Antimicrobial susceptibility and genotyping of Mycoplasma pneumoniae isolates in Beijing, China, from 2014 to 2016

Background The presence of macrolide-resistant Myocplasma pneumoniae has been frequently reported in recent years, especially in China. In this study, we investigated the antimicrobial susceptibility and genotype against M. pneumoniae isolates from 2014 to 2016, Beijing. Methods We investigated the activities of four antibiotics against 81 M. pneumoniae isolates in vitro. All isolates were amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments. All isolates were genotyped with duplex real-time PCR, MLVA and VNTR detection in p1 gene. Results The macrolide resistance rate was 65.4% (53/81). Each of the macrolide-resistant M. pneumoniae isolates was resistant to erythromycin (Minimum Inhibitory Concentration, MIC, ≥256 μg/ml) and azithromycin (MIC, 2–64 μg/ml), but susceptible to tetracycline and levofloxacin in vitro. Fifty two macrolide-resistant isolates harbored the A2063G mutation, and only 1 macrolide-resistant isolates harbored the A2064G mutation in domain V of the 23S ribosomal RNA gene. The C162A, A430G, and T279C mutations in the L4 and L22 ribosomal protein genes were not responsible for macrolide resistance, but they were related to the particular genotype of M. pneumoniae. 95.7% of type 1 isolates (45/47) were macrolide-resistance, and 23.5% of the type 2 isolates (8/34) were macrolide-resistance. Type 2 M. pneumoniae macrolide-resistance rate was 50.6% higher than that of the previous reports in China. The eight macrolide-resistant type 2 M. pneumoniae isolates were belong to 3/5/6/2 and 3/5/7/2 MLVA genotypes. Conclusion To our knowledge, this phenomenon likely resulted from a combination of genotype shifting from type1 to type 2 and antibiotic selection pressure in M. pneumoniae in China in recent years. The increase of resistance in type 2 is not due to the spread of same clone. However, the relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.


