

agar supplemented with myco-bactin J(MJ). Mycobacterialbrothmedia alsosupportedthe slowgrowthof subcultures fromourisolates. In this report, we describe the results ofa variety of identification

tech-niques for the newly recognized opportunist "M.

genavense," including whole-cell fatty acid analyses by gas-liquidchromatography(GLC), mycolicacidanalyses by

TLC andhigh-performanceliquidchromatography(HPLC),

and conventional biochemical tests employing MJ-supple-mented media forgrowth.

MATERIALSANDMETHODS

Strains. The 17isolates of "M.genavense"characterized inthis study included 15 from blood and 1 each from spleen

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and bonemarrow. Reference strains included

recently

pur-chased cultures of M. agri ATCC

27407,

M. avium ATCC 25291,M. bovis ATCC27290,M.

fortuitum

ATCC

6841,

M. gordonae ATCC 14470, M. malmoense ATCC 29571, M.

obuense ATCC27023, andM. simiaeATCC25275. Routine mycobacterial cultures. Theroutinemycobacterial blood culture mediumwas 13Abroth, which contains 30ml ofmodifiedMiddlebrook7H12 broth with

[14CJpalmitic acid,

catalase, and bovine serum albumin

(Becton

Dickinson Diagnostic InstrumentSystems, Sparks, Md.). Blood

spec-imens from Harborview Medical Centerpatientswith

sus-pected mycobacteremia were inoculated

directly

into 13A vials, and specimens from outside laboratorieswere

trans-ferred into 13A vials from VACUTAINER tubes

containing

sodium polyanetholesulfonate

(Becton

Dickinson and

Co.,

Rutherford, N.J.). Spleen tissue and bone marrow were

stained as unconcentrated

specimens

and inoculated onto

Lowenstein-Jensen

(UJ)

slants andM7H11 agarslants

(Pre-pared Media Laboratory, Inc.,

Tualatin, Oreg.). Spleen

tissuewasadditionally inoculated intoaBACTEC12B

(12B)

vial and the bone marrow

specimen

was inoculated into a 13A vial.

All cultures were incubated at 35 to 37°C. Media were

routinely incubated for 8weeks, with the

exception

of 12B

vials, whichwere heldfor 6 weeks.Vialswere incubated in an aerobic atmosphere and solid media in a5 to 10%

CO2

incubator. Vials were read for release of

14Co2

in the

BACTEC

460 instrument twice

weekly

for thefirst2weeks andthenweekly.Afteravialhadaradiometric

growth

index

of215,itwasconsidereda

suspected

positive

andwasread

daily until the growthindexreached

20,

atwhich

point

itwas

stained with an auramine acid-fast stain and Gram stain.

When solid media had visible

growth,

it was stained with Kinyoun's acid-fast stain. Positive

primary

cultures were

routinely subcultured onto

double-pour

(40 ml)

M7H11

plates and incubated in 100%

polyethylene

C02-permeable

bags at37°C in 5 to 10%

CO2

for 4 weeks.

Growth studies. Growth of "M.

genavense"

in 13A vials was subcultured to the following broth media: 12B,

BACTEC pyrazinamidase

testmedium

(pH 5.9), Septi-Chek

AFB medium (Roche Diagnostic Systems,

Nutley,

N.J.),

and Middlebrook7H9broth. The

supplemented

solid media used in this

study

included M7H11 agar

containing

2 Fgof MJ(Allied

Laboratories,

Fayette,

Mo.)

per ml

(M7H11-MJ);

UJ

with 50 ,ug of MJ added to the surfaces of the

slants;

M7H11-13Aagar, whichcontained M7H11 agarwith half of thewater

replaced by

13A

broth;

M7H11-13Aagar with5%

human

blood;

and M7H11 agar

supplemented

with 10%

yeastextract.

Supplemented

brothmedia included 13Aand 12Bvialswith MJ (2

,ug/ml).

Other subculture mediawere brain heart infusion agar with 5%

sheep blood,

chocolate agar, and buffered charcoal yeast extract agar

(Prepared

Media

Laboratory,

Inc.).

