Vol. 28, No. 12JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1990, p. 2726-27320095-1137/90/122726-07$02.00/0Copyright © 1990, American Society for Microbiology

Cefotaxime-Resistant Nocardia asteroides Strains Are Isolates ofthe Controversial Species Nocardia farcinica

RICHARD J. WALLACE, JR.,1* MICHIO TSUKAMURA,2 BARBARA A. BROWN,' JUNE BROWN,3VINCENT A. STEINGRUBE,' YANSHENG ZHANG,' AND DONALD R. NASH'

Department of Microbiology, The University of Texas Health Center, Tyler, Texas 757101; The National ChubuHospital, Obu-Aichi, Japan2; and Division of Mycotic Diseases, Center for Infectious Diseases,

Centers for Disease Control, Atlanta, Georgia 303333

Received 22 June 1990/Accepted 18 September 1990

A recent study of Nocardia asteroids revealed that 95% of clinical strains had one of five antibiotic resistancepatterns. We found the pattern of resistance to cefotaxime and cefamandole in 19% of 200 clinical N. asteroidesisolates. Isolates with this drug resistance pattern were from numerous geographic sources and were associatedwith significant clinical disease (56% of patients had disseminated infections). Phenotypic studies revealed thatthese isolates were relatively homogeneous and matched previous descriptions and reference strains of thecontroversial species N. farcinica. Growth at 45°C, acid production from rhamnose, ability to utilize acetamideas a nitrogen and carbon source, and resistance to tobramycin and cefamandole were features of N. farcinicathat could be tested in the clinical laboratory and allowed their distinction from N. asteroids. The seriousnature of disease due to N. farcinica and its resistance to the newer cephalosporins suggest a clinical need forlaboratory identification of this species. (Current tests used in clinical laboratories do not distinguish N.farcinica from N. asteroidss) This is the first recognition that N. farcinica has a specific drug resistance patternand confirms the previously described concept that drug resistance patterns of N. asteroids may be associatedwith specific taxonomic groups.

A recent study of the antimicrobial susceptibility patternsof 78 clinical isolates of Nocardia asteroides identified fivemajor patterns that included 95% of the strains. One of thesegroups, involving 17% of the strains, was unique in thatisolates within the group were resistant to cefotaxime andceftriaxone (28). These two newer broad-spectrum cephalo-sporins are of clinical interest for treatment of nocardiosisbecause of excellent (in vitro) activity against N. asteroidesand N. brasiliensis (5, 10, 26, 28), excellent central nervoussystem penetration, and for ceftriaxone, prolonged half-lifein serum that makes long-term parenteral therapy feasible.The N. asteroides isolates resistant to these cephalosporinswere also remarkably homogeneous in their patterns ofresistance to other antimicrobial agents, including aminogly-cosides, quinolones, macrolides, and other beta-lactam an-tibiotics.Because of these similarities, we elected to study isolates

of N. asteroides with this drug resistance pattern (referred toas a type 5 pattern) (28) by detailed biochemical studies todetermine whether they might be taxonomically similar aswell. These isolates were remarkably similar when studiedby these methods and matched previous descriptions andlaboratory strains, including the type strain of N. farcinica.

MATERIALS AND METHODS

Organisms. Isolates of N. asteroids submitted to theMycobacteria/Nocardia Laboratory of the University ofTexas Health Center for susceptibility testing between 1983and 1989 and 30 random isolates submitted to the Microbi-ology Laboratory of the Mayo Clinic, Rochester, Minn.,between 1979 and 1983 were screened for isolates resistantto cefotaxime and ceftriaxone. Clinical information on the

* Corresponding author.

resistant isolates was obtained by correspondence, by tele-phone conversations with the primary physicians, or bychart review, with attention to the underlying diseases, thetypes of clinical diseases, and whether the isolates wereclinically significant. In addition, 40 clinical isolates thatincluded examples of the other four major drug resistancepatterns of N. asteroides identified in our previous study (28)and the type strain of N. asteroides, ATCC 19247, werechosen as controls. These isolates had previously beenidentified as N. asteroides by standard methods (15), mostby the Mycology Section of the Texas Department ofHealth, Austin. Following initial testing, the isolates werestored at -70°C in tryptic soy broth with 15% glycerol forlater study.

Six reference strains were studied. The already mentionedtype strain of N. asteroides, ATCC 19247, was obtainedfrom the American Type Culture Collection (ATCC), Rock-ville, Md. The type strain of N. farcinica, ATCC 3318, wasprovided by M. Tsukamura (The National Chubu Hospital,Obu-Aichi, Japan), who had originally received the strainfrom R. Gordon (Waksman Institute of Microbiology, Rut-gers University, Piscataway, N.J.) in 1967.