Background
Mycoplasma pneumoniae is a common pathogen that causes human respiratory tract infections, especially in community-acquired pneumonia, which accounts for 10-40% of cases [1][2][3]. Although most of the infections are mild or self-limited, severe clinical symptoms are increasingly occurring. Furthermore, M. pneumoniae infections may lead to more severe extra-pulmonary conditions, such as erythema multiforme, myocarditis, and meningitis neuritis [4][5][6]. It is well known that M. pneumoniae is one of the smallest self-replicating organisms and lacks a cell wall, which makes it non-susceptible to β-lactam antibiotics. Therefore, macrolides are the primary drugs of choice for the treatment of M. pneumoniae infections in clinical settings [7].
Excessive or inappropriate use of antibiotics provides selective pressure for the development of macrolide resistance in M. pneumoniae. Since 2000, the emergence of macrolide-resistant M. pneumoniae strains has become a growing global problem. Macrolide resistance rates were reported to be approximately 3% in Germany [8], 10% in France [9], 2% in Switzerland [10], 26% in Italy [11], 3% in Denmark [12], and 11% in the United States [13]. Finally in Spain, the presence of a macrolide-resistant M. pneumoniae isolate causing community-acquired pneumonia was first reported in 2014 [14]. Although Europe and the United States have lower rates of macrolide resistance, Asiatic countries have the highest rates. After the first macrolide-resistant strain of M. pneumoniae was isolated in 2001 [15], Japan reported a dramatic increase in macrolide-resistant M. pneumoniae, and the macrolide resistance rate exceeded 90% in 2011 [16][17][18][19]. In South Korea, the prevalence of macrolide-resistant M. pneumoniae increased from 0 to 63% within 10 years [20,21]. In China, the infection rate of macrolide-resistant M. pneumoniae has always been high, ranging from 69 to 100% [22][23][24] in recent years. Given the distinct increase in the prevalence of macrolide-resistant M. pneumoniae, it is crucial to detect macrolide-resistant M. pneumoniae in a timely fashion and facilitate targeted treatment adjustments for M. pneumoniae-induced infections.
The resistance mechanism employed by M. pneumoniae involves point mutations in domain V of the 23S rRNA gene. Specifically, mutations at positions 2063 or 2064 lead to high-level resistance, whereas mutations at positions 2067 or 2617 are associated with low-level resistance to macrolides [25,26]. Furthermore, point mutations that occur in the L4 and L22 ribosomal protein genes seem to have no impact on resistance to macrolides in M. pneumoniae [22,26]. Until now, many erythromycin ribosome methylation (erm) and efflux pump (mef ) genes introduced via transposons or plasmids have been reported in many strains of pneumococci and enterobacteria [27], but these genes have still not been detected in M. pneumoniae [28]. The existence of any other macrolide resistance mechanisms of M. pneumoniae remains unclear and requires further study. Although early reports have found some potential correlation between macrolide-resistance and genotype in China, most type 2 isolates are macrolide-susceptible [22-24, 28, 29]. The data are relatively limited, and the association of the macrolide-resistance with genotype does not exist abroad reports [14,26]. Therefore, macrolide-resistance and genotypes of M. pneumoniae need to do further research.
In this study, 81 M. pneumoniae isolates were obtained from 271 throat swabs that were collected between 2014 and 2016 in Beijing, China, to evaluate the prevalence of macrolide-resistant M. pneumoniae and characterize its mechanisms of resistance, and analyze the correlation between genotype and macrolide resistance observed in M. pneumoniae in China. M. pneumoniae isolates, culturing, and genomic DNA extraction From January 2014 to December 2016, total 271 throat swabs were collected from the Beijing Centers for Disease Control and Prevention and Beijing Children's Hospital. None of the patients were immunocompromised, patients with neutropenia, and patients receiving immunosuppressive chemotherapy were excluded. All specimens were collected from patients with respiratory tract infections according to clinical symptoms. Each throat swab was cultured in Mycoplasma selective liquid media (OXOID) at 37°C. When the color of the media changed from red to yellow, approximately 0.1 ml of the suspension was transferred onto agar to subculture and purify the bacteria using the filtration-cloning technique for M. pneumoniae clinical isolates. Genomic DNA from each obtained isolate was extracted with the QIAamp DNA MINI kit (QIAGEN) using the protocol for blood and body fluids, and the identity of each isolate was verified using real-time PCR [30].

Antimicrobial susceptibility testing of M. pneumoniae
Minimum inhibitory concentrations (MICs) of four antibiotics, including erythromycin, azithromycin, levofloxacin and tetracycline (Sigma), were determined via micro-dilution methods using SP4 broth (Remel). M. pneumoniae M129 (ATCC 29342) and ICDC P028 (Clinical isolate) were used as macrolide-susceptible and -resistant controls, respectively. The MIC operation was carried out according to the latest version of CSLI M43-A. Serial two-fold dilutions of each antibiotic prepared in SP4 broth containing about 10 5 colony-forming units (CFU)/ml of M. pneumoniae were plated in 96-well micro-plates. The micro-plates were sealed with sterilized liquid paraffin oil and incubated at 37°C for 5 days. Each antimicrobial susceptibility test was performed in triplicate. The MIC was determined as the lowest concentration of antimicrobial agent that induced a color change in the control media.
Amplification and sequencing domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein genes Amplification of domains II and V of the 23S rRNA gene and the L4 and L22 ribosomal protein fragments were performed using the primers described previously [26]. Each reaction was performed in a final volume of 20 μl, containing 2 μl 10× Ex Taq Buffer (Mg 2+ plus), 0.4 μM primers, 1.6 μl dNTP mixture (each 2.5 μM), 0.2 μl TaKaRa Ex Taq (5 U/μl), 1 μl of template DNA, and nuclease-free water to achieve a 20-μl final volume. The cycling conditions were as follows: 95°C for 3 min, followed by 30 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 60 s with a final extension step of 72°C for 5 min. All amplification products were sequenced bidirectionally by Sangon Biotech (Beijing) Co. Ltd.

