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J Clin Microbiol. Feb 2007; 45(2): 290–293.
Published online Nov 8, 2006. doi:  10.1128/JCM.01653-06
PMCID: PMC1829018

Prevalence and Antibacterial Susceptibility of mef(A)-Positive Macrolide-Resistant Streptococcus pneumoniae over 4 Years (2000 to 2004) of the PROTEKT US Study[down-pointing small open triangle]

Abstract

In the United States, approximately 30% of Streptococcus pneumoniae isolates are macrolide (erythromycin [ERY]) resistant (ERSP), most commonly due to expression of the mef(A) gene previously associated with lower-level ERY resistance (ERYr; MIC = 1 to 4 μg/ml). The data from the PROTEKT US surveillance study were analyzed to evaluate the prevalence and antibacterial susceptibility of mef(A)-positive ERSP. In all, 26,634 isolates of S. pneumoniae were collected in the United States between 2000 and 2004 from centers common to all years. ERYr was stable at approximately 29% over the 4 years, but the proportion of ERSP isolates positive for mef(A) alone decreased (year 1 [2000 to 2001], 69.0%; year 4 [2003 to 2004], 60.7%), with the sharpest declines seen in isolates from patients from 0 to 2 years of age. Conversely, the proportion isolates positive for both erm(B) and mef(A) increased over the duration of the present study (year 1, 9.3%; year 4, 19.1%), a change that was again most marked in patients aged ≤2 years. The majority of ERSP isolates expressing mef(A) alone exhibited higher than previously reported levels of ERYr (MIC90 = 16 μg/ml). However, the ketolide antibacterial telithromycin consistently demonstrated in vitro activity against these isolates over the 4 years of the study (MIC90 = 0.5 to 1 μg/ml).

Streptococcus pneumoniae is one of the most frequent causes of community-acquired respiratory tract infections (RTIs). Given that the antibacterial treatment of community-acquired RTIs is generally empirical, the high levels of antibacterial resistance among isolates of S. pneumoniae have become a global concern, and surveillance of antibacterial resistance patterns at national and local levels is of increasing importance.

In the United States, ca. 30% of S. pneumoniae isolates are resistant to macrolide antibacterials (erythromycin, clarithromycin, roxithromycin, and azithromycin) (7a, 10). Macrolide resistance in S. pneumoniae is mediated by two main mechanisms: methylation of ribosomal macrolide target sites [encoded by the erm(B) gene] and drug efflux [encoded by the mef(A) gene]. Isolates expressing the erm(B) gene have typically been found to exhibit high-level resistance; i.e., macrolide MIC90 values of ≥64 μg/ml, whereas those expressing the mef(A) gene have been characterized by lower-level resistance (MIC90s of 4 to 8 μg/ml) (6, 13). Currently, mef(A) is the predominant resistance mechanism in the United States (7a), whereas erm(B)-positive isolates are more prevalent throughout most of the rest of the world (5a).

Pneumococcal macrolide resistance is of increasing concern in the clinical setting (8). In recent years, a number of reports have been published linking occurrences of macrolide treatment failure (sometimes resulting in hospitalization with breakthrough bacteremia) with infection by macrolide-resistant strains of S. pneumoniae in patients with community-acquired RTIs. It is notable that clinical failures have been reported in patients infected with pneumococcal strains expressing mef(A)-encoded macrolide resistance, as well as in patients infected with strains with erm(B)-mediated resistance (9, 14).

PROTEKT US (an acronym for prospective resistant organism tracking and epidemiology for the ketolide telithromycin in the United States) is a longitudinal surveillance study of antibacterial resistance among key respiratory tract pathogens. It was initiated in 2000 to track the susceptibility of common bacterial respiratory tract pathogens to telithromycin (the first ketolide antibacterial approved for clinical use) and other antibacterial agents. This analysis of data from the PROTEKT US study evaluates the prevalence and antibacterial susceptibility of mef(A)-positive strains of macrolide-resistant S. pneumoniae collected between 2000 and 2004.