Five additional laboratory isolates of N. farcinica wereidentified among isolates of Nocardia spp. kindly providedby the ATCC for evaluation in the current study. These wereATCC 13781, ATCC 23825, ATCC 23826, ATCC 3308, andATCC 6846 (formerly Actinomyces blackwellii). The firsttwo isolates are listed by the ATCC as examples of N.farcinica, while the third isolate is listed as N. paratubercu-losis (a suggested but not accepted replacement name for N.farcinica). ATCC 3308 is the Preceptrol culture made avail-able as an example of N. asteroides, while ATCC 6846 isalso included as N. asteroides. Both were found to beexamples of N. farcinica only after evaluation in this study.

Susceptibility testing. Susceptibility testing was performedby broth microdilution using cation-supplemented Mueller-

2726

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

CEFOTAXIME-RESISTANT N. ASTEROIDES 2727

Hinton broth and 96-well plates. The plates contained two-fold dilutions of amikacin, kanamycin, gentamicin, tobramy-cin, erythromycin, ciprofloxacin, ampicillin, cefamandole,cefotaxime, and ceftriaxone which were prepared fromdiagnostic powders and dispensed with the Mini QuickSpense Il System (Dynatech Inc., Chantilly, Va.). Plateswere inoculated with a disposable inoculator, with a result-ant final organism concentration of approximately 104 CFU/ml. The plates were incubated for 72 h at 35°C in room airbefore being read. Control strains were Escherichia coliATCC 25922, Staphylococcus aureus ATCC 29213, and thetype strain of N. asteroides, ATCC 19247. Details of theprocedure are presented elsewhere (28). The definitions ofsusceptibility and resistance used were those of the NationalCommittee for Clinical Laboratory Standards for organismsthat grow aerobically (16).

Disk diffusion susceptibility testing was performed byusing Mueller-Hinton agar and commercial disks of cefaman-dole and tobramycin as previously described (27). Plateswere incubated for 72 h at 35°C, after which the diameters ofthe clear zones of inhibition (including the size of the 6-mmdisk) were measured.

Biochemical testing. Of the 40 clinical isolates of N.farcinica, 20 were chosen for extensive biochemical studies,as were ATCC reference strains of N. farcinica and the 40comparative strains of N. asteroides. Standard hydrolysistests were performed for tyrosine, xanthine, adenine, casein,and hypoxanthine. Resistance to lysozyme was determined,as was acid production from the 23 carbohydrates glucose,mannitol, lactose, sucrose, maltose, salicin, glycerol, starch,arabinose, xylose, inositol, rhamnose, trehalose, galactose,mannose, sorbitol, dulcitol, raffinose, adonitol, fructose,erythritol, cellobiose, and melibiose. All results were readafter 14 days.The isolates of N. farcinica and N. asteroides were tested

for 112 growth and enzymatic characteristics as previouslydescribed by Tsukamura for Nocardia spp. and rapidlygrowing mycobacteria (22). This included evaluation forresistance to multiple agents and drugs, amidase production,and ability to utilize numerous amines, carbohydrates, andglycols as nitrogen or carbon sources or both.Four tests were then chosen that, on the basis of the

previous results, appeared to separate N. farcinica from N.asteroides and could be performed with commercially avail-able media. The selected tests were (i) growth at 45 versus35°C after 3 days on tryptic soy agar slants (Difco Labora-tories, Detroit, Mich.), (ii) acid production on CTA Mediumwith rhamnose with phenol red as the indicator (BBLMicrobiology Systems, Inc., Cockeysville, Md.), (iii) utili-zation of acetamide as a nitrogen and carbon source withprepared acetamide agar slants and phenol red as the indi-cator (BBL), and (iv) resistance to tobramycin and cefaman-dole. Positive growth at 45°C was interpreted as confluent,mature growth comparable to growth at 35°C. For rhamnose,the test result was considered positive if the upper portion ofthe agar changed from red to bright yellow. For acetamide,the result was considered positive if the entire slant changedfrom pale yellow to a dark pink that matched the colorchange obtained with a control organism. The latter twotests were read at 24, 48, and 72 h and 1 and 2 weeks, andresults were considered positive when the above-describedcolor changes occurred. Early readings obtained with rham-nose were important, since some test results became nega-tive with prolonged incubation.