Duplex real-time PCR assay for genotyping of M. pneumoniae
Amplification of the genotype specific regions from all obtained isolates was performed with the duplex real-time PCR assay described previously [31]. Each PCR mixture was prepared in a total volume of 25 μl and contained the following per reaction: 12.5 μl Platinum Quantitative PCR SuperMix-UDG (Life Technologies-Invitrogen), 1.5 μl MgCl 2 (50 mM), 0.5 μM final concentration of each primer, 0.2 μM final concentration of each probe, 1.25 U Platinum Taq DNA polymerase(5 U/ μl; Life Technologies-Invitrogen), 1 μl PCR nucleotide mix (10 mM), 5 μl nucleic acid extracted from each specimen, and nuclease-free water to achieve a 25 μl final volume. Real-time PCR for each target was performed in the CFX96 Real-time PCR Detection System (Bio-Rad, Hercules, CA, USA) under the following conditions: predenaturation at 95°C for 2 min, followed by 45 cycles at 95°C for 15 s and 56°C for 15 s. The data were analyzed with the CFX Manager Software (version 2.1; Bio-Rad).

MLVA genotyping and VNTR detection in p1 gene
Extracted nucleic acids from all 81 isolates were used as the template for PCR amplification of the four loci selected for multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), as described previously [32]. Furthermore, all nucleic acids were also as the template for PCR amplification of the high resolution VNTR sequence in the p1 gene [24], The PCR products were sequenced by the Sangon Biotech (Beijing) Co., Ltd.

Results
Clinical M. pneumoniae culture and identification A total of 81 M. pneumoniae isolates were obtained from 271 patients. Of the 81 isolates, 27 were isolated in 2014, 19 were isolated in 2015, and 35 were isolated in 2016. Genomic DNAs from all 81 obtained isolates was identified by real-time PCR, which indicated that each isolate was indeed M. pneumoniae.

Antimicrobial susceptibility of M. pneumoniae
Of the 81 clinical isolates, 53 (65.4%) were erythromycin resistant (MIC, ≥256 μg/ml). These erythromycin-resistant isolates, as well as the reference strain ICDCP028, also showed resistance for the azithromycin. The MIC for the 15-member macrolide, azithromycin (2-64 μg/ ml), was lower than that of erythromycin. The other 28 isolates, as well as the reference strain, M129, were macrolide-susceptible with each having an MIC of ≤0.008 for erythromycin and azithromycin. All 81 clinical isolates were susceptible to tetracycline and levofloxacin used in this study. (Table 1).

Clinical patient data
Clinical data for M. pneumoniae-related cases of pneumonia were available for the 81 patients, 53 (65.4%) of whom had pneumonia caused by macrolide-resistant isolates. None of patients had coinfection with Streptococcus pneumoniae (as defined by positive results of sputum culture, blood culture, and urinary antigen testing for S. pneumoniae) or Legionella pneumophila (as defined by urinary antigen testing). Demographic characteristics and clinical presentations were similar between the group infected with macrolide-resistant isolates (53/ 81) and the group infected with macrolide-susceptible isolates (28/81) (  Table 1.

Macrolide-resistant isolates with genotyping from 2014 to 2016
The total Macrolide-resistant rate was 59.3, 73.7 and 65.7% from 2014 to 2016, respectively. However, the  (1) ≤0.008 ≤0.008 0.25-1 0.032-0.5 The MIC of each agent was defined as the lowest concentration of each antibiotic that prevented the color change ERY erythromycin, AZM azithromycin, LVX levofloxacin, TET tetracycline