Mediawere incubated in

CO2

for up to 6 months or until desiccated. To allow

prolonged

incubation, all variations of M7H11 agar

plates

contained

30-mlvolumesandwereincubatedin

C02-permeable

plastic

bags.

Lipid analyses. Short-chainfattyacid

analysis

wasdone

by

GLC by

using

the Microbial Identification

System

(MIS)

(Microbial ID, Inc.,

Newark,

Del.).

The MIS includes a 5890A gas

chromatograph

witha

capillary

column andflame

ionization detector, an automatic sampler, an

integrator

3392A, and a microcomputer

(Hewlett-Packard,

Palo

Alto,

Calif.).

Peakswere

automatically

integrated, fatty

acid iden-tities and percentages were

calculated,

and the

species

identifications were determined by

using

the MIS MYCO

data base

(33, 34).

The manufacturer's

mycobacterial

proto-colwas followed for all stages of the

saponification,

meth-ylation, extraction,

and GLC

procedures

except that all

cultures, including

reference

strains,

were harvested from M7H11-MJ agarinstead of7H10 agarmedium.

Mycolic

acid

analysis by

TLCfollowedalkaline

methanol-ysis

asdescribed

by

Minnikinetal.

(24).

The

mycolic

acids

were

separated

by

using

one-dimensional TLCwith three

different solvent systems:

petroleum

ether-acetone

(95:5

(vol/vol]),

toluene-acetone

(97:3

[vol/vol]),

and dichloro-methane-petroleum ether

(95:5

[vol/vol]).

Two-dimensional TLC was done with two different solvent combinations:

petroleum

ether-acetone

(95:5

[vol/vol])

inthefirst direction followed

by

toluene-acetone

(97:3

[vol/vol])

in the second

direction and

dichloromethane-petroleum

ether

(95:5

[volt

vol])

in the first direction followed

by petroleum

ether-acetone

(95:5

[vol/vol])

in the second direction. All petro-leumether-acetone

separations

were

triple developments

for

good

resolution. Forboth

fatty

acid and

mycolic

acid

anal-yses,the referencestrains and

patient

isolateswerecultured

on M7H11-MJ

plates

and harvested when there was

suffi-cient cellmass

(a large loopful

of

cells)

for reliable

analyses.

For HPLC,

growth

from Middlebrook 7H9 broth was

pelleted,

washed once with

phosphate-buffered saline,

and

shipped

on

dry

ice to the Centers for Disease Control.

Mycolic

acids extracted from

saponified

cellswereexamined

asp-bromophenacyl

esterswith theHPLC Beckman

System

Gold

(Altex

Division Beckman

Instruments, Inc.,

San Ra-mon,

Calif.)

as

previously

described

(5).

Commercial DNA

probes.

Cell

pellets

from 30 ml of 13A brothwere tested with

125I-labeled

DNA

probes

for

identi-fication ofM.

tuberculosis,

M. avium,andM. intracellulare

according

to Gen-Probe

(San Diego,

Calif.) specifications.

M.

paratuberculosis

probe

testing

was done in the

Veteri-nary

Diagnostic

Laboratory,

Iowa State

University.

The X

probe (Syngene, Inc.,

San

Diego,

Calif.)

for M. avium

complex

was

performed

at the Nichols Institute

(San

Juan

Capistrano, Calif.).

Biochemical tests.

Urease,

Tween

hydrolysis,

3- and

14-day arylsulfatase,

semiquantitative catalase,

heat-stable

cat-alase,

and

7-day pyrazinamidase

tests were

performed

as

specified by

Kent and Kubica

(16).

For tests that

required

growth

onLJmedium

(niacin

and

semiquantitative

catalase),

50 ,ug of

MJ

wasaddedtothe surfaces oftheslantsor

deeps

prior

toinoculation. Variations of theniacinandnitratetests were

attempted

asfollows. Niacin and nitrateweretested

by

the combined methodfrom

growth

on

M7H11-MJ

plus

0.1%

potassium

aspartate

(16).

Niacinwastested

by

the conven-tional chemical reagent methodwith a 2-h extraction

time,

and nitrate was

additionally

tested

by

the classical method with the

crystalline

reagent

(16).

