A study of community-acquired Mycoplasma pneumoniae in Yantai, China

Abstract Introduction: Community-acquired pneumonia (CAP) is a global disease responsible for a large number of deaths, with significant economic impact. As diagnostic tools have increased in sensitivity, understanding of the etiology of CAP has begun to change. Mycoplasma pneumoniae is one of the major pathogens causing CAP. Macrolides and related antibiotics are first-line treatments for M. pneumoniae. Macrolide resistance has been spreading for 15 years and now occurs in worldwide. We undertook the first study on macrolide resistance of M. pneumoniae in Yantai. This may be helpful to determine the appropriate therapy for CAP in this population. Objective: To investigate the rate and mechanism of macrolide resistance in Yantai. Methods: Pharyngeal swab samples were collected from adult CAP patients. Samples were assayed by polymerase chain reaction (PCR) and cultivated to test for M. pneumoniae. Nested PCR was used to specifically amplify M. pneumoniae 23S rRNA gene fragments containing mutations, and amplicons were analyzed by CE-SSCP for macrolide resistance mutations. Results were confirmed by sequencing. Twenty-seven strains of M. pneumoniae were isolated and the activities of nine antibiotics against M. pneumoniae were tested in vitro. Results: Out of 128 samples tested, 27 were positive for M. pneumoniae. Mycoplasma 100% macrolides resistance to Mycoplasma pneumoniae. The mechanism of macrolides resistance was A2063G point mutation in the sequence directly binding to macrolides in the 23S rRNA V domain in vitro. The mean pyretolytic time for the fluoroquinolone group was 4.7 ±2.9 d, which was significantly shorter than 8.2 ±4.1 d for the azithromycin group. Conclusions: Macrolides are not the first-line treatment for M. pneumoniae respiratory tract infections in Yantai.


Introduction
In recent years, researchers worldwide have identified Mycoplasma pneumoniae as one of the most common pathogens causing adult community-acquired pneumonia (CAP). In North America, Europe, Latin America, and Asia, M. pneumoniae pneumonia (MPP) accounts for 22%, 28%, 21%, and 20% of all pneumonia cases, respectively 1 . In China, M. pneumoniae is responsible for 6.8%-38.9% of CAP etiology [2][3][4] . Although MPP symptoms are mild in most adult patients, recent research shows that the incidence of severe MPP is increasing [5][6][7][8] . Resistance of MPP to macrolide treatment is becoming a serious issue worldwide [9][10][11][12] . Therefore, it is necessary to investigate drug resistance and drug resistance mechanisms of MPP, to determine the best treatment for CAP in the local community.

Materials and Methods
Inclusion criteria Our study recruited CAP patients from the outpatient clinic and hospital admissions at Yantai Yuhuangding Hospital and its Laishan branch between January 2015 and January 2016. The inclusion criteria for this study were: 1) age >18 years; 2) meeting the diagnostic criteria for CAP issued by the American Thoracic Society and Infectious Diseases Society of America in 2007 13 ; and 3) diagnosis of M. pneumoniae infection by positive pharyngeal swab polymerase chain reaction (PCR) test, according to the British Thoracic Society CAP guidelines 14 . Patients were excluded if they: 1) presented with lung disease, such as lung abscess, aspiration pneumonia, or obstructive pneumonia; 2) were immunosuppressed, including AIDS patients, organ transplantation patients, patients taking two or more types of immunosuppressive drugs such as azathioprine, cyclosporine, and cyclophosphamide, or patients with malignant tumors who were actively receiving chemotherapy, radiotherapy, or surgical treatment; 3) were readmitted within 14 days from hospital discharge; 4) were pregnant or within 6 weeks after childbirth; 5) were previously enrolled in a similar study, were unable to provide medical history, or were unwilling to participate in this study.