MATERIALS AND METHODS

Isolates of S. pneumoniae were collected over the 4 years of the PROTEKT US study (year 1, 2000 to 2001; year 2, 2001 to 2002; year 3, 2002 to 2003; year 4, 2003 to 2004) from the 135 centers common to all 4 years of the study. For the purposes of geographic analysis, centers were assigned to one of six regions: Northwest (Alaska, Idaho, Montana, Oregon, Washington, and Wyoming), Northeast (Connecticut, Delaware, Indiana, Maryland, Massachusetts, Michigan, New Jersey, New York, Ohio, Pennsylvania, Rhode Island, Vermont, and Washington, DC), North-central (Illinois, Iowa, Kansas, Minnesota, Missouri, Nebraska, North Dakota, South Dakota, and Wisconsin), Southwest (Arizona, California, Colorado, Nevada, New Mexico, Utah), Southeast (Florida, Georgia, Kentucky, North Carolina, Puerto Rico, South Carolina, Virginia, and West Virginia), and South-central (Alabama, Arkansas, Louisiana, Oklahoma, Tennessee, and Texas).

Respiratory tract isolates of S. pneumoniae, deemed pathogenic on isolation, were collected from adult and pediatric outpatients with community-acquired RTIs (otitis media, pneumonia, acute bacterial exacerbations of chronic bronchitis, acute exacerbations of chronic obstructive pulmonary disease, and sinusitis). Isolates cultured from material collected within 48 h of admission from patients hospitalized with these infections were also included. Eligible culture sources were blood, sputum, bronchoalveolar lavage, middle-ear fluid (sampled by tympanocentesis), nasopharyngeal swab or aspirate, and sinus aspirate. Excluded were samples from patients with nosocomial RTIs or cystic fibrosis, duplicate strains, and strains originating from existing collections.

Antibacterial susceptibility testing.

MICs of antibiotics commonly prescribed for treatment of community-acquired RTIs were determined at a central laboratory (CMI, Wilsonville, OR) by using the Clinical and Laboratory Standards Institute (CLSI) broth microdilution method (1). CLSI MIC interpretive criteria were used to define susceptibility and resistance (2).

Genotyping analysis.

Isolates resistant to erythromycin (MIC ≥ 1 μg/ml) were analyzed for the presence of erm(B), erm(A) subclass erm(TR), and mef(A) macrolide resistance genes. In year 1, isolates were analyzed by using multiplex rapid-cycle PCR with Microwell-format probe hybridization, as described previously (3). In years 2 to 4, isolates were analyzed by using a multiplex TaqMan PCR assay (Applied Biosystems, Foster City, CA) that was validated against the previous PCR method (12).

Statistical analysis.

Statistical analysis was performed by using the χ2 and Fisher exact tests and InStat software (GraphPad Software, Inc., San Diego, CA).

RESULTS

A total of 26,634 isolates of S. pneumoniae were collected over the 4 years of the PROTEKT US study from centers common to all 4 years: year 1, 7,314; year 2, 6,052; year 3, 6,831; and year 4, 6,437. The rates of erythromycin resistance remained stable at ca. 29% throughout this period: year 1, 31.3%; year 2, 27.6%; year 3, 28.4%; and year 4, 28.9%.