Niacin and nitrate reactions were determined after extended incubation for 4 to 12 weeks. A

28-day

arylsulfatase

test wasdonewiththe

14-day

broth substrate.

Pigmentation

studies were

performed

with

M7H11-MJ

plates

because theconventional

IJ

slants didnot support

growth.

Three different

light

exposure methods included a

single

4-h and adouble 4-h exposureon consec-utive

days

and an

overnight

light

exposure

(39).

Control

organisms

were handled in

exactly

thesame manner as the

patient isolates,

with the

exception

of those for the nitrate tests, whichwere doneonyoung cultures.

16S rRNA sequence

analysis.

Cultures of three repre-sentative isolates from 13A vials andM7H11-MJ

plates

were

subjected

to sequence

analysis.

DNA was extracted and

amplified by polymerase

chain reactionas described

previ-ously

(3). Briefly,

the bacterial cell

pellet

was dissolved in

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100 ,ul of H20, heated for 10 min at80°C toinactivate the

mycobacteria, and then transferred to a 1.5-ml screw-top

plastic microcentrifuge tube containing glass beads with a diameter of 100p,m(Sigma).Atissuedisintegratorwasused

to disrupt the cells (H. Mickle, Gomshall, Surrey, United Kingdom). A5-,ulvolume of supernatantwasused in

poly-merase chain reaction. The relevant fragment from the

hypervariable region of the 16S rRNA gene was amplified withprimer246(AGAGTT TGA TCC TGGCTCAG) and

primer 264 (TGC ACA CAG GCC ACA AGG GA) as

describedpreviously (3). TheamplifiedDNAfragmentwas

analyzed for the characteristic signature sequence of "M.

genavense" bydirect sequence determination (9, 29).

RESULTS

Original cultures. Between December 1988 and January

1991, seven patients withAIDS had slowly growing, acid-fast bacilli in 13A blood culturevialsthatfailedtogrowwhen subcultured onto M7H11 agar plates. The clinical back-groundsof thesepatientshavebeen describedbyWaldetal.

(38).

Therewas atotal of15positivebloodculturesfromthe

seven

patients,

who each had 1 to 5

positive

cultures. The meanandmediantimesto radiometric detection ofpositive blood cultures from these patients were 38 and 42 days, respectively,whichwere24and 28days longerthanforM.

avium blood cultures in our laboratory (1). Six of the 15

positive blood cultures failed to grow within the

manufac-turer-recommended 6 weeks of incubation, and 2 did not growwithin 8 weeks. The range for time to positiveblood cultureswas 14to67 days.

Twopatients had positive mycobacterial cultures froma second site. One hadrareacid-fast bacilli inadirectsmearof bonemarrowwhichyielded pinpointcolonies after 60days

onM7H11platesand failedtogrowin eitheraprimary13A vial or an LJ slant after 70 days. A spleen specimen from anotherpatient containednumeroussmall acid-fast bacilli in

the directsmear andwas

positive

inthe 12Bvialon

day

4 and on the M7H11

plate

on

day

25. The LJ slant fromthe spleenwasdiscarded after it failedtogrowwithin the routine 8-week incubation period. The

microscopic

morphology of

growth from all of these fastidious isolates in 13A vials

revealedsmall, clumped, acid-fast coccobacilli.

Growth studies. The

only

solid medium tested in our

laboratory that consistently yielded colonies of "M.

genavense"wasM7H11-MJ. Themeantime for the appear-ance of

tiny,

transparent,

dysgonic

coloniesonM7H11-MJ

plates subcultured from growth in 13A vials was 42 days (range, 18 to 53 days). However, themeantime forvisible

colonies to develop from consecutive subcultures on

M7H11-MJ

plates

was 13

days

(range,

5to67

days).

As agar

cultures

aged, they

usually produced

variant

eugonic

colo-nies whichwereeither dense and creamyorflatanddry. In

general, after prolongedincubation the cultures resembled those of M. avium. Subcultures of all colony types were

similar,

but the eugonic colonies usually yielded a larger proportionofeugoniccolonies. Subcultures from 13Avials yielded slow growth in all the mycobacterial broth media tested: 13A and12B,BACTECpyrazinamidasetestmedium

(pH

5.9), Septi-ChekAFB medium, and Middlebrook 7H9 broth.

