Tobramycin-Resistant in the Sputum of Patients w/ CF Part 1

[Guest guest]

Journal of Clinical Microbiology, January 2002, p. 26-30, Vol. 40, No. 1

Copyright ? 2002, American Society for Microbiology. All Rights Reserved.

Comparison of Two Culture Methods for Detection of Tobramycin-Resistant

Gram-Negative Organisms in the Sputum of Patients with Cystic Fibrosis

Jill M. Van Dalfsen,1 R. Stapp,2 Phelps,1,

,1, and Jane L. Burns2*

Chiron Corporation, Department of Pediatrics, University of

Washington,Children?s Hospital and Regional Medical Center, Seattle,

Washington,1Division of Infectious Disease, Department of Pediatrics,

University ofWashington, Children?s Hospital and Regional Medical Center,

Seattle,Washington2 A culture method utilizing quantitative plating on

antibiotic-containing

media has been proposed as a technique for the detection of

tobramycin-resistant organisms that is more sensitive than standard

methods. Typical sputum culture methods quantitate the relative amounts

of each distinct morphotype, followed by antibiotic susceptibility

testing of a single colony of each morphotype. Sputum specimens from 240

cystic fibrosis patients were homogenized, serially diluted, and

processed in parallel by the standard method (MacConkey agar and OF basal

medium with agar, polymyxin, bacitracin, and lactose) and by plating on

antibiotic-containing media (MacConkey agar with tobramycin added at 25

?g/ml [MAC-25] and 100 ?g/ml [MAC-100]). MICs of tobramycin were

determined for all Pseudomonas aeruginosa isolates by broth

microdilution. Growth of P. aeruginosa on MAC-25 was considered to be

equivalent to a tobramycin MIC of 16 ?g/ml, and growth on MAC-100 was

considered to be equivalent to a tobramycin MIC of 128 ?g/ml. Analysis of

method-specific detection rates showed that tobramycin-containing medium

was more sensitive than the standard method for the detection of

tobramycin-resistant P. aeruginosa, Stenotrophomonas maltophilia, and

Achromobacter xylosoxidans but was less sensitive for the detection of

Burkholderia cepacia than the standard method. When MICs for P.

aeruginosa that grew on tobramycin-containing medium were tested by broth

microdilution, the MICs for 28 of 121 strains (23%) growing on M AC-25

and 22 of 56 strains (39%) growing on MAC-100 were MICs <16 and <128

?g/ml, respectively. Addition of a tobramycin-containing MacConkey plate

to the routine media for sputum culture may provide additional,

clinically relevant microbiologic

Patients with cystic fibrosis (CF) typically harbor multiple

morphologically distinct strains of Pseudomonas aeruginosa in their

sputum. These morphotypes contribute in different degrees to the total

sputum density of P. aeruginosa, and each may have a different level of

antibiotic susceptibility. Typical CF sputum culture methods quantitate

the relative amounts of each distinct morphotype, followed by antibiotic

susceptibility testing of a pure culture of each morphotype. This

methodology assumes that the MIC result for a single clone accurately

represents the susceptibility of the entire population of that morphotype

and that all morphotypes will be represented even if present in small

numbers relative to the predominant morphotype. It is possible that this

methodology underrepresents the number of antibiotic-resistant P.

aeruginosa clones within the sputum sample.

Following the reported efficacy of inhaled tobramycin in CF patients (3,

14) the question of increasing tobramycin resistance among sputum

isolates of P. aeruginosa and other gram-negative organisms gained

importance. If antibiotic selection occurs related to the chronic use of

inhaled drug, the ability to detect even small numbers of

tobramycin-resistant isolates in sputum may be significant. As noted

above, the methods currently in use may underrepresent tobramycin

resistance. Thus, development of a technique that would sample the

resistance phenotype of all of the organisms present in a given sputum

sample was proposed. A previous study by Maduri-Traczewski et al.

utilized a culture method in which each P. aeruginosa clone was tested

individually for susceptibility, by directly plating sputum to

antibiotic-containing medium (12). They concluded that utilization of

antibiotic-containing primary media accurately and promptly

detectedantibiotic-resistant organisms compared to standard quantitative

culturesfrom which colonies were selected for subsequent MIC testing. The

currentstudy specifically examines the utility of tobramycin-containing

primarymedia for the detection of tobramycin-resistant organisms from the

sputumof CF patients. (This work was presented in part at the 14th Annual

North American CysticFibrosis Conference, 9 to 12 November 2000,

Baltimore, Md.)

Specimen collection and processing. Sputum specimens were collected

from240 CF patients from seven CF clinics across the United States

during1999. All participants and their families gave informed consent

inaccordance with experimental guidelines of the US Department of

Healthand Human Services and the institutions at which the study was

performed.To be eligible for the study, patients were required to be able

toproduce sputum, to have a history of P. aeruginosa in the

lowerrespiratory tract, and, if they were currently receiving

aerosolizedantibiotics, the specimen had to be collected at least 12 h

after theirlast dose. Sputum was processed as described in Fig. 1.