RESULTS

Organisms. Between 1983 and 1989, 170 consecutive iso-lates identified as N. asteroides by standard hydrolysismethods underwent susceptibility studies in the Universityof Texas Department of Microbiology laboratory. Of 33isolates that were resistant to cefotaxime and/or ceftriaxone,31 (18%) had the type 5 drug resistance pattern (28). Anadditional 30 random clinical isolates submitted to the Mi-crobiology Laboratory of the Mayo Clinic from 1979 to 1983were also assessed. Seven (23%) of these isolates werecefotaxime resistant, and all had the type 5 drug resistancepattern. Overall, 19% of the 200 unselected isolates obtainedover this 10-year period had this drug resistance pattern andhereafter are referred to as isolates of N. farcinica. Oneadditional isolate from 1975 and one from 1982 were alsoidentified in the University of Texas Department of Micro-biology laboratory and used in the study.

Clinical histories were available for 30 of the 40 patientsfrom whom N. farcinica had been recovered. The isolatesfrom all but one patient were considered clinically significantand the cause of disease. Most of the patients had underlyingimmunosuppressive disorders. Ten (33%) of the patientswere receiving corticosteroids and/or other chemotherapeu-tic agents. An additional nine (30%) of the patients hadreceived a renal or cardiac transplant. Five (17%) of thepatients with primary cutaneous diseases had a history oftrauma. Only two (7%) of the patients had no knownunderlying disease. Of the 30 patients in whom the extent ofdisease was known, 17 (57%) had disseminated diseases and10 of these had meningitis and/or a brain abscess. Theremaining 13 patients had localized diseases. The isolatescame from 11 states (Alabama, Arizona, Connecticut, Flor-ida, Idaho, Iowa, Minnesota, Mississippi, Nebraska, Penn-sylvania, and Texas), and most (50%) were from Texas. Twoisolates from France (N5 and N9) were kindly provided byM. D. Kitzis and L. Gutmann (Hospital Saint Joseph, Paris,France) and have previously been included in a susceptibil-ity study (10).

Susceptibility patterns. Because MICs were determined atthe time of initial recovery of the organism and because theantimicrobial agents considered for therapy for disease dueto Nocardia infection have changed with time, not allisolates were tested against all of the drugs. The MICs of thedrugs tested against the clinical and five reference strains ofN. farcinica are shown in Table 1.The clinical isolates were highly drug resistant, with

resistance MICs of cefamandole (100%), cefotaxime (100%),ceftriaxone (80%), ampicillin (95%), kanamycin (100%), gen-tamicin (100%), tobramycin (100%), and erythromycin(96%). The isolates were usually susceptible to imipenem(82%) and ciprofloxacin (88%), and all strains were suscep-tible to sulfamethoxazole and amikacin. Interestingly, two ofthe reference strains of N. farcinica were more susceptibleto all P-lactam antibiotics than were the 40 clinical strains(Table 1). The MIC50 (MIC for 50% of the strains tested),MIC.., mode, and range of MICs of 12 of the antimicrobialagents for 13 of the clinical isolates were reported in theinitial study of drug resistance patterns of N. asteroides (28).

Disk diffusion testing of susceptibility to tobramycin andcefamandole was performed on the 40 clinical strains of N.farcinica and the 40 strains representing all of the drugresistance patterns recognized in N. asteroides (28). With azone diameter of growth inhibition of 20 mm around the diskas the breakpoint, 37 of 38 isolates with zone sizes of lessthan 20 mm in response to cefamandole were isolates of N.

VOL. 28, 1990

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

2728 WALLACE ET AL.

TABLE 1. MICs for clinical and reference isolates of N. farcinica

MIC (,ug/ml) for reference strain: Clinical strains

Drug ATCC ATCC ATCC ATCC ATCC No. MIC (,ug/ml) %23826 23825 3318 3308 6846 tested 50% 90% Mode Range Susceptible'

Gentamicin >16 >16 >16 >16 >16 24 >16 >16 >16 >16 0Tobramycin >16 >16 16 >16 >16 11 >16 >16 >16 16->16 0Kanamycin >16 >32 >32 >32 >32 24 >32 >32 >32 >32 0Amikacin 0.25 <0.25 0.5 1 2 40 1 2 1 0.5-4 100

Erythromycin >16 16 >16 8 >16 35 >16 >16 >16 4->16 3Sulfamethoxazole 2 2 4 '1 2 40 4 16 4, 8 <32 100Ciprofloxacin 1 4 1 1 4 40 1 4 1 0.5->4 88