Nogrowthoccurredon

UJ

with 50 ,ug of MJ addedtothe surface of the slantsor on13A agarwith5%human blood. A few subculturesoccasionallygrewminimallyonM7H11-13A agar, M7H11 agar with 10% yeast extract, and buffered charcoal yeast extract agar. Supplementing 13A and 12B

vials with MJ(2 ,ug/ml) either partially or completely inhib-ited growth of "M. genavense."

Identification tests. Fourteen of the 17 fastidious isolates wereanalyzed for whole-cell fatty acids by GLC in the MIS, which identified all as M. fortuitum, withsimilarity indices that are considered diagnostic for cells harvested from standardizedgrowth conditions.Table 1shows the distribu-tion of the fatty acid components from representative cul-turesfrom each of the sevenpatients and the type strains of M. avium, M.fortuitum,andM. simiae. With the exception

of minor peaks found in samples prepared from relatively largequantitiesofcells, all clinical isolates appeared to have the samefattyacidcomposition in theGLC analysis. The M. simiae strain consistently differed from the clinical isolates

in two fatty acids, cis-11-hexadecenoic acid (present) and cis-10-hexadecenoic acid (absent). The MIS results from

growthonthe routine M7H11 agarwereessentially identical tothose from M7H11-MJ agar for the three reference strains. The mycolic acid patterns of isolates from the seven

patients were indistinguishable from each other and from

those of the typestrainsof M. simiae and M. malmoensein bothone- andtwo-dimensional TLC in all solvent systems. Figure 1 illustrates the two-dimensional patterns from a

representative clinical isolateaswell as reference strains of M. simiae, M.fortuitum,and M. avium. Seven isolatesfrom four patientswere analyzed by HPLC at the Centers for DiseaseControl(byJ. 0. K.),wherethey closely resembled M. simiaeasshown inFig.2. Nodifferencesweredetectable in the mycolic acid patterns of dysgonic and eugonic colony typesineither TLCorHPLCanalyses.

Theinitial blood isolatefrom eachpatient and the spleen and bonemarrowisolateswerepelletedfrom60 ml of 13A broth and tested with the commercial DNAprobes for M.

tuberculosiscomplex,M. avium,andM.intracellulare, and

the results were negative in all tests. Because these fastidi-ousisolates exhibitedgrowthstimulation onsolid media in the presence ofMJ,arepresentative culture from each of the sevenpatientswastested with aDNAprobe forM.

paratu-berculosis, whichwasunreactive withall tested isolates. One isolate from eachpatientwastested ineight conven-tionalbiochemicaltestsand forpigmentation (Table2). The isolates had strong activity in the urease test, which was

positive within an hour. Both of the catalase tests were

stronglypositive, althoughtherewas noevidence ofgrowth

on the LU deeps for the semiquantitative catalase. The pyrazinamidasetest waspositive within 30 min. Theniacin, nitrate,Tweenhydrolysis, andarylsulfatase tests were neg-ative. The negative biochemical tests and the failure to

produce pigment could not be interpreted with complete

confidence because of the poor growth rate of "M.

genavense."

The sequences of the hypervariable regions of the 16S rRNA gene from three representative patient isolates har-vested from both 13A broth and M7H11-MJ agar were identical to those described for "M. genavense" (4).

DISCUSSION

This is the first report of the proposed species "M. genavense" growingonany culture medium other than 13A broth. Growth on solid media makes it possible for these fastidiousorganisms tobebiochemicallycharacterized and

presumptively identified in mycobacteriology laboratories for which HPLCor nucleic acid analysis technologyis not available. The M7H11-MJ medium isaverysimplemedium toprepare; however, the slow growth of "M. genavense"

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a

CA

D E

,F

c

F

M

FIG. 1. Two-dimensional TLC of alk ple development was done with petr4 [vol/vol])inthefirstdirection followed b! toluene-acetone (97:3 [vol/vol]) in the s(

tions:A,a-mycolate; A',a'-mycolate;I boxymycolate; E, alcoholshomologousN

mycolate;F,nonhydroxylatedfatty acid ATCC 25291; (b)M. simiae ATCC252, 6841;(d)"M.genavense"isolate 2289.

requires 4 to8weeks for sufficienti icaltests.

