Bordetella pertussis Isolates with a Heterogeneous Phenotype for Erythromycin Resistance

Erythromycin is currently being used for both prophylaxis andtreatment of pertussis infections. Erythromycin resistance wasfirst recognized in Bordetella pertussis in Arizona in 1994,and since then, three additional resistant isolates have beenidentified in the United States. To better assess the potentialpublic health impact of erythromycin-resistant B. pertussis,we used the disk diffusion assay to evaluate the frequency oferythromycin resistance among 1,030 recently circulating U.S.isolates and found the rate of occurrence to be <1%. We alsodescribe a novel heterogeneous phenotype, with erythromycin-resistantcolonies appearing only after a 7-day incubation period. Tooptimize patient management, we recommend that clinicians bealert to potential treatment failures and that laboratoriansuse a 7-day incubation period when screening for resistance.Our ongoing national surveillance will continue to monitor forresistant B. pertussis isolates and their potential associationwith changing pertussis epidemiology.

INTRODUCTION

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Introduction

Erythromycin has been used in the United States for over 45years for treatment of patients with pertussis and is currentlyrecommended for prophylaxis as well (1). To date, erythromycinresistance in Bordetella pertussis has been reported in onlythe United States, with the first resistant isolate detectedin Arizona in 1994 (7). Three additional resistant isolateshave since been reported from patients in Minnesota, Utah, andCalifornia (5, 6; J. Bartkus, B. A. Juni, K. Ehresman, C. Miller,G. N. Sanden, P. K. Cassiday, M. Saubolle, B. Lee, J. Long,and J. M. Besser, Abstr. 41st Intersci. Conf. Antimicrob. AgentsChemother., abstr. C1-1817, p. 102, 2001). Due to the low frequencyof resistance and the lack of a standardized screening method,very few clinical laboratories currently perform susceptibilitytesting of B. pertussis (3, 8). Reference laboratories thatdo perform susceptibility testing primarily use the disk diffusionassay.

To assess the frequency of erythromycin resistance in the UnitedStates, we tested B. pertussis isolates collected through anenhanced pertussis surveillance program, as well as a subsetof those already in our strain collection. The enhanced surveillanceprogram involves several states (California, New York, Minnesota,Massachusetts, Illinois, Arizona, and Georgia) and aims to associatevariations in the circulating B. pertussis population with relevantchanges in pertussis epidemiology, such as overall burden ofdisease, disease severity, and treatment or vaccine failures(2). This evaluation of over 1,000 B. pertussis isolates resultedin the detection of a novel erythromycin-resistant phenotypein B. pertussis that is unlikely to be recognized by the currentdisk diffusion protocols used to screen for erythromycin resistance.

MATERIALS AND METHODS

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Materials and Methods

B. pertussis strains. We used two previously reported erythromycin-resistant isolatesfrom Arizona (A228) and California (C310) as resistant controlsand B. pertussis ATCC 9797 as the erythromycin-susceptible control(6, 7). We tested 1,030 B. pertussis isolates for erythromycinresistance: 776 were obtained from 1994 to 2000 through ourenhanced pertussis surveillance program in California, New York,Minnesota, Massachusetts, Illinois, Arizona, and Georgia; theremaining 254 isolates were a convenience sample from the B.pertussis collection at the Centers for Disease Control andPrevention and were isolated from 1958 to 2001.

Erythromycin disk diffusion assay. Since no universally standardized and validated macrolide susceptibilityassay exists for B. pertussis, we used a disk diffusion assayadapted from previously published guidelines specific for B.pertussis as follows (3, 4, 8). Isolates were grown at 37°Cunder high humidity for 3 to 5 days on plates of charcoal agar(Oxoid, Unipath, Ltd., Basingstoke, Hampshire, England) supplementedwith 10% defibrinated horse blood (Lampire Biological Laboratories,Pipersville, Pa.). The cells were harvested and suspended in2 ml of phosphate-buffered saline (pH 7.4), and the turbiditywas adjusted to the 0.5 McFarland standard. The cell suspensionwas applied with a cotton swab over an entire charcoal-horseblood agar plate surface (100 by 15 mm), and a 15-µg erythromycindisk (Becton Dickinson, Baltimore, Md.) was placed in the centerof the plate. The plates were incubated at 37°C under highhumidity, and zones of growth inhibition were measured on days3, 5, and 7.