The distribution of macrolide resistance mechanisms among erythromycin-resistant isolates of S. pneumoniae collected during years 1 to 4 of the study is shown in Fig. Fig.1.1. Overall, mef(A) was the most prevalent resistance gene; however, the proportion of isolates expressing mef(A) alone showed a significant decrease over the 4 years of the study: year 1, 69.0%; year 2, 67.5%; year 3, 62.5%; and year 4, 60.7% (Table (Table1,1, [P = 0.0313]). Conversely, the proportion of S. pneumoniae isolates that were positive for both erm(B) and mef(A) genes increased over the 4 years (year 1, 9.3%; year 2, 11.9%; year 3, 17.3%; year 4, 19.1%), while the proportion with erm(B) alone showed little change (from 17.1% in year 1 to 16.7% in year 4). This shift in the prevalence of mef(A)-positive S. pneumoniae isolates over the 4 years of the study varied according to the age of the patient (Table (Table1).1). The sharpest declines were seen in isolates from the youngest patients, aged 0 to 2 years (from 71.8% in year 1 to 53.7% in year 4 [P = 0.0479]). Conversely, patients in the 0- to 2-year-old age group showed the greatest rise in erm(B)+mef(A) prevalence, from 11.0% in year 1 to 34.2% in year Y4 [the respective year 1 and 4 prevalences of erm(B)+mef(A) in the other age groups were 10.0 and 21.7% (3 to 14 years); 9.2 and 14.6% (15 to 64 years); 7.1 and 12.7% (>64 years)].

FIG. 1.
Distribution of macrolide resistance mechanisms in erythromycin-resistant S. pneumoniae isolates collected during years 1 to 4 of the PROTEKT US study.
TABLE 1.
Proportion of erythromycin-resistant isolates of S. pneumoniae positive for the mef(A) gene alone, by U.S. region and by age group, during years 1 to 4 of the PROTEKT US study

The proportions of isolates positive for the mef(A) gene collected in six U.S. regions over the 4 years of the study are summarized in Table Table1.1. The South-central and Southeastern regions displayed the highest proportion of mef(A)-positive isolates in each year. The greatest decline in mef(A) prevalence over the 4 years occurred in the Northeast region (year 1, 69.4%; year 4, 53.8% [P = 0.0211]). In contrast, the prevalence of mef(A) was more stable in the other regions, with no statistically significant trends over the 4 years.

The in vitro activities of a range of antibacterials against isolates of erythromycin-resistant S. pneumoniae positive for mef(A) are presented in Table Table2.2. A large proportion of mef(A)-positive strains showed a higher than previously reported resistance to erythromycin (MIC50 = 8 μg/ml; MIC90 = 16 μg/ml). The MIC50 and MIC90 values against these strains were also higher than previously reported for the other macrolides, azithromycin (8 and 32 μg/ml, respectively) and clarithromycin (4 and 16 μg/ml, respectively). In contrast, telithromycin retained consistent activity against mef(A)-positive strains, with respective MIC50 and MIC90 values of 0.25 and 0.5 μg/ml and a susceptibility of 99.6% across the 4 years of the study. S. pneumoniae strains expressing erm(B) also exhibited a high rate of susceptibility to telithromycin [erm(B), 98.6% (1,293 of 1,312); erm(B)+mef(A), 99.0% (1,093 of 1,104)].

TABLE 2.
Comparative in vitro activity of selected antibacterials against ERYr S. pneumoniae isolates positive for mef(A)a

The in vitro mode MIC and MIC ranges for the same panel of antibacterials against macrolide-resistant S. pneumoniae isolates positive for mef(A) are shown in Table Table3.3. These data indicate that whereas the MIC ranges have remained largely stable, the mode MICs have decreased for a number of the antibiotics tested between years 1 and 4 of the PROTEKT US study. The mode MIC of erythromycin decreased from 16 μg/ml in year 1 to 4 μg/ml in year 4, whereas that for azithromycin decreased from 32 to 8 μg/ml over this period. In both instances these mode MICs show that the S. pneumoniae isolates expressing the mef(A) genotype maintained their low-level resistance to these macrolides. The mode MIC for telithromycin also decreased slightly from 0.5 μg/ml in year 1 to 0.12 μg/ml in year 2, a reduction in MIC that was maintained up to and including year 4.