Disscussion
In general, macrolides are the primary therapeutic agent for treating M. pneumoniae infections in both children and adults, but the macrolide resistance rate has been shown to be very high in clinical isolates of M. pneumoniae in China in recent years [22][23][24]31]. Basic clinical data demonstrated that the demographic characteristics, clinical presentations, and biochemical blood indices were similar between the group infected with macrolideresistant isolates and the group infected with macrolide-susceptible isolates, which is consistent with our previous report [22]. This suggests that the pathogenicity of the macrolide-susceptible and macrolide-resistant isolates is unremarkable. However, our previous study found that patients infected with macrolideresistant M. pneumoniae required significantly longer durations of antibiotic therapy and needed more time to recuperate from fevers [22]. Unfortunately, we did not obtain further clinical treatment data in this study. Although, Lluch-Senar [33] found that type 2 strains show higher expression levels of CARDS toxin, a protein recently shown to be one of the major factors of inflammation. It proposed that type 2 strains could be more toxigenic than type 1 strains of M. pneumoniae. Our basic clinical data still have not found some features in common for the patients with different genotypes. This suggests that the pathogenicity may also unremarkable for M. pneumoniae with genotypes. Therefore, the influence of the macrolide-resistance and the genotypes of M. pneumoniae isolates on the effect of the clinical outcomes requires further investigation. Macrolide resistance in M. pneumoniae is strongly associated with mutations in the 23S rRNA gene [34]. Several common mutations in the 23S rRNA gene were found at positions 2063, 2064, and 2617 [15,35,36]. Among these genetic loci, the A2063G and A2064G mutations are responsible for high levels of macrolide resistance in M. pneumoniae. Notably, the A2063G mutation in domain V of the 23S rRNA gene is the most prevalent in macrolide-resistant M. pneumoniae isolates in China [22,28,31,37,38]. In this study, 98.1%(52/53) macrolide-resistant clinical isolates harbored this A2063G mutation. Only one (1/53) macrolide-resistant clinical isolates harbored the A2064G mutation, and no other mutations in the 23S rRNA gene were observed. These findings coincided with the results from the antimicrobial susceptibility tests. Based on our antimicrobial susceptibility results, each of the A2063G isolates was responsible for high-level resistance to erythromycin (≥256 μg/ml), and azithromycin (2-64 μg/ml) in vitro. The susceptibility data also revealed that all M. pneumoniae isolates were susceptible to tetracycline and fluoroquinolones. Taken together, these findings suggest that these antibiotics have the potential to be used as alternatives for treating M. pneumoniae infections in adults with cases of high macrolide resistance, but this class of antibiotics is not ordinarily recommended for children, except in particular cases.
Furthermore, mutations in the L4 and L22 ribosomal proteins are related to macrolide resistance in other species [39]. In this study, we found that the C162A and A430G mutations in the L4 ribosomal protein gene and the T279C mutation in the L22 ribosomal protein gene were observed in all isolates classified as type 2 for the p1 gene, which is consistent with our previous report [22]. An intensive study using whole genome sequence analysis of 20 Chinese clinically isolated strains (unpublished data) and M. pneumoniae genomes available in NCBI database [33,40], identified the C162A, A430G, and T279C mutations in the ribosomal protein gene that were detected in all type 2 isolates. We reasoned that the three mutations in L4 and L22 were not responsible for macrolide resistance, but rather influenced the particular genotype of M. pneumoniae.
This study showed a comparatively low macrolide resistance rate of 65.4% in China, in recent 10 years [22-24, 37, 38, 41-45]. Interestingly, 95.7% (45/47) of the type 1 isolates were resistant to macrolides, whereas only 23.5% (8/34) of the type 2 isolates were resistant to macrolides in this study. To our knowledge, this interesting phenomenon has not been reported in other counties [26,46]. Due to the relatively low resistance rate of type 2 isolates, the overall rate of macrolides resistance is lower than previously reported in China [23,24,28,29,37,38,47]. In recent publication, we found that M. pneumoniae type 1 was the predominate genotype present during 2008-2012 in Beijing, and a genotype shift from