Definitions. A susceptible isolate exhibited a zone of growth inhibitionof >40 mm in diameter. A resistant isolate showed no zoneof growth inhibition, i.e., growth was observed all the wayup to the disk. An isolate with the novel heterogeneous phenotypeexhibited an initial zone of growth inhibition of >40 mmin diameter after 3 days of incubation; resistant colonies appearedwithin the initial zone of inhibition after the extended 5-to 7-day incubation.

Testing heterogeneous phenotype isolates. The confluent growth outside the zone of inhibition was harvestedwith an inoculating loop and streaked on a charcoal-horse bloodagar plate for isolation. Following a 3-day incubation at 37°Cunder humid conditions, four individual colonies were subculturedand retested by the disk diffusion assay described herein. Thesame 0.5 McFarland suspension used in the disk diffusion assaywas also used to perform the Etest according to the manufacturer'sinstructions. Etest MICs were recorded on days 3 and 7.

Resistant colonies appearing within the zone of growth inhibitionwere counted after 7 days. Four resistant colonies from eachculture were selected and subcultured onto a charcoal-horseblood agar plate; these colonies were retested by the disk diffusionassay described above. The same 0.5 McFarland suspension usedin the disk diffusion assay was also used to perform the Etest.Etest MICs were recorded on days 3 and 7.

Testing for degradation of erythromycin in the disk diffusion assay. Erythromycin disks were placed in the center of 45 fresh charcoal-horseblood agar plates. These plates were incubated at 37°C underhigh humidity for up to 14 days before testing. On days 0, 3,5, 7, 10, and 14, 0.5 McFarland suspensions of the susceptible(ATCC 9797), resistant (C310), and heterogeneous phenotype (B715)isolates were prepared from 3-day cultures and 100 µlof each suspension was inoculated in triplicate around the erythromycindisks on the previously incubated charcoal-horse blood agarplates. These plates were incubated for an additional 10 days,and zones of inhibition were measured after 3, 5, 7, and 10days. As a control, a disk diffusion assay using a fresh charcoal-horseblood agar plate and erythromycin disk was performed with suspensionsof strains representing each erythromycin susceptibility phenotypeon days 0, 3, and 7.

RESULTS

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Results

All 1,030 test isolates showed zones of inhibition of >40mm by the disk diffusion assay after a 3-day incubation; accordingly,they were all initially defined as susceptible. However, after5 to 7 days of incubation, 5 of the 1,030 isolates (0.5%) demonstratedthe heterogeneous phenotype, as isolated colonies appeared insidethe initial zone of growth inhibition (Fig. 1). All five isolateswere collected through the enhanced surveillance program; fourwere from Arizona (1994 to 1995) and one was from Georgia (1999).

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FIG. 1. Disk diffusion assay and Etest for screening of B. pertussis for erythromycin resistance. (A) B. pertussis ATCC 9797 susceptible control displaying a zone of inhibition of >40 mm. (B) B. pertussis C310 resistant control (Calif., 1999) showing growth confluent to the disk. (C) B. pertussis B715 (Ariz., 1994) displaying a heterogeneous phenotype after 7 days of incubation. (D) B. pertussis B715. Four colonies from both inside and outside the zone of growth inhibition were subcultured and retested by the disk diffusion assay and Etest. Colonies outside the zone of inhibition were reproducibly of the heterogeneous phenotype, while colonies inside the zone were resistant (MIC > 256 µg/ml).

The number of resistant colonies appearing within the zone differedfor each of these five strains, ranging from approximately 10for each of the four Arizona strains to 100 for the Georgiastrain. When these resistant colonies were tested by the diskdiffusion assay, all displayed only the resistant phenotypewithin the standard 3- to 5-day incubation and MICs of >256µg/ml were obtained by the Etest. When the colonies outsideof the initial zone of inhibition for the same five strainswere subcultured and retested by the disk diffusion assay, theheterogeneous phenotype was consistently reproduced. Comparableresults were seen by using the Etest, e.g., MICs for the coloniesoutside the initial zone in the disk diffusion assay were <0.06µg/ml, while the resistant colonies growing within theinitial zone of inhibition in the disk diffusion test also grewinside the Etest inhibition zone (Fig. 1). No decreases in thesize of the zone of inhibition were observed between the platesthat contained fresh and preincubated erythromycin disks.

