Tobramycin-resistant P. aeruginosa Part 2

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Density of tobramycin-resistant P. aeruginosa. Seventy of 121

(58%)patients with resistant P. aeruginosa detected on the MAC-25 plates

hadcolony counts of less than 106 CFU/g of sputum compared with 5 of the

58patients (9%) with resistant P. aeruginosa (tobramycin MIC, 16

¾g/ml)detected by the standard method (Fig. 2A). Colony counts of

resistant P.aeruginosa were higher when it was detected on both MAC and

MAC-25 (38 of55 patients [69%] had counts of >106 CFU/g) than when it was

detected on

MAC-25 only (13 of 69 patients [19%]). Similar findings were noted forthe

MAC-100 plates (Fig. 2B).   FIG. 2. (A) Density of resistant P.

aeruginosa detected by the standardmethod and on MAC-25 (defined as

tobramycin MIC of 16 ¾g/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

standardmethod and by MAC-100 (defined as tobramycin MIC of 128 ¾g/ml by

brothmicrodilution for standard method and as growth on plate for

MAC-100).

False-positive rates on antibiotic-containing media. P. aeruginosa

thatgrew on either MAC-25 or MAC-100 had confirmatory broth

microdilutionsusceptibility testing performed. On MAC-25, 93 of 121

isolates hadconfirmatory tobramycin MICs of 16 ¾g/ml, and on MAC-100, 34

of 56isolates had confirmatory tobramycin MICs of 128 ¾g/ml,

forfalse-positive rates of 23 and 39%, respectively. Laboratory notes.

There were several notable findings regarding thetechniques used for this

study. First, drug-containing media (MAC-25 andMAC-100) required longer

incubation (at least 72 h) for organisms to growadequately for

identification and quantitation. Second, colonies weremuch smaller and

more difficult to differentiate on the drug-containing

media. Third, on plates where undiluted sputum was plated,

instancesoccurred where the mucus in the sputum appeared to protect the

organismsfrom the antibiotic in the plate, resulting in false-positive

growth.

This study demonstrates that the addition of tobramycin to primaryplating

media increases the sensitivity for detection of

tobramycin-resistant P. aeruginosa from CF sputum. Resistance

totobramycin was much higher in this group of patients than would

beexpected. Fifty-two percent of patients had a P. aeruginosa isolate

witha tobramycin MIC of 16 ¾g/ml (as detected by either method) in

thecurrent study, compared with the 13% of patients observed from a

similarCF population at baseline in the inhaled-tobramycin clinical

trials (3).This high number may reflect the fact that 59.6% of patients

had received

either intravenous or inhaled aminoglycosides within 8 weeks of

providinga sputum sample for the study. However, when only the data from

thestandard method was considered, 24% of patients had P.

aeruginosaisolates for which the tobramycin MICs were 16 ¾g/ml. This

number iscomparable to the 23% of patients reported to have

tobramycin-resistantP. aeruginosa following 6 months of inhaled treatment

with tobramycin(3). CF patients typically harbor multiple phenotypically

distinct P.

aeruginosa organisms, and these organisms frequently have

differingantibiotic susceptibility patterns (3, 7, 15, 18). In this

study, therelative density of the resistant P. aeruginosa compared to the

totalcolony count of P. aeruginosa was very small for many of the

patientsfrom whom tobramycin-resistant P. aeruginosa was isolated. The

clinicalsignificance of small numbers of slowly growing

tobramycin-resistant P.aeruginosa is unknown. Several published studies

of antibiotic treatmentfor acute exacerbation in CF patients have shown

that patients withorganisms classified as resistant to the antibiotics

they were givenresponded to treatment as well as those patients with

organisms that wereclassified as susceptible (13, 17; R. Davey, D.