type 1 to type 2 began to occur in 2013. Here, the percentage of type 2 M. pneumoniae isolates was low to19.5% (34/349) before 2012, and was 26.3% (8/41) during 2012, but raised to 39.4% (29/66) during 2013-2014 [31]. In this study, genotyping results support this earlier research findings. The percentage of type 2 M. pneumoniae isolates was 42.0% (34/81) during 2014-2016. These indicates that the genotype of M. pneumoniae isolates in Beijing is shifting from type 1 to type 2. Based on earlier work [48], which reported that M. pneumoniae genotype shifts occur every 8-10 years and that a shift from one type to another requires 2-3 years. Our publication deduced that type 2 will likely become the predominate genotype in Beiijing over the next few years [31]. However, in this research, the M. pneumoniae genotype in Beijing still did not become the absolute dominant type 2 till 2016, but entered the stage where the ratio of type 1 and type 2 was nearly closed. This is not consistent in the M. pneumoniae genotype shift cycle reported in publication [48]. Maybe the genotype shift cycle takes more longer in Beijing, and perhaps there is no very regular periodic genotype shifting about M. pneumoniae [14,26,49]. After all, M. pneumoniae genotyping research is less than 15 years in China, and requiring researchers to do longer-term monitoring and research.
Although the macrolide resistance rate of type 2 M. pneumoniae isolates is not very high in this study. Compared with the previous data, we found that the macrolide resistance rate of type 2 isolates is also increased synchronously with the increase of the proportion of type 2 isolate (Table 3). No macrolide resistance M. pneumoniae of type 2 isolates was found in 2008 in Beijing. The proportion of macrolide resistance M. pneumoniae of type 2 isolates increased from 15.6% before 2012 to 23.5% in this study. Macrolide resistance rate of type 2 M. pneumoniae increased by 50.6% within 9 years in China. In contrast, although the proportion of type 1 isolates is decreasing gradually, the macrolide resistance rate still remains above 90.0%. We deduced that this phenomenon may be related to the irregular use of antibiotics in China. The widespread overuse of antibiotics is very common in China, especially in some district hospitals. Under tremendous antibiotic selection pressure, almost all of the predominant type 1 M. pneumoniae strains isolated in China could certainly turn into macrolide-resistant isolates. Relatively speaking, the inferior type 2 M. pneumoniae strains with little chance to suffer from antibiotic selection pressure might still remain macrolide-susceptible in early years. With the increasing of the proportion of type 2 isolates in recent year, the chances of exposure to macrolide antibiotics increase greatly. Under the excessive selection of antibiotic pressure, macrolide -resistant type 2 isolates appear and spread rapidly, resulting in an increase in the macrolide -resistant rate of type 2 isolates in recent years in Beijing. The MLVA and the VNTRs in the p1 gene results both indicated that the increase of resistance in genotype 2 is not due to the spread of same clone. We will continue to monitor the status of the genotypes and macrolide resistance rates of M. pneumoniae in Beijing over the next few years to verify our hypotheses.
Our study had limitations. This was a single-center study conducted over 3 year, and only 81 M. pneumoniae isolates were studied. The statistical power may have been insufficient to assess other clinical outcomes.

Conclusions
In summary, macrolide-resistant M. pneumoniae in Beijing, China, has been present at high levels in recent years. The A2063G transition in domain V of the 23S rRNA gene was identified in all macrolide-resistant M. pneumoniae isolates, and we noted that the mutations in L4 and L22 were not responsible for macrolide resistance but rather influenced the particular genotype of M. pneumoniae. We deduced that the significant increased macrolide-resistant type 2 isolates may be due to the genotype shifting of M. pneumoniae in Beijing. The relationship between genotype shifts and macrolide resistance in M. pneumoniae needs to be further verified with more extensive surveillance data.

Availability of data and materials
The data sets analyzed during the current study are available from the corresponding author.
Authors' contributions ZF, LJR, SWX and ZJZ designed the study. ZF, LLY, HF and ZSY performed the experimental work. LJR, HF, and ZSY collected the data. ZF, LJR, SWX and ZJZ analysed the data. All authors read and approved the final manuscript.