For the five heterogeneous phenotype strains, all tested colonies,both within and outside of the initial zone of inhibition, wereconfirmed to be B. pertussis by direct fluorescent-antibodytesting (8).

DISCUSSION

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Discussion

We report here for the first time on five Bordetella pertussisisolates that exhibit a heterogeneous phenotype for erythromycinresistance. These isolates have a zone of inhibition characteristicof erythromycin susceptibility (>40 mm) after 3 days of incubation,but after extended incubation, resistant colonies appeared insidethis zone with Etest MICs of >256 µg/ml. An incubationperiod greater than the commonly used 3 to 5 days is necessaryfor the detection of this heterogeneous phenotype; this extendedincubation period might be a reason that no such B. pertussisisolates have been reported to date.

The frequencies of resistant and heterogeneous phenotypes detectedamong the 1,030 B. pertussis isolates in our study were low.However, our results also suggest a significant potential forthese phenotypes to negatively affect patient management throughtheir apparent distribution and persistence in diverse B. pertussispopulations. Erythromycin treatment failures have been associatedwith resistant phenotypes isolated from 1994 to 2000 in Arizona,California, and Minnesota (6, 7; Bartkus et al., 41st ICAAC).The Georgia heterogeneous phenotype isolate was cultured froma pediatric AIDS patient after erythromycin treatment. Unfortunately,we were unable to retrospectively evaluate the treatment outcomesof the four Arizona patients whose cultures yielded the remainingheterogeneous phenotype isolates. But because these four cultureswere isolated over a period of 2 years from different infantsin a single county, they were probably maintained in the localB. pertussis population and could have been transmitted duringthis period of time. Isolation of both the resistant and heterogeneousphenotypes from distant geographic areas and over a 5-year periodmay represent geographic and temporal dissemination. Alternatively,these last observations may reflect the independent emergenceof resistant phenotypes in response to local selective pressures.

The appearance of resistant colonies only after extended incubationraised the possibility that they might have been selected forby the disk diffusion assay as the erythromycin concentrationsdecreased with degradation over time. However, we have shownthat erythromycin did not degrade in the disk diffusion assay;its activity was maintained for over 14 days at 37°C. Preliminarydata also suggest the molecular basis for erythromycin resistancein B. pertussis isolates with the heterogeneous phenotype, andmore extensive studies are under way to fully elucidate themechanism (Bartkus et al., 41st ICAAC; K. Wilson, P. K. Cassiday,J. M. Bartkus, F. Doucet-Populaire, and G. N. Sanden, Abstr.41st Intersci. Conf. Antimicrob. Agents Chemother., abstr. C1-1818,p. 102, 2001).

Our study identified B. pertussis isolates with a heterogeneousphenotype comprised of both erythromycin-susceptible and -resistantcolonies. Laboratorians are advised to incubate the disk diffusionassay for at least 7 days in order to identify all strains witheither the erythromycin-resistant or heterogeneous phenotypes.At the same time, clinicians should be cognizant of the potentialfor erythromycin treatment failures due to an infection withthe heterogeneous phenotype and the need to obtain posttreatmentcultures in order to identify alternative antimicrobial therapies.Rapid communication between clinicians and laboratorians andsubsequent therapy changes could substantially reduce transmissionof these isolates.

On the national level, the ongoing enhanced surveillance programis needed to identify resistant and heterogeneous B. pertussisphenotypes in order to monitor their appearance, dissemination,and potential association with changes in pertussis epidemiology.Correlation of resistant phenotypes with genetic subtypes, includingpulsed-field gel electrophoresis patterns and pertactin andpertussis toxin gene sequence types, will allow assessment oftheir clonality and genetic relatedness to B. pertussis isolatescurrently circulating in the United States.

FOOTNOTES

* Corresponding author. Mailing address: Epidemiologic Investigations Laboratory, Meningitis and Special Pathogens Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Rd., MSD11, Atlanta, GA 30333. Phone: (404) 639-1373. Fax: (404) 639-4421. E-mail: gnsl{at}cdc.gov.

REFERENCES

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