Peckham, C. therington,

and S. Conway. Abstr. 23rd Eur. Cystic Fibrosis Conv., abstr. 409,

2000).This phenomenon is likely the result of multiple factors. First,

bothsusceptible and resistant populations of organisms are present within

thelungs of CF patients (18). Second, most aminoglycoside-resistant

P.aeruginosa strains from CF patients lack

aminoglycoside-modifyingenzymes, instead falling into a resistance type

termed " impermeability " (11). There is some in vitro evidence that mutants

within theimpermeability class often show impaired growth in vitro and

reducedvirulence in animal models (2, 5, 9), suggesting that such strains

may beat a competitive disadvantage in the human lung. Finally,

antibioticshave been shown to inhibit virulence factors at concentrations

below theMIC (4, 6, 8, 10). There were some patients who would have

falsely been reported to havetobramycin-resistant P. aeruginosa had only

the results from thetobramycin-containing media been available. These

false positives mayhave been the result of adaptive or transient

resistance reverting tosusceptibility after a single pass on drug-free

media, since a 24 hsubculture to blood agar was always performed prior to

preparation of theinoculum suspension for the confirmatory broth

microdilution MIC test.The phenomenon of adaptive resistance to

tobramycin in P. aeruginosa fromCF lung infections was reported

previously by Barclay et al. (1).Alternatively, when sputum was plated

directly on theantibiotic-containing medium, the mucus may have protected

the organismsfrom the antibiotic in the plate, allowing them to grow.

This study focused solely on tobramycin resistance. The previous study

byMaduri-Traczewski et al. found that primary culture plates

containingticarcillin and azlocillin increased the sensitivity for

detection ofresistance to these antibiotics as well (12). Direct

susceptibilitytesting on media containing multiple antibiotics has also

been used as arapid screening test to test for synergy in CF patients;

lower counts ondrug-containing media compared with non-drug-containing

media wereconsidered an indication of synergy (J. Caracciolo, S. Riddell,

S.

, A. Kerr, and P. Gilligan, Abstr. 96th Gen. Meet. Am.

Soc.Microbiol. 1996, abstr. C-309, 1996). It is likely that many CF

patientsharbor small subpopulations of P. aeruginosa that are resistant

to manyof the antibiotics that are routinely and repeatedly used for

treatment. Before a decision to add antibiotic-containing plates to the

primaryplating is made, the costs in both material and technical time

should becarefully weighed against the possible benefit of increased

detection ofresistant organisms. Currently tobramycin-containing

MacConkey agar isnot available commercially, so plates must either be

prepared in-house or

be specially ordered. Importantly, because B. cepacia was not detected

aswell on the tobramycin-containing media and given the significance

ofthis pathogen in the CF patient population, the need to plate to a

B.cepacia selective agar would not be obviated by the addition

oftobramycin-containing MacConkey agar. In addition, the longer

incubationtime required for growth on the tobramycin-containing plates

and thesmall, difficult-to-differentiate colonies would increase

laboratorytechnologist time for reading each culture. Finally, because of

thefalse-positive results observed with directly plated sputum, dilution

ofthe sputum in saline may be required prior to plating.

This study was sponsored by Chiron Corporation, Seattle, Wash. We thank

the following principal investigators (PI) and study

coordinators for their support of this study: H. Eigen (PI), M.

Blagburn,and D. Terrill, Indiana University and Medical Center; R. Anbarm

(PI), D.Lindner, and L. Grabowski, SUNY Health Science Center; S. Nasr

(PI) andE. Sakmar, University of Michigan; C. Ren (PI) and L. Maffia,

UniversityHospital, Children?s Medical Center at Stonybrook; R. Gibson

(PI), S.McNamara, and P. Joy, Children?s Hospital and Regional Medical

Center,Seattle, Wash.; P. Hiatt (PI), L. , and D. Treece, Baylor

College

of Medicine/Texas Children?s Hospital; M. McCarty (PI) and D.

Harrington,Deaconess Medical Center, CF Clinic, Spokane, Wash. Most

importantly, wethank the patients who volunteered to participate in the

study.

* Corresponding author. Mailing address: Division of Infectious Disease,

Children?s Hospital and Regional Medical Center, 4800 Sand Point Way

N.E., CH-32, Seattle, WA 98105. Phone: . Fax: (206)

527-3890. E-mail: jburns@....

Present address: ICOS Corporation, Seattle, Wash.

Becki

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