Cefotaxime >64 >64 4, 16 8 >64 40 >64 >64 >64 64->64 0Ceftriaxone >64 64 2, 8 4 >64 32 >64 >64 >64 32->64 20Cefamandole >64 >64 8, 16 8 32 30 >64 >64 >64 64->64 0

Ampicillin >32 >32 8 >32 16 40 >32 >32 >32 16->32 5Imipenem 8 1 O0.5 o0.5 c0.5 40 4 16 2 1-32 82

Cl Isolates with MICs less than or equal to the moderate-susceptibility breakpoint defined by the National Committee for Clinical Laboratory Standards for brothdilution testing (16).

farcinica. With the same breakpoint of 20 mm for bothtobramycin and cefamandole, 37 (93%) of the 40 clinical N.farcinica isolates were resistant to both drugs, comparedwith none of the 40 N. asteroides isolates (Table 2). Inter-estingly, four of the five ATCC strains of N. farcinica hadzones of inhibition of 20 to 27 mm in response to cefaman-dole and hence were considered susceptible.

Biochemical results. In the standard hydrolysis assays, the20 clinical isolates of N. farcinica (chosen from the original40 isolates for more extensive study) failed to hydrolyzeadenine, casein, xanthine, hypoxanthine and tyrosine, andall but two were resistant to lysozyme. By these tests, the N.farcinica isolates were indistinguishable from the 40 isolatesof N. asteroides.The 20 clinical isolates of N. farcinica were reAatively

inactive when tested for fermentation of the Gordon sugars(Table 3). Of the 23 carbohydrates against which they were

tested, acid production occurred with only glucose (100%),glycerol (95%), and rhamnose (85%). The N. asteroidesisolates were also relatively inactive. They also fermentedglucose (88%) and glycerol (100%) but were unable to utilizerhamnose (0%).On the basis of the test characteristics of Tsukamura, the

20 clinical strains of N. farcinica were remarkably homoge-neous (Table 3). They grew at 45°C (100%), were inhibitedby 0.1% NaNO2 (100%), utilized acetamide (85%) and mono-ethanolamine (100%) but not serine (0%) as nitrogen andcarbon sources, were unable to use numerous carbohydratesas carbon sources, and failed to reduce nitrate. These results

TABLE 2. Disk zone sizes of N. asteroides and N. farcinica in response to tobramycin and cefamandole with commercial drugdisks and a Kirby-Bauer susceptibility testing method (27)

No. of isolates with or presence of the following diskNo. of zone sizes with: No. of isolates

Strain isolates Tobramycin Cefamandole with both disktested __zones <20 mm

<20 mm -20 mm <20 mm .20 mm

N. asteroidesClinical strainsType 1 8 1 7 1 7 0Type 2 2 0 2 0 2 0Type 3 7 6 1 0 7 0Type 4 7 7 O O 7 OType 6 14 0 14 0 14 0Miscellaneous 2 0 2 0 2 0

Reference strain ATCC 19247T - + - +

N. farcinicaClinical strains 40 40 0 37 3 37Reference strainsATCC 3318T + - - +ATCC 3308 + - - +ATCC 6846 + - - +ATCC 23825 + - - -ATCC 23826 + - + -

a See reference 28 for details of the drug resistance groups.

J. CLIN. MICROBIOL.

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

CEFOTAXIME-RESISTANT N. ASTEROIDES 2729

TABLE 3. Growth characteristics and biochemical test results of 20 current N. farcinica isolates compared with those of four referencestrains, 40 N. asteroides isolates, and the original description of the species

N. farcinica% of N. asteroids

Test % of clinical ATCC ATCC ATCC ATCC % Positive in clinical isolates"

psositive 23826 23825 3318 3308 original positivepositive description'

Growth45°C (3 days)0.1% NaNO20.2% NaNO2

Nitrate reduction (24 h)

Utilization as N and C sourceAcetamideMonoethanolamineSerine

Utilization as C sourceArabinose2,3-Butylene glycol1,3-Butylene glycolCitrateEthanolFructoseGlucoseInositolMannoseMannitolRhamnoseSorbitolSucroseTrehaloseXylose

Amidase production from:AcetamidaseBenzamidaseNicotinamidasePyrazinamidaseUrease

Resistance to 5-fluorouracil (20 ,ug/ml)

a-EsteraseP-EsteraseArylsulfatase (14 days)

Acid production from:ArabinoseGalactoseGlucoseGlycerolInositolMannitolMannoseRhamnoseSorbitolTrehalose

