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Journal of Clinical Microbiology, October 2003, p. 4808-4811, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4808-4811.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

National Department of Defense Surveillance Data for Antibiotic Resistance and emm Gene Types of Clinical Group A Streptococcal Isolates from Eight Basic Training Military Sites{dagger}

Christopher P. Barrozo,1 Kevin L. Russell,1* Tyler C. Smith,1 Anthony W. Hawksworth,1 Margaret A. K. Ryan,1 and Gregory C. Gray2

Department of Defense Center for Deployment Health Research, Naval Health Research Center, San Diego, California,1 Department of Epidemiology, College of Public Health, University of Iowa, Iowa City, Iowa2

Received 8 April 2003/ Returned for modification 23 May 2003/ Accepted 28 July 2003


   ABSTRACT
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Antibiotic resistance and emm gene types were examined from 692 Group A streptococci isolates from eight United States military basic training sites between 1998 and 2001. Macrolide resistance was associated with geographic sites and emm type. These data are useful for vaccine development initiatives and antimicrobial treatment considerations.


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Group A streptococci (GAS; Streptococcus pyogenes) are responsible for a variety of illnesses that affect humans (14). In the 1980s both United States civilian and military personnel experienced numerous outbreaks of acute rheumatic fever, the first for the United States military in over 20 years (4, 30, 31). Epidemiological data suggest that these epidemics may have been due to emergent, potentially more virulent GAS strains (3, 12, 27).

In response to these concerns, GAS surveillance was established at eight military basic training sites throughout the United States (1, 5, 26). Between January 1998 and December 2001, GAS pharyngeal culture isolates from symptomatic military recruit trainees were systematically collected along with basic demographic data (Table 1).


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  		TABLE 1. Characteristics of military trainees and multivariable logistic regression modeling of antibiotic resistance among GAS isolates

 
This research has been conducted in compliance with all applicable federal regulations governing the protection of human subjects in research, under protocol no. 31230.

Training sites preserved GAS specimens in tryptic soy broth with 15% glycerol at -70°C. Isolates were reconfirmed as GAS by colony morphology, bacitracin sensitivity, and a positive latex agglutination test (Hardy Diagnostics, Santa Maria, Calif.). MICs of penicillin, erythromycin, clindamycin, tetracycline, levofloxacin, and vancomycin were performed on confirmed isolates with E-test methodology (AB Biodisk, Piscataway, N.J.). Plates were incubated for 18 to 24 h at 36°C with 5% CO2. National Committee for Clinical Laboratory Standards (NCCLS) breakpoints for agar dilution of streptococci were used to interpret MIC results and to define criteria for resistant, intermediate susceptible, and susceptible isolates (21, 22). For quality control, Streptococcus pneumoniae ATCC 49619 was tested with each batch of samples.

GAS isolates were emm typed by using procedures adapted from those of the Centers for Disease Control and Prevention (2, 6). Sequencing products were analyzed with an ABI Prism 3100 sequencer as described by the manufacturer (Applied Biosystems, Foster City, Calif.). DNA sequences were submitted to a BLAST search, requiring 95% or greater homology with a reference emm gene sequence for emm type assignment (2).

Univariate analyses were initially performed to assess possible associations between demographic variables and antibiotic resistance or emm type. Variables associated with the outcome of interest (P <= 0.15) were included in subsequent exact multivariable logistic regression model analyses. Analyses and final models were conducted by using SAS and included only those variables independently associated with the outcome of interest with P values of <=0.05.

From January 1998 to December 2001, 692 GAS samples were received from eight military basic training sites (Table 1). Susceptibility to penicillin, levofloxacin, and vancomycin was seen in 100% of samples. Forty-four isolates (6.4%) were resistant to erythromycin, 34 (4.9%) were resistant to tetracycline, 4 (0.58%) were resistant to clindamycin, and 10 (1.45%) were multidrug resistant. Sustained temporal trends in antibiotic resistance over the duration of the study were not seen (data not shown).

Gender and training site were associated with antibiotic resistance (Table 1). Women were more likely to be resistant to tetracycline in multivariable modeling. In addition, isolates from the Air Force site were much more likely to be resistant to erythromycin, while isolates from Army sites were more likely to be resistant to tetracycline.

