Abstract
MICs of eight antibiotics were detected with 40 Chinese Mycoplasma pneumoniae isolates. Thirty-eight isolates (95%) were macrolide resistant. Each macrolide-resistant isolate harbored an A2063G or A2064G point mutation in the 23S rRNA gene. All 40 isolates (100%) were type I strains, but they might have originated from different clones.
TEXT
Mycoplasma pneumoniae is a common pathogen causing community-acquired pneumonia (CAP) (4, 9, 12). Macrolides are the drugs of primary choice for the treatment of M. pneumoniae infections. Macrolide resistance rates of M. pneumoniae have increased rapidly in recent years, especially in Asia (3, 10, 11). In 2009, studies from China found that 83% (44/53) and 92% (46/50) of M. pneumoniae strains isolated from pediatric patients in Shanghai (6) and Beijing (14) were resistant to macrolides, respectively. In 2010, the macrolide resistance rate of M. pneumoniae isolated from ambulatory adult patients was 69% (46/67) in Beijing (1).
In this study, 40 M. pneumoniae isolates were collected from CAP patients in Beijing, China. The antibiotic resistance patterns of this pathogen were surveyed with eight agents, and the mechanisms of resistance for macrolide-resistant isolates were investigated with 23S rRNA gene analysis.
M. pneumoniae strains.
Forty M. pneumoniae isolates were obtained from 182 CAP patients in the Beijing, Dongcheng, and Xicheng Centers for Disease Control and Prevention from January 2011 to June 2011. The clonalities of all M. pneumoniae isolates were determined by the filtration-cloning technique and identified by colony morphology and real-time PCR assays (2).
Antimicrobial susceptibility testing of isolates.
The MICs of eight antibiotics were determined by broth microdilution methods with SP4 broth (Remel). M. pneumoniae reference strain M129 (ATCC 29342) was tested as an antibiotic-sensitive control. Thirty-eight (95%) isolates were macrolide resistant. The MIC50 values of isolates for erythromycin, clarithromycin, azithromycin, and josamycin were greater than 256 μg/ml, 256 μg/ml, 32 μg/ml, and 4 μg/ml, respectively. The MIC90 values of the isolates with the four macrolides above were greater than 256 μg/ml, 256 μg/ml, 32 μg/ml, and 8 μg/ml, respectively. All M. pneumoniae isolates were susceptible to tetracycline and fluoroquinolones (Table 1). Gatifloxacin was more active than ciprofloxacin and levofloxacin. The MIC90 of gatifloxacin (0.064 μg/ml) was much lower than those of ciprofloxacin (1 μg/ml) and levofloxacin (1 μg/ml).
Table 1.
Genotype characteristics and MIC ranges of eight antimicrobial agents against 40 M. pneumoniae clinical isolates
| Sequence type of p1 gene | Mutation in 23S rRNA gene | No. of isolates | MICa (μg/ml) |
|||||||
|---|---|---|---|---|---|---|---|---|---|---|
| ERY | CLR | AZM | JOS | TET | CIP | LVX | GAT | |||
| Type I | A2063G | 37 | 256–>256 | 64–>256 | 8–64 | 2–8 | 0.032–0.125 | 0.5–2 | 0.25–2 | 0.032–0.125 |
| Type I | A2064G | 1 | >256 | >256 | 32 | 4 | 0.125 | 1 | 1 | 0.125 |
| Type I | None | 2 | 0.008–0.016 | <0.008 | <0.008 | 0.064 | 0.125 | 0.5–1 | 1 | 0.125 |
MICs were determined by microdilution methods using SP4 broth. The MIC of each agent was defined as the lowest concentration of each antibiotic preventing the color change. ERY, erythromycin; CLR, clarithromycin; AZM, azithromycin; JOS, josamycin; TET, tetracycline; CIP, ciprofloxacin; LVX, levofloxacin; GAT, gatifloxacin.
Sequencing analysis of the 23S rRNA gene.
Genomic DNA of each isolate was extracted with the QIAamp DNA minikit (Qiagen). Domains II and V of the 23S rRNA gene were amplified by methods described previously (8). All the amplicons were sequenced by the Beijing Genomics Institute (BGI). Thirty-seven isolates harbored an A2063G mutation in domain V of the 23S rRNA gene, while one isolate harbored an A2064G mutation in domain V of the 23S rRNA gene. No mutations were found in domain II (Table 1).