100 + + + +O NDd _ _ +0 ND - - +

15

85 + + + +100 + + + +

O

O100

5o

95100100

5355

905osO

+ - + _

+_

+ - + ++ + + ++ + + _

+ +

+ + + +

+ +

100 + + + +55 + + + -95 + + + _95 + + + +100 + + + +

100

O100

O

+ + + +

+ + + +

15 ND ND15 ND ND

100 ND ND95 ND NDS ND ND0 ND ND

20 ND ND85 ND +0 ND NDS ND ND

- ND- ND+ NDND ND- ND- ND- ND+ +- ND+ ND

a Reference 19.b n = 40.C n = 38 (number of isolates in original description [191).d ND, Not determined.e Not in original description (19); characterized in a later study (20).

were similar to results reported for prior isolates of N.farcinica by Tsukamura (19, 20) (Table 4).

Three of the four ATCC reference strains obtained asexamples of N. farcinica gave results with the Gordonsugars and the test of Tsukamura that were comparable tothose of the 20 clinical isolates and historical controls. These

were ATCC 3318; the type strain of N. farcinica, ATCC23825; and ATCC 23826. The fourth strain maintained in theATCC as an example of N. farcinica, ATCC 13781, was acidfast, was unable to grow at 45°C, failed to produce acid fromrhamnose, was arylsulfatase positive at 3 days, and wasidentified as a rapidly growing mycobacterium. One addi-

92NDND

3310088

O 90

9797o

o97946e

9710097O

71O89o3

26o

13155

31513481883100

315333o283

10071948797

ND

oe77eo

5513633570

80

283013

OO

100NDoo3

89O18

NDNDNDNDNDNDNDNDNDND

VOL. 28, 1990

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

2730 WALLACE ET AL.

TABLE 4. Results of four tests chosen from Table 3 because of commercial availability of materials and ability to distinguish40 N. farcinica strains and 40 N. asteroides strains

N. farcinica N. asteroides

Test Reference strainsTest % of clinical % of clinical ATCCisolates positive ATCC ATCC ATCC ATCC ATCC isolatesa positive 19247

23826 3318 6846 3308 23825

Growth at 45°C for 3 days 100 + + + + + 33 -

Utilization of acetamide as N 80 + + + + + 13 -

and C sourceAcid production from rhamnose 80 + + + + + 3 -

Resistance to tobramycin and 93 + -C _C C C 0 -

cefamandolea n = 40.b +, Positive; -, negative.c Four of the five laboratory strains were resistant to tobramycin but borderline susceptible to cefamandole.

tional strain, ATCC 3308, carried as the Preceptrol culture ofN. asteroides, was tested with both techniques and hadbiochemical features typical of N. farcinica. The results ofthese four laboratory strains confirmed as N. farcinica (i.e.,ATCC 3318, ATCC 3308, ATCC 23825, and ATCC 23826) onthe basis of the test characteristics of Tsukamura are in-cluded with those of the clinical isolates in Table 4.On the basis of the previous results, four tests that could

separate N. farcinica from N. asteroides and could beperformed with available commercial media, enzymatictests, or susceptibility disks were chosen. The results ofthese tests of the 40 clinical and 5 reference N. farcinicastrains and the 40 N. asteroides strains are shown in Table 4.Growth at 45°C at 3 days combined with positive results fortwo of the three remaining tests (rhamnose, acetamide, orresistance to tobramycin and cefamandole) correctly identi-fied 38 (95%) of 40 of the clinical strains and all 5 referencestrains of N. farcinica. None of the 40 N. asteroides isolateswere positive for more than two of the characteristics.

DISCUSSIONN. farcinica as a species and as a name has a confusing

and storied past. The Nocardia strain isolated by veterinar-ian Edmond Nocard in 1888 was characterized 1 year laterby Trevisan and named N. farcinica (13). In 1954, it wasmade the type species of the genus Nocardia. Unfortu-nately, examples of Nocard's original strain (known as N.farcinica Trevisan) kept in two different national culturecollections proved to be markedly different, with one strain(ATCC 3318) containing mycolic acids characteristic ofNocardia spp. and the other (NCTC 4524) containing my-colic acids and mycosides characteristic of mycobacteria(12). Taxonomic studies by Gordon and Mihm in 1962 (8)could not distinguish ATCC 3318 from strains of N. asteroi-des, and thus, they grouped the two together. At theirrecommendation, the name N. asteroides was finally chosenin 1980 to replace N. farcinica as the type species of thegenus Nocardia (actually against taxonomic rules) on thebasis of the facts that the status of N. farcinica and its typestrain was uncertain (12) and that the name N. asteroideshad come into common usage while N. farcinica had essen-tially disappeared from the literature (7, 8). A new typestrain of N. asteroides (ATCC 19247) was also chosen.