Intheoriginalreportby Hirschel yieldednogrowthonavariety ofso

addedanumber of supplements, inc

however, the study did not incl mented M7H11agar,whichwefoun

growth ofourisolates. It is well ki

lot-to-lot variation in the ability of richmentsto supportthe growth ofrn

mycobacteria (6).Althoughmany Ic

acid-albumin-dextrose-catalase enri

over the course of our studies, it

C.)

C n0 2 (0

D)

7.0 8.0

(min.) FIG. 2. Representativepatternsofbr colicacidsdetermined by HPLC fromA patientisolatesof"M. genavense."

b batches of M7H11-MJ will support growth of "M.

genavense." It is interesting that our routine M7H11 agar * * yielded growth of thesefastidious mycobacteria when inoc-A F ulated with concentratesof thespleen andbone marrowbut CA' did not supportgrowth when inoculated with subculturesof these specimens. This may reflect a nutritional factor that was carried over from the specimen or it could simply bedue to the large biomass in these smear-positive specimens,

particularly those ofthe spleen, which contained very large numbers of acid-fast bacilli.

The fact that "M. genavense"did not grow onsolidmedia d without

mycobactin

initially

raised

questions

about its

pos-sible relationship to species known to exhibit mycobactin

dependence,

such as M. avium subsp.

paratuberculosis

(M. paratuberculosis),M. avium subsp.silvaticum (wood pigeon

'A F

mycobacteria),

and M. avium

subsp.

avium

(35).

However,

C

A"

DNA probes, whole-cell fatty acid analyses, mycolic acid

analyses, andbiochemical reactions ruled outpossible

iden-tity with the M. avium group. Furthermore, the growth dependence of "M. genavense" on mycobactin was not evident with 13A broth, in which the mycobactin inhibited growth. It is possible that the role of mycobactin in the agar kaline methanolysates. Tri- medium maynotbeas achelator ofironbutas achelatorof

oleum ether-acetone (95:5 some other trivalent metal ion which is potentially toxic for ya single development with "M. genavense."

econd direction. Abbrevia- Without the benefit of 16S rRNA sequencing, the most

C,

ketomycolate;

D,w-car- objective evidence against our patients' isolates simply being

with

2-eicosanol;

M, epoxy- dysgonic variants of M. simiae was the results from

whole-methylesters.(a)M. avium cell

fatty

acid

analysis

in the MIS. Previous studies also 75; (c)M.fortuitumATCC found that mycobacterial isolates could be more conclu-sively identified when fatty acids and mycolic acids were analyzed by a combination of GLC and TLC (8, 17, 37). A visual inspection of the GLC patterns presented in Table 1

noculation

for

biochem-

shows two consistent differences between M. simiae and our

eta

.genavense"

isolates of "M. genavense." The

cis-10-hexadecenoic

acid

etal., "M.

genavere

peak was missing in

M.

simiae, and the cis-11-hexadecenoic lid media to which were acid was missing in the patients' isolates. On the other

hand,

luding

mycobactins(14);

the whole-cell fatty acid patterns did suggest that "M.

ude mycobactin-supple- genavense" is closely related to M. simiae. Both "M.

dnt

consistently support genavense" and the type strain of M. simiae had major nown that there is great peaks corresponding to palmitic acid and oleic acid as well as albumin-containing en- moderate amounts of myristic acid and tuberculostearic outine clinical isolates of acid. It may be more than coincidental that our isolates of

)tsofMiddlebrookoleic

"M.

genavense" were misnamed

M.

fortuitum in the GLC chment have been used analysis in view of the fact that sequencing of 16S rRNA has is possible that not all shown that

M.

simiae occupies a phylogenetically interme-diate position between the slowly growing and fast-growing mycobacteria (30).

