Comparison of two culture methods for detection of tobramycin-resistant gram-negative organisms in the sputum of patients with cystic fibrosis

Abstract

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 microg/ml [MAC-25] and 100 microg/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 > or = 16 microg/ml, and growth on MAC-100 was considered to be equivalent to a tobramycin MIC of > or = 128 microg/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 MAC-25 and 22 of 56 strains (39%) growing on MAC-100 were MICs < 16 and < 128 microg/ml, respectively. Addition of a tobramycin-containing MacConkey plate to the routine media for sputum culture may provide additional, clinically relevant microbiologic data.

FIG. 1.

Diagram of the processing of sputum from 240 patients at seven CF centers in the United States. All samples were sent to a single laboratory (Children’s Hospital and Regional Medical Center, Seattle, Wash.), where they were processed by both the standard method and the antibiotic-containing-medium method (MAC-25 and MAC-100). Tobramycin MICs were determined for all morphologically distinct P. aeruginosa isolates and for all other non-lactose-fermenting gram-negative bacilli.

FIG. 2.

(A) Density of resistant P. aeruginosa detected by the standard method and on MAC-25 (defined as tobramycin MIC of ≥16 μg/ml by broth microdilution for standard method and as growth on plate for MAC-25). (B) Density of high-level-resistant P. aeruginosa detected by the standard method and by MAC-100 (defined as tobramycin MIC of ≥128 μg/ml by broth microdilution for standard method and as growth on plate for MAC-100).