Unfortunately (for the taxonomists), this was not the endof the story. Although it is unknown which of the two culturecollection strains (i.e., a nocardia or a mycobacterium) isrepresentative of the original organism reported by Nocard,

ATCC 3318 is definitely a Nocardia species and studies by anumber of investigators, beginning with Tsukamura in 1969,have shown that collections of N. asteroides contain asubgroup of isolates that biochemically and immunologicallymatch this strain and are distinct from other isolates identi-fied as N. asteroides (4, 11, 14, 18-21, 24, 25). Because ofthis, the International Judicial Committee has agreed tocontinued use of the name N. farcinica (12), although itsstatus as a species or a biovariant of N. asteroides and theclinical need for this separation remain controversial. Inanticipation that this name might ultimately be rejected bythe International Judicial Committee, Tsukamura has recom-mended the name N. paratuberculosis and submitted a newtype strain (ATCC 23826) (22). However, this change innomenclature has not been accepted and N. farcinica re-mains the legitimate name for this controversial Nocardiagroup.

Differentiation of the three major human Nocardia species(N. asteroides, N. brasiliensis, and N. otitidis-caviarum) inclinical laboratories is done on the basis of the pattern ofdecomposition of casein, hypoxanthine, tyrosine, and xan-thine. Unfortunately, these tests, as well as acid productionfrom all carbohydrates except rhamnose, produce identicalresults with N. farcinica and N. asteroides. These tests werethe basic taxonomic tools used by Gordon and co-workers intheir historic studies (7, 8, 15) of the genus Nocardia (theyare still often referred to as the Gordon tests), and they helpexplain why those researchers did not believe that N.farcinica was a valid taxonomic group. Contributing to thisconfusion was the finding that the type strain of N. farcinica,ATCC 3318, was negative for acid production from rham-nose on initial testing (8), although subsequent studies (1,21), including ours, found it to be positive.An example of the inability to separate N. farcinica from

N. asteroides by standard diagnostic testing is ATCC 3308.This strain is the Preceptrol culture made available by theATCC as an example of N. asteroids but was identified inthis study as N. farcinica.

Before addressing the issue of the ability to distinguish N.farcinica from N. asteroides, some reasons for providingthis separation should be discussed. Does N. farcinica causeclinical disease? On the basis of the taxonomic tests ofTsukamura, Holm reported that 7 of 12 isolates causinghuman nocardiosis in Denmark were N. farcinica (9). Tsuka-mura reported that three of seven isolates causing pulmo-nary disease in Japan were N. farcinica (21). In the oneprevious study from the United States that attempted to

J. CLIN. MICROBIOL.

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

CEFOTAXIME-RESISTANT N. ASTEROIDES 2731

correlate taxonomic groups of N. asteroides with clinicaldisease, Berd reported that 26 (40%) of a series of 65 clinicalisolates collected by the Centers for Disease Control (Atlan-ta, Ga.) were acetamidase positive. Eighteen (69%) of these26 isolates produced acid from rhamnose. Some of theseisolates were responsible for clinical disease and were likelyisolates of N. farcinica (1). In this study of 200 isolates, 19%were N. farcinica. This is the largest number of clinicalisolates assessed for the presence of N. farcinica and is thefirst study to show the prevalence of this species in theUnited States. Most of these isolates are significant, andmany are associated with disseminated disease.Are there additional clinical reasons to distinguish isolates

of N. farcinicba? Our initial study of 12 isolates (28) and thisstudy involving 40 isolates demonstrate that isolates of N.farcinica are highly drug resistant, with 18% of the strainsresistant to imipenem and all strains resistant to the newercephalosporins cefotaxime and cefamandole. They are theonly group of isolates (formerly) included within N. asteroi-des that are resistant to the latter two drugs (28). We believethat these two factors, the frequent presence of the speciesas a cause of serious human disease and its high degree ofdrug resistance, warrant attempts to separate N. farcinicafrom N. asteroides in clinical laboratories.