Briefly, eachsputum sample was homogenized using dithiothreitol

(Sputolysin;Calbiochem-Behring, La Jolla, Calif.) and serially diluted in

sterilephysiologic saline, and 0.1-ml aliquots of each dilution were

plated to

MacConkey agar (MAC), MacConkey agar with tobramycin at 25 ?g/ml

(MAC-25)and 100 ?g/ml (MAC-100), and a selective agar for Burkholderia

cepacia(OFPBL [19]) (media were purchased from Remel, Lenexa, Kans.). All

plateswere incubated at 37°C for at least 72 h. Quantitation of

allgram-negative bacilli was performed on all medium types.

Eachmorphologically distinct isolate (texture [mucoid, rough versus

smooth],colony size, color) of P. aeruginosa was enumerated separately

and

subcultured for MIC determination. Broth microdilution tobramycin (0.12to

512 ?g/ml) susceptibility testing was performed on all

gram-negativebacilli using a semiautomated system (Sensititre; Trek

Diagnostics,Westlake, Ohio) and manual reading of end points following 18

to 24 h ofincubation (3). Organisms were identified by standard

techniques,including the use of a biochemical panel for the

identification of non-P.aeruginosa, gram-negative, non-lactose fermenters

(16).

FIG. 1. Diagram of the processing of sputum from 240 patients at sevenCF

centers in the United States. All samples were sent to a singlelaboratory

(Children?s Hospital and Regional Medical Center, Seattle,Wash.), where

they were processed by both the standard method and

theantibiotic-containing-medium method (MAC-25 and MAC-100). Tobramycin

MICswere determined for all morphologically distinct P. aeruginosa

isolatesand for all other non-lactose-fermenting gram-negative bacilli.

Statistical analysis. Preliminary range finding experiments using

strainsof P. aeruginosa with known tobramycin MICs showed that MacConkey

agarcontaining tobramycin at 25 ?g/ml would inhibit the growth of 93% of

P.aeruginosa isolates for which the tobramycin MICs were <16 ?g/ml

andsupport the growth of more than 75% of P. aeruginosa isolates for

whichthe tobramycin MICs were 16 ?g/ml. Similarly, MAC-100 would suppress

thegrowth of most P. aeruginosa isolates for which the tobramycin MICs

are<128 ?g/ml while supporting the growth of most P. aeruginosa isolates

forwhich the tobramycin MICs are 128 ?g/ml. Thus, growth of P. aeruginosa

onMAC-25 was considered to be equivalent to a tobramycin MIC of 16

?g/ml,and growth of P. aeruginosa on MAC-100 was considered to be

equivalent toa tobramycin MIC of 128 ?g/ml. The method-specific detection

rate for

each method (standard method [MAC and OFPBL], MAC-25, and MAC-100)

wascalculated by dividing the number of patients with a given

organismdetected by that method by the number of patients with that

organismdetected by all methods.

The percentage of patients whose sputum grew tobramycin-resistant

P.aeruginosa detected by the standard method (broth microdilution MIC,

16?g/ml) was compared with the percentage of patients whose sputum grew

P.aeruginosa on MAC-25 by using a two-sided, one-sample McNemar test.

Patient population. The study included an equal number of male and

femalepatients; the mean age was 20.4 years. Overall, 59.6% of

patientsreceived aminoglycosides by either the intravenous or aerosol

routewithin the 8 weeks prior to providing sputum for this study (Table

1).Two hundred eighteen of 240 patients had P. aeruginosa detected

fromtheir sputum on MacConkey agar without added tobramycin. Mean

P.aeruginosa density in sputum for these patients was 7.1 log10

CFU/g(standard deviation, 1.5 log10 CFU/g).

Detection of tobramycin-resistant organisms. By all methods, 124 of

240(52%) patients had a tobramycin-resistant P. aeruginosa

detected.However, using standard methodology alone, only 58 of 240 (24%)

had P.aeruginosa isolates for which the MIC was 16 ?g/ml. For the 58

patientswith resistant P. aeruginosa detected on MAC, the resistant

coloniesrepresented, on average, 70% of the total CFU count (median, 97%;

range,0.2 to 100%). Tobramycin-containing medium was much more sensitive

forthe detection of resistant P. aeruginosa at both the 16-?g/ml

level(MAC-25 versus MAC, 98 versus 47%) and the 128-?g/ml levels (MAC-100

versus MAC, 100 versus 21%) (Table 2). Stenotrophomonas maltophilia

andAchromobacter xylosoxidans were also detected more frequently

ontobramycin-containing media. Conversely, B. cepacia was detected

morefrequently by the standard method (MAC or OFPBL) than on either

MAC-25 or

MAC-100.

Becki

YOUR FAVORITE LilGooberGirl

YOUNGLUNG EMAIL SUPPORT LIST

www.topica.com/lists/younglung

Pediatric Interstitial Lung Disease Society

http://groups.yahoo.com/group/InterstitialLung_Kids/