Over 30 different emm types were identified, with the most prevalent being emm29 (18.0%), emm3 (15.2%), emm6 (13.5%), emm44/61 (9.1%), emm2 (7.3%), emm75 (6.4%), and emm1 (4.8%) (Table 2). Univariate analyses demonstrated that training site was statistically associated with emm type. The Navy's Illinois site had proportionally more emm6 and emm44/61. Both emm29 and emm3 types were more prevalent at the Marine's site in South Carolina. The Air Force's site had a predominance of emm75 among its GAS isolates. Additionally, the prevalence of emm types varied from year to year (Fig. 1). An association between emm types and antibiotic resistance was also observed. Erythromycin resistance was strongly associated with emm75 isolates (P < 0.001). Tetracycline resistance was associated with emm29 and emm1 isolates.


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  		TABLE 2. Factors associated with emm gene type of GAS isolates

 


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  		FIG. 1. Most common emm types of GAS isolates identified in military basic training surveillance, 1998-2001.

 
Discussion. The growth of antibiotic resistance in GAS is a major concern for both military and civilian populations (1, 5, 19). We noted a prevalence of 6.4% erythromycin resistance and 4.9% tetracycline resistance among our population. Mass penicillin prophylaxis among recruits has proven effective in decreasing the frequency of outbreaks (11, 28). This practice is seasonal and/or dynamic and is modified depending upon local surveillance indicators. Prior research has demonstrated that prophylaxis of the penicillin-allergic individuals with erythromycin is useful in further reducing outbreak occurrences (9). However, this practice is site specific and variable. Due to the observed erythromycin resistance rate, antibiotic prophylaxis among the recruits at the training sites was examined. No apparent correlation with erythromycin resistance and a history of erythromycin use at the sites was noted; however, other researchers have shown the reality of this concern (10, 25).

From the molecular perspective, several GAS virulence factors are suggested to be associated with disease prevalence (13, 20, 24). The M protein is one of these virulence factors that offers GAS several mechanisms of defense against the host system (7, 8, 23, 29). Many common emm types reported by the Centers for Disease Control and Prevention's Active Bacterial Core Surveillance for invasive GAS were found in our surveillance of noninvasive illnesses: emm1, emm3, emm12, emm28, and emm89 (see Table 2). These emm types from noninvasive illnesses are comparable to those seen in civilian populations and reaffirm the concern that virulent strains of GAS are circulating in more benign presentations and potentially could escalate into invasive disease trends and mortality if not properly managed (16, 18). Furthermore, the heterogeneity and shifts of emm types observed reflects the importance of considering multiple emm types in designing GAS vaccines (17). Since the M protein is presently described as a major virulence factor in GAS infections, a number of present vaccine constructs are composed of multivalent M protein sequences specific to particular diseases or geographic regions (15). Our surveillance data may help to guide these constructs.

In conclusion, surveillance for GAS isolates among United States military trainees has revealed a significant prevalence of macrolide resistance. The emm type distribution varied across military training sites, with emm75 strongly correlated with erythromycin resistance at the Air Force site. Use of alternative antibiotics in penicillin-allergic individuals should be considered at sites with a background of increased macrolide resistance. Continued laboratory-based monitoring of GAS infections is important for the appropriate use of antibiotics and development of vaccines, thus minimizing streptococcal disease morbidity in both civilian and military populations.


   ACKNOWLEDGMENTS
 
This work represents report 03-01, supported by the Department of Defense, under work unit no. 6609.

The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Navy, Department of Defense, or the United States government.

This surveillance is largely a credit to the personnel of our Streptococcal pyogenes Surveillance Group at the following institutions: Naval Recruit Training Center, Great Lakes, Ill.; Marine Corps Recruit Depot, San Diego, Calif.; Marine Corps Recruit Depot, Parris Island, S. C.; Army Basic Training Centers in Fort Jackson, S. C., Fort Knox, Ky., Fort Leonard Wood, Mo., and Fort Sill, Okla.; and Lackland Air Force Base, Tex. We also are grateful for the support and guidance of Ed Kaplan and Dwight Johnson of the University of Minnesota; Pat Kelley and Joel Gaydos of the Department of Defense Global Emerging Infections Surveillance program; Gary Gackstetter and Tonie Hooper of USUHS; and Mike Dove and the team at DMDC.


   FOOTNOTES
 
* Corresponding author. Mailing address: Naval Health Research Center, Department of Defense Center for Deployment Health Research, P.O. Box 85122, San Diego, CA 92186-5122. Phone: (619) 553-0576. Fax: (619) 553-7601. E-mail: russell{at}nhrc.navy.mil. Back

{dagger} This research was presented in part at the American Society of Microbiology General Meeting, Salt Lake City, Utah, May 2002. Back


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Journal of Clinical Microbiology, October 2003, p. 4808-4811, Vol. 41, No. 10
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.10.4808-4811.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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