For clinical separation of N. farcinica from N. asteroides,we chose four tests that use readily available commercialmedia, enzyme assays, or susceptibility tests. Although notest was 100% sensitive and 100% specific, the combination ofthese tests readily separated the two species. Growth at 45°Cin 3 days and expected results with any two of the remainingthree tests should establish an isolate as N. farcinica.The one different taxonomic test included was growth

inhibition by tobramycin and cefamandole. A previous studyin our laboratory demonstrated that cefamandole was usefulin separating N. brasiliensis and N. otitidis-caviarum fromN. asteroides (29). Orchard and Goodfellow, using labora-tory-prepared disks containing approximately 2 ,ug of tobra-mycin, had previously shown isolates of N. farcinica to beresistant while most subgroups of N. asteroides were sus-ceptible (17). The current study used commercial 10-,igtobramycin disks (BBL) with comparable results. The use ofantimicrobial agents for taxonomic separation of species orbiovariants has been described for both nocardia and myco-bacteria (2, 6, 17, 23, 29).We confirmed the taxonomic status of three isolates

identified as N. farcinica (or N. paratuberculosis) in theATCC and identified two additional strains (ATCC 3308 andATCC 6846) currently identified only as N. asteroides.ATCC 3318, ATCC 3308, ATCC 6846, ATCC 23825, andATCC 23826 were typical examples of the species, except forincreased susceptibility to some ,-lactam antibiotics, includ-ing cefamandole, a finding that we suspect relates to changesassociated with long-term laboratory passage. One additionalstrain listed as N. farcinica, ATCC 13781, failed to grow at45°C, was unable to produce acid from rhamnose, utilizedmannitol as a sole carbon source, and had the features of arapidly growing mycobacterium. This strain was submittedby the Pasteur Institute, Paris, France, as strain IP 378. Ithad originally been isolated from a case of bovine farcy inDakar, Senegal (18). Previous work with this strain by Cham-oiseau and Asselineau had shown that it contained mycolicacids, typical of mycobacteria, and this strain, the confusingstrain NCTC 4524, and additional African strains from casesof bovine farcy have now been recognized as rapidly grow-ing mycobacteria and not Nocardia spp. and have beengiven the name Mycobacterium senegalense (3, 17).

Our results support a previous postulate that the fivemajor drug resistance patterns identified within N. asteroi-des would correlate with specific taxonomic groups (28). Theisolates with the type 5 drug resistance pattern (i.e., N.farcinica) are the first group to be reported in detail. Wehave completed preliminary work with a second group (type3 drug resistance pattern), and these isolates also appear tobe biochemically homogeneous, easily separated from N.farcuinica isolates, and identical to the type strain of N. nova(R.J.W.. unpublished data). These taxonomic studies areongoing.

ACKNOWLEDGMENTS

We acknowledge the kind help of Glenn Roberts of the Microbi-ology Laboratory, Mayo Clinic, Rochester, Minn., in providingsome of the isolates used and clinical information and EdwardSeptimus, whose early interest in susceptibility testing helpedprompt these studies.

This study was supported in part by Roche Laboratories, Nutley,N.J.

LITERATURE CITED1. Berd, D. 1973. Nocardia asteroides. A taxonomic study with

clinical correlations. Am. Rev. Respir. Dis. 108:909-917.2. Boiron, P., and F. Provost. 1988. In-vitro susceptibility testing of

Nocardia spp. and its taxonomic implication. J. Antimicrob.Chemother. 22:623-629.

3. Chamoiseau, G. 1979. Etiology of farcy in African bovines:nomenclature of the causal organisms Mvcoba(cteriurn farcino-genes Chamoiseau and Mvcobacteriun senegalense (Chamoi-seau) comb. nov. Int. J. Syst. Bacteriol. 29:407-410.

4. Franklin, A. A., Jr., and N. M. McClung. 1976. Heterogeneityamong Nocardia asteroides strains. J. Gen. Apple. Microbiol.22:151-159.

5. Gombert, M. E., and T. M. Aulicino. 1983. Synergism ofimipenem and amikacin in combination with other antibioticsagainst Nocardia asteroides. Antimicrob. Agents Chemother.24:810-811.

6. Goodfellow, M., and V. A. Orchard. 1974. Antibiotic sensitivityof some nocardioform bacteria and its value as a criterion fortaxonomy. J. Gen. Microbiol. 83:375-387.

7. Gordon, R. E., and J. M. Mihm. 1957. A comparative study ofsome strains received as nocardiae. J. Bacteriol. 73:15-27.

