Antimicrobial Susceptibility Of And Macrolide Resistance Characterization In Mycoplasma Pneumoniae Isolates

Mycoplasma pneumoniae, which belongs to mycoplasmas of Mollicutes, represents one of the smallest self-replicating organisms, in both cellular dimensions and genome size, which are capable of cell-free existence. It is a common cause of community acquired upper and lower respiratory tract infections in persons of all ages and may be responsible for up to 40% or more of all cases of community-acquired pneumonia (CAP) and as many as 18% of cases requiring hospitalization in children.Culture of M. pneumoniae from the respiratory tract and other body sites is laborious and expensive, requiring serial blind passages, specialized and expensive growth media, and incubation periods of up to several weeks. However the small genome of M. pneumoniae and its limited biosynthetic capabilities are responsible for many of the biological characteristics and requirements for complex medium supplementation in order for the organism to be cultivated in vitro. Therefore, culture is rarely used for routine diagnosis and/or management of M. pneumoniae infections. But obtaining purified M. pneumoniae isolates are very important to perform the susceptibility testing and to do pharmacodynamic study of new antimicrobials.In this study, M. pneumoniae clinical strains were collected consecutively and the library of isolates was established. Broth microdilution method using SP4 broth was performed to determine the minimal inhibitory concentrations (MICs) of macrolides, tetracyclines and fluoroquinolones against M. pneumoniae isolates. The mechanisms of resistance for macrolides-resistant stains were investigated. The type of M. pneumoniae clinical isolates was determined by restriction fragment length polymorphism (PCR-RFLP) typing of the P1 gene and multiple ocus variable number tandem repeat analysis (MLVA) typing assay. Cycleave PCR technique was developed as a novel method to detect erythromycin-resistant strains harboring A2063G and A2064G transition mutation in the 23s rRNA gene. In addition, the clinical significance of macrolides-resistant M. pneumoniae infections was preliminarily evaluated.In this part of study, M. pneumoniae clinical strains were collected consecutively and the library of isolates was established. Culture of M. pneumoniae was carried out as described by Waites et al. Detection of M. pneumoniae by culture is predicated on its characteristic hydrolysis of glucose, with a resultant acidic shift, after 4 days or more of incubation in broth containing a phenol red PH indicator. Initial specimens, as well as broths with color change and blind subcultures, should be transferred to SP4 agar, incubated, and examined under a stereomicroscope at regular intervals to look for development of spherical colonies of up to 100μm in diameter. Incubation of M. pneumoniae broth cultures under atmospheric conditions at 37℃is satisfactory, but agar plates will yield the best colonial growth if 5% CO2 is provided and plates are sealed to prevent loss of moisture during prolonged incubation. One hundred and fifty-two M. pneumoniae isolates were obtained from bronchial aspirations (each specimen collected from one patient) at Shanghai Children's Hospital from Nov.2005 to July 2009. PCR amplification of P1 gene was done to identify M. pneumoniae. The total culture positive rate was 4.8%.Broth microdilution is the most widely used method of susceptibility testing of human mycoplasmas and ureaplasmas. Broth microdilution method using SP4 broth (Remel, Santa Fe Drive Lenexa, KS, USA) was performed as described previously by Waites et al to determine the minimal inhibitory concentrations (MICs). Some modifications were made to improve the accuracy of the testing. Ten antimicrobials belonging to three classes were included:macrolides (erythromycin, azithromycin, clarithromycin and josamycin), tetracyclines (tetracycline, doxycycline and minocycline) and fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin). Susceptibility testing on each isolate was repeated three times.All 152 M. pneumoniae isolates were susceptible to the 3 tetracyclines and 3 fluoroquinolones tested. Moxifloxacin was more active than ciprofloxacin and levofloxacin. Both MIC50 and MIC90 of moxifloxacin were 0.06 mg/L, much lower than those of ciprofloxacin and levofloxacin (1 and 0.5 mg/L, respectively). Among 152 M. pneumoniae strains,90.1%(137/152) were resistant to erythromycin. Of 137 erythromycin-resistant strains,135 (98.5%) had MICs of>128 mg/L to either erythromycin or clarithromycin, while two strains had MICs of 64 mg/L for erythromycin. Azithromycin had lower MICs than that of erythromycin and clarithromycin with MIC50 and MIC90 of 32 and 64 mg/L, respectively. The 16-membered macrolide josamycin possessed lower MICs than the 14-and 15-membered macrolides with MICs 1 to 8 mg/L, except one strain had MIC>128 mg/L. All 15 macrolide-susceptible M. pneumoniae strains had MICs≤0.015 mg/L for four macrolides tested. The resistance rates of M. pneumoniae isolates from 2005 to 2009 were 60.0%(3/5),82.4%(14/17),100%(24/24),80.8%(42/52) and 100% (54/54), respectively.The resistance rate of M. pneumoniae isolates was very high. It is important to differentiate between the spread of a single macrolide resistant clone or multiple isolates. In this part PCR-RFLP and MLVA were carried out to determine what is going to with regard to the spread of macrolide-resistant M. pneumoniae.PCR-RFLP of the amplified P1 gene was used in this part of study to type 152 M. pneumoniae isolates. One hundred and thirty-eight clinical isolates were classified into P1-typeⅠ, twelve were classified into P1-typeⅡ, and the other two isolates could not be classified by this method. Thus, typeⅠwas predominant among the isolates tested.131 (94.9%) from 138 typesⅠstrains were