TABLE 3.
Mode MIC and MIC range of selected antibacterials against erythromycin-resistant S. pneumoniae isolates positive for mef(A) from years 1 to 4 of the PROTEKT US study

DISCUSSION

Results from this analysis of PROTEKT US indicate that, over a 4-year period, the prevalence of S. pneumoniae strains expressing mef(A) alone has declined in the United States from 69.0 to 60.7%. The decrease in prevalence of mef(A)-positive isolates coincided with an increase in the prevalence of strains expressing both mef(A) and erm(B) resistance genes, both of which were most marked in isolates from pediatric patients aged ≤2 years. An increase in the prevalence of the erm(B)+mef(A) genotype in the United States since 2000 has been reported previously (4). It has also been shown that the vast majority of erm(B)+mef(A) isolates belong to a small group of clonal strains that exhibit multidrug resistance (4). There is evidence that these clones have an evolutionary advantage over strains with the single resistance determinant (5), which may explain why erm(B)+mef(A) strains are increasing at the apparent expense of the mef(A) genotype.

Another key finding of the present analysis was that the majority of mef(A)-positive isolates exhibited levels of macrolide resistance higher than those reported in previous surveillance studies. Although erythromycin MIC90 values of 4 to 8 μg/ml have previously been reported for mef(A)-positive isolates collected in a U.S. study (1994 to 1995) (13) and a Canadian study (1998 to 1999) (6), the MIC90 for isolates collected during PROTEKT US (2000 to 2004) was 16 μg/ml. Such increases in macrolide MICs for mef(A)-positive isolates may impact the ability of macrolide antibacterials to eradicate these strains at sites of infection in patients with community-acquired RTIs. Indeed, using an in vitro pharmacodynamic model of clinically achievable epithelial lining fluid (ELF) concentrations of clarithromycin, Noreddin et al. (11) demonstrated that clarithromycin was unable to eradicate mef(A)-producing S. pneumoniae isolates with MICs of ≥16 μg/ml. Furthermore, in a similar study using azithromycin, whereas serum, ELF, and middle-ear fluid concentrations of the drug rapidly eradicated macrolide-susceptible S. pneumoniae, they did not eradicate macrolide-resistant S. pneumoniae, regardless of the resistance genotype (15). A study using a murine model of pneumococcal pneumonia (7) also showed that clarithromycin was only effective in treating infections caused by macrolide-susceptible strains or strains with low-level mef(A)-mediated resistance (MICs of 0.5 to 1 μg/ml), whereas azithromycin was ineffective against all macrolide-resistant strains.

Possible mechanisms that could account for the observed increase in MIC90 values among mef(A) isolates include changes in expression of the mef(A) gene and underlying ribosomal mutations. However, further investigations are needed to elucidate the precise mechanism involved.

Despite the failure of macrolides to eradicate resistant strains of S. pneumoniae in the laboratory, empirical treatment with these antibacterials continues to be a mainstay of therapy for community-acquired RTIs. Published examples of clinical failure are rare; however, infections with S. pneumoniae isolates that are resistant based on the expression of either mef(A) or erm(B) have been reported to result in hospitalization in a number of patients treated with macrolide antibacterials (9, 14). The apparent scarcity of treatment failure in community-acquired RTIs caused by macrolide-resistant S. pneumoniae to date may be due to the accumulation of high concentrations of these antibacterials in ELF, allowing them to be clinically effective even when challenged by pathogenic isolates of moderate resistance (16). However, increased levels of macrolide resistance in S. pneumoniae isolates expressing the mef(A) gene, which has been traditionally associated with lower-level resistance, may result in a rise in the number of clinical failures associated with pathogenic strains with this genotype.

In summary, the findings from this analysis of the PROTEKT US study from 2000 to 2004 emphasize that continual surveillance of genotype distribution and antibacterial resistance in S. pneumoniae is essential to guide the effective use of empirical treatment options for community-acquired RTIs.

Acknowledgments

We acknowledge the support of coworkers in the United States for the supply of bacterial isolates as part of the PROTEKT US study, and the G.R. Micro, Ltd., and CMI PROTEKT teams who performed the MIC determinations and genotyping.

The PROTEKT US study is supported by Sanofi-Aventis US, Inc.

Footnotes

[down-pointing small open triangle]Published ahead of print on 8 November 2006.

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