Because M. avium infections have been thought to origi-nate in the

intestine,

it is

interesting

to

speculate

that "M.

genavense" sepsis may have a similar origin. Portaels et al. used a MJ-containing medium to recover 18 isolates of unidentified fastidious mycobacteria from intestinal tissues of patients with a variety of intestinal diseases (27). The recent culture survey of intestinal tissues by Markesich et al. suggested that fastidious acid-fast bacteria are relatively common in patients who have bowel biopsies or resections

*, (23). When intestinal samples from patients with and without

\'I inflammatory bowel disease were cultured for prolonged ___________ periods on MJ-containing medium, 28 to 32% yielded uni-dentified spheroplasts and fastidious mycobacteria, respec-9.0 10.0 tively, of which

very

few could be identified by standard

biochemical methods.

romophenacyl esters of my- To our knowledge, there is no other report ofdysgonic

W.simiae ATCC 25275 and mycobacteria resembling M.simiaefrompatientswith AIDS inthe United States. We have no good explanation for why

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TABLE 2. Biochemical reactions of "M. genavense"and reference strains

Result"with strain

Test M. avium M.fortuitum M.

malnoense

M.sirniae "M navense"b

ATCC 25291 ATCC6841 ATCC29571 ATCC25275 ge

Pigment N N N P N

Niacin - - +

Nitrate - +

Semiquantitative catalase - + + +

68°C catalase + + + + +

Tweenhydrolysis - + +

Arylsulfatase - +

Urease - + + + +

Pyrazinamidase + + + + +

aN,nonchromogenic; P,photochromogenic.

bOneisolate from each of thesevenpatients was used.

our laboratory has recovered so many isolates of "M.

genavense." This amount of recovery may be due to

envi-ronmental influencesin the Seattleregionor tothefact that we incubate all 13A vials for 8 weeks, which is 2 weeks

longer thanthetime recommendedinthe BACTEC manual.

However, growth was detected before 6 weeks in blood cultures from four of the seven

patients

in this

study.

Regional differences in the incidence of nontuberculous mycobacterialdisease have beenrecognizedfor manyyears

(41). These regional differences are particularly striking in the caseofM. simiae,whichwasthemostfrequentatypical mycobacterium isolatedfrom sputumspecimens in Tel Aviv, Israel (20), and from stool specimensfrom healthy volun-teers in one Europeanstudy

(27).

In contrast,M. simiaeis

extremelyrareinmostpartsof the UnitedStates, composing only0.7% of all nontuberculous

mycobacterial

isolates

(25),

whereasin southTexas,it isarelativelycommonisolate

(2).

Ourisolates of"M.genavense" are thefirst strains

resem-bling M. simiae that our

laboratory

has recovered from a

specimen.The stateof

Washington

receivesreportsof

only

one or twoisolates ofM.simiaeeach year. A report that M. simiae wasisolated fromwatersuggests that

regional

differ-encesmay be due tothe

prevalence

ofthis

species

inlocal water supplies (19). Eveninregions whereit iscommonly isolated,M. simiaeisreportedtobearare causeofdisease (40).

The incidence of"M.genavense" inthe Seattleareahas varied with time.Althoughwehad 17positivecultures from seven patientsbetween December 1988 and January

1991,

wefoundno subsequent isolatesuntil December 1991. This sharpdropin the recovery of"M.

genavense"

is

particularly

strikinginviewof the fact that9.1% of the77

patients

with mycobacteremia in 1989 and 1990 carried this fastidious

organism. Ourfailure to recover"M.

genavense"

between January andDecember1991 may be dueto some

changes

in the Seattle water supply, 13A broth, or the therapy of

patientswith AIDS.

Some laboratories in the United States have replaced

brothmedia formycobacterialbloodcultureswitha combi-nation oflysis-centrifugation and solid media. Our

experi-ence with "M. genavense" from patients with AIDS sug-gests that all blood cultures formycobacteriashould include abroth medium which is incubated foratleast 8 weeks.

ACKNOWLEDGMENTS

Wegratefullyacknowledgethededicatedefforts ofthe technolo-gistsin theMycobacteriologyLaboratory,especiallySusanStiglich, MollyDougherty, andCheryl McMillan.

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