resistant to erythromycin, however, only 4 (33.3%) out of 12 typeⅡstains was resistant to erythromycin. A strong association between the P1 gene PCR-RFLP type and the macrolide resistance was demonstrated (P<0.01).Because the P1 gene based typing systems have a limited power of discrimination, MLVA is employed for epidemiological studies to assess the genetic relatedness of M. pneumoniae clinical isolates. A total of 154 M. pneumoniae strains, including two reference strains, were divided into 18 MLVA types.137 macrolide-resistant isolates were clustered into 15 MLVA types, and 8 MLVA types, including more than 10 macrolide-resistant strains each, accounted 84% of the 137 isolates. The overall discriminatory index (HGI) of the MLVA method was 0.917. As a complementary analysis, a minimum spaning tree (MST) modeling showed the diversity among the tested isolates but each type was closely related to each other meanwhile no distinct clusters were found.Macrolide resistance in M. pneumoniae is due to mutations in 23S rRNA. The point mutation in the peptidytransferase region of 23S rRNA results in the loss of binding ability of the macrolides to the 23S rRNA components of the ribosome and was the major cause of macrolides resistance. Several point mutations in 23S RNA have been reported and different mutations cause different level of resistance to 14-,15-and 16-member macrolides. The A2063G mutation is found to be most prevalent from macrolide-resistant M. pneumoniae isolates, followed by the A2064G mutation. Other mutations like A2063C were very rare.PCR assay for 23S rRNA were performed to amplify and sequence the full length of 23S rRNA fragment. Of 137 macrolide-resistant strains, the A2063G transition in domainⅤof 23S rRNA genes was found in 135 (98.5%) isolates which resulted in high erythromycin MIC (>128 mg/L). A2064G mutation was detected in one strain with erythromycin 64μg/ml and josamycin>128 mg/L. Both A2063G and A2064G transitions can cause high level resistance to erythromycin and azithromycin (MIC of >64 mg/L and 32-64 mg/L, respectively) in M. pneumoniae, although it seems A2064G is associated with higher josamycin MIC than that of A2063G (>64-256 mg/L vs.8-16 mg/L). A new transition of A2063T was identified in this study, which may be responsible for high MIC of erythromycin (64 mg/L), but relatively low MICs of josamycin (8 mg/L) and azithromycin (1 mg/L). In addition, an A1290G transition mutation was found in all 152 tested strains regardless sensitive or resistant to erythromycin except the reference strain Mp FH, which indicates change of A1290G is not related to macrolide-resistance.A rapid and specific assay should been developed for the detection of the resistance of M. pneumoniae. The cycling probe technology is a unique nucleic acid-based method that detects single nucleic acid polymorphisms (SNPs) in a target DNA sequence by using a probe-adapted real-time PCR. The cycling probe method involves a reaction between a chimeric fluorescenceand quencher-labeled DNA/RNA oligonucleotide probe (Cycling probe) and RNase (RNase H).In this part of study, we developed a rapid assay using a chimera probe-adapted real-time PCR, or the cycling probe method, to detect macrolide-resistant M. pneumoniae with the A2063G/A2064G mutaton in the 23S rRNA. The primers and cycling probe sets were designed to detect two single nucleotide mutation, A2063G and A2064G, in the 23S rRNA gene of M. pneumoniae. By using recombinant plasmids containing 23S rRNA gene fragments,152 M. pneumoniae clinical isolates, 150 bronchial aspirations from pediatric patients with low respiratory tract infections and common respiratory tract pathogens, the specificity and the sensitivity of the Cycleave PCR method were evaluated. PCR amplification and sequence analysis of 23S rRNA genes were performed for all M. pneumoniae strains and M. pneumoniae positive specimens to confirm the results of the Cycleave PCR method. Of 151 clinical isolates, one hundred and thirty-five was resistant to erythromycin and all macrolide-resistant M. pneumoniae strains harbored an A2063G (135/136) or A2064G (1/136) transition mutation in 23S rRNA genes. Twenty-five was M. pneumoniae positive detected by Cycleave PCR in 150 bronchial aspirations. The Cycleave PCR results were consistent with those of routine PCR and sequencing. The testing limits for both reactions were 10 copy/PCR reactions.It was reported by Suzuki et al that macrolides were less effective in the treatment of patients who were infected with macrolide-resistant M. pneumoniae than those infected with macrolide-susceptible strains, e.g., febrile period was longer in patients with resistant strains. Though the macrolide resistance in M. pneumoniae is quite high in China, macrolides are still the most common antimicrobials used in the treatment of M. pneumoniae infections in children. The impact of the resistance to macrolides on the efficacy of treatment with macrolides is thus needed to be investigated.A comparative study was performed to determine whether or not macrolide-resistant M. pneumoniae influences the clinical outcome in patients treated with macrolides. The clinical courses of 9 patients with macrolide-resistant M. pneumoniae infection treated with macrolides were compared with those of 48 patients with macrolide-susceptible M. pneumoniae infection. The number of febrile days during macrolide administration and the length of stay (LOS) were longer in the macrolide-resistant patients than in the MS patients (mean of 7.31 days versus mean of 3.00 days [P=0.004] and 11.19 days versus 8.22 day [P=0.005], respectively). Despite the fact that the febrile period was prolonged in MR patients given macrolides, the fever resolved even when the initial prescription was not changed. These results show that macrolides are certainly less effective in macrolide-resistant patients.