Typing of the p1 gene.
All 40 M. pneumoniae isolates from CAP patients were genotyped by the PCR method described previously (5). PCR analysis of the p1 genes showed that all (100%) isolates were characteristic of type I. The variable-number tandem repeat (VNTR) sequence in the p1 gene (15) was amplified using the primer pair VNTR-F (5′-GATACCGCTACCGTACCTCG-3′) and VNTR-R (5′-TGAGAATAGCAGCAAACAAGGA-3′). The reaction conditions were 30 cycles of 98°C for 15 s, 56°C for 15 s, and 72°C for 30 s. PCRs were performed using the PrimeSTAR Kit (TaKaRa). Sequence analysis showed that the stable “AGT” VNTR in the p1 gene of each M. pneumoniae isolate appeared 4 to 10 times (Table 2).
Table 2.
VNTRs in all 40 M. pneumoniae isolates
| No. of “AGT” VNTRs | No. detected |
|---|---|
| 4 | 2 |
| 5 | 6 |
| 7 | 16 |
| 8 | 11 |
| 9 | 2 |
| 10 | 3 |
In general, macrolides are used as the first-choice therapeutic agent for treating M. pneumoniae infections in children, as well as in adults. The macrolide resistance rate has been shown to be very high in M. pneumoniae strains isolated in China in recent years (1, 6, 14). This study showed a macrolide resistance rate of 95% in Beijing. Our previous study found that adult and adolescent patients infected with macrolide-resistant M. pneumoniae required significantly longer durations of antibiotic therapy and needed longer times to recuperate from fever (1). Although M. pneumoniae isolates were susceptible to fluoroquinolones, this class of drugs is not ordinarily recommended for children, except in particular cases.
Macrolide resistance in M. pneumoniae is strongly associated with mutations in the 23S rRNA gene (7, 13). In fact, A2063G and A2064G mutations have been shown to be responsible for high-level macrolide resistance in M. pneumoniae (8). The A2063G mutation in domain V of the 23S rRNA gene is recognized to be the most prevalent in macrolide-resistant M. pneumoniae isolates (8). More than 95% of the reported macrolide-resistant M. pneumoniae isolates in China had this mutation, including those in this study. Only one isolate with the A2064G mutation was detected in this study; this mutation locus is rarely reported.
Since 2007, the predominant M. pneumoniae strains isolated from China have been type I (1, 6, 14). Most macrolide-resistant isolates have also been found to belong to this type. In this study, all of the macrolide-resistant isolates belong to type I strains. In a previous study, a shift from type II to type I was found in Japan from 2000 to 2004, and macrolide-resistant isolates were also found in both types (8). It has been suggested that the macrolide resistance for M. pneumoniae is not associated with a specific p1 gene type but the current predominant isolate. Although all macrolide-resistant isolates belong to type I in this study, different numbers of stable “AGT” VNTRs (15) in the p1 gene of each isolate appeared (Table 2). Based on the VNTR data, these type I macrolide-resistant M. pneumoniae isolates spread in Beijing belonged to different resistant clones. Since the numbers of stable “AGT” VNTRs in the p1 gene are different among the macrolide-resistant isolates, the question mentioned in previous studies (1, 6) that asks whether or not all M. pneumoniae isolates identified with the same p1 type are derived from the same clone has now been answered. This also may be a better locus for the multilocus VNTR analysis (MLVA) typing method.
In summary, 95% (38/40) of M. pneumoniae isolates from CAP patients were macrolide resistant. Each macrolide-resistant isolate harbored a point mutation in the 23S rRNA gene. All M. pneumoniae isolates in this study were type I, which is the predominant type in Beijing. Based on the different numbers of stable “AGT” VNTRs in the p1 gene, these macrolide-resistant M. pneumoniae isolates spread in Beijing were shown to belong to different resistant clones.
ACKNOWLEDGMENT
This study was supported by the Major Infectious Diseases Such as AIDS and Viral Hepatitis Prevention and Control Technology Major Projects (grant no. 2008ZX10004-002).
Footnotes
Published ahead of print 21 November 2011
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