8. Gordon, R. E., and J. M. Mihm. 1962. The type species of thegenus Nocardia. J. Gen. Microbiol. 27:1-10.

9. Holm, P. 1975. Seven cases of human nocardiosis caused byNocardia farcinica. Sabouraudia 13:161-169.

10. Kitzis, M. D., L. Gutmann, and J. F. Acar. 1985. In vitrosusceptibility of Nocardia asteroides to 21 beta-lactam antibi-otics, in combination with three beta-lactamase inhibitors, andits relationship to the ,-lactamase content. J. Antimicrob.Chemother. 15:23-30.

11. Kurup, P. V., and J. A. Schmitt. 1973. Numerical taxonomy ofNocardia. Can. J. Microbiol. 19:1035-1048.

12. Lechevalier, H. A. 1986. Nocardioforms, p. 1458-1471. In J. G.Holt (ed.), Bergey's manual of systematic bacteriology, vol. 2.The Williams & Wilkins, Co., Baltimore.

13. Magnusson, M. 1976. Sensitin tests as an aid in the taxonomy ofNocardia and its pathogenicity, p. 236-265. In M. Goodfellow,G. H. Brownell, and J. A. Jerrano (ed.), The biology of thenocardiae. Academic Press, Inc. (London), Ltd., London.

14. Magnusson, M., and F. Mariat. 1968. Delineation of Nocardiafarcinica by delayed type skin reactions on guinea pigs. J. Gen.Microbiol. 51:151-158.

15. Mishra, S. K., R. E. Gordon, and D. A. Barnett. 1980. Identifi-cation of nocardiae and streptomycetes of medical importance.J. Clin. Microbiol. 11:728-736.

16. National Committee for Clinical Laboratory Standards. 1985.Methods for dilution antimicrobial susceptibility testing forbacteria which grow aerobically. Approved standard M7-A.National Committee for Clinical Laboratory Standards, Vill-

VOL. 28. 1990

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from

J. CLIN. MICROBIOL.

anova, Pa.17. Orchard, V. A., and M. Goodfeliow. 1980. Numerical classifica-

tion of some named strains of Nocardia asteroides and relatedisolates from soil. J. Gen. Microbiol. 118:295-312.

18. Ridell, M. 1975. Taxonomic study of Nocardia farcinica usingserological and physiological characters. Int. J. Syst. Bacteriol.25:124-132.

19. Tsukamura, M. 1969. Numerical taxonomy of the genus Nocar-dia. J. Gen. Microbiol. 56:265-287.

20. Tsukamura, M. 1977. Extended numerical taxonomy study ofNocardia. Int. J. Syst. Bacteriol. 27:311-323.

21. Tsukamura, M. 1982. Nocardiae that recently caused lunginfection in Japan-Nocardia asteroides and Nocardia farcini-ca. Microbiol. Immunol. 26:341-345.

22. Tsukamura, M. 1982. Numerical analysis of the taxonomy ofnocardiae and rhodococci. Microbiol. Immunol. 26:1101-1119.

23. Tsukamura, M. 1988. Differentiation between genera Rhodo-coccus and Nocardia by susceptibility testing to kanamycin andsome other antituberculosis agents. Microbiol. Immunol. 32:441-445.

24. Tsukamura, M., and M. Ohta. 1980. Nocardia farcinica as apathogen of lung infection. Microbiol. Immunol. 24:327-341.

25. Tsukamura, M., H. Shimoide, K. Kaneda, R. Sakai, and A.Seino. 1988. A case of lung infection caused by an unusual strainof Nocardia farcinica. Microbiol. Immunol. 32:541-546.

26. Wallace, R. J., Jr., D. R. Nash, W. K. Johnson, L. C. Steele, andV. A. Steingrube. 1987. P-Lactam resistance in Nocardia bra-siliensis is mediated by ,-lactamase and reversed in the pres-ence of clavulanic acid. J. Infect. Dis. 156:959-966.

27. Wallace, R. J., Jr., and L. C. Steele. 1988. Susceptibility testingof Nocardia species for the clinical laboratory. Diagn. Micro-biol. Infect. Dis. 9:155-166.

28. Wallace, R. J., Jr., L. C. Steele, G. Sumter, and J. M. Smith.1988. Antimicrobial susceptibility patterns of Nocardia asteroi-des. Antimicrob. Agents Chemother. 32:1776-1779.

29. Wallace, R. J., Jr., K. Wiss, R. Curvey, P. H. Vance, and J.Steadham. 1983. Differences among Nocardia spp. in suscepti-bility to aminoglycosides and ,B-lactam antibiotics and theirpotential use in taxonomy. Antimicrob. Agents Chemother.23:19-21.

2732 WALLACE ET AL.

on June 15, 2020 by guesthttp://jcm

.asm.org/

Dow

nloaded from