Patients
One hundred and thirty CAP patients were enrolled in this study. Two patients were lost to follow-up. Among the 128 cases finally enrolled, 27 were positive for M. pneumoniae (21.1%), including 11 males and 16 females aged between 18 and 72 years (average age 36 ±5.7).

Mycoplasma pneumoniae isolation and identification
Patients' clinical information was recorded. Pharyngeal swabs were obtained from each patient for M. pneumoniae identification and isolation. Pharyngeal swabs were either inoculated within 24 h after collection into 1 mL of CM403 medium (Oxoid, UK) with Mycoplasma Supplement G (Oxoid, UK) and incubated at 37° C, or preserved at -20° C. After 6 weeks, if the liquid medium changed from red to yellow, suggesting utilization of glucose, 0.2 mL of the culture was spread onto agar medium and incubated at 37° C with 5% CO 2 for 7-14 days. A single colony was then isolated and subcultivated three times until "fried-egg" colonies, typical of M. pneumoniae, were observed. Identification of M. pneumoniae strains was performed using a molecular method. Genomic DNA was extracted from 2.0 mL of culture with single "fried-egg" colonies using the QIAamp DNA Mini Kit (QIAGEN, Germany), according to the manufacturer's instructions. The M. pneumoniae-specific 16s rRNA gene fragment was amplified from the extracted DNA by PCR with the primer sequences used by Morozumi et al 15 . The appearance of the target band on an electrophoresis gel indicated a positive M. pneumoniae infection.
Antimicrobial susceptibility of M. pneumoniae In vitro antimicrobial susceptibility tests were performed using erythromycin, azithromycin, moxifloxacin, levofloxacin, and tetracycline. The procedures and interpretation of results were conducted according to the "Methods for Antimicrobial Susceptibility Testing for Human Mycoplasma; Approved Guideline" 16 . M. pneumoniae reference strain FH (ATCC15531) was used as drug-sensitive control. Standard drugs were purchased from China Pharmaceutical Biological Products Analysis Institute.
Detection of macrolide resistance-related genes Domain V of the M. pneumoniae 23s rRNA gene was amplified by nested PCR (nPCR) of extracted DNA from clinical isolates, using previously described protocols and primer sequences 15 . The nPCR products, including the reference strain, were sequenced (Sangon Biotech Co, Ltd., Shanghai, China). DNA sequences were compared to the M. pneumoniae strain FH sequence (GenBank accession no. CP002077.1) using BLAST.
Statistical analysis Data were analyzed by a t-test using SPSS 19.0 software. p: <0.05 suggested a statistically significant difference.
This study was conducted in accordance with the declaration of Helsinki. This study was conducted with approval from the Ethics Committee of Qingdao University. Written informed consent was obtained from all participants.

Results
Antimicrobial susceptibility Eight of the 27 patients testing positive for M. pneumoniae used antimicrobial drugs 72 h prior to enrollment into the study. This included two patients using cephalosporin (7.4%), two patients using clindamycin (7.4%), three patients using macrolides (11.1%), and one patient using quinolones. All 27 strains (100%) of M. pneumoniae isolated from patients in this study were found to be resistant to macrolides. No quinolone-or tetracycline-resistant strains were identified ( Table 1).
The MICs of erythromycin and clarithromycin (64-256 mg/L) were higher than that of azithromycin (16-128 mg/L). All 27 clinical isolates were susceptible to tetracyclines (tetracycline and minocycline) and fluoroquinolones (levofloxacin, ciprofloxacin, moxifloxacin, and gatifloxacin). Comparison between the clinical efficacy of azithromycin and fluoroquinolones Among the 27 macrolide-resistant clinical strains, 9 cases were treated with azithromycin and 18 cases were treated with fluoroquinolones (10 cases with levofloxacin and 8 cases with moxifloxacin). The mean pyretolytic time for the fluoroquinolone group was 4.7 ±2.9 d, which is significantly shorter than 8.2±4.1 d for the azithromycin group (p <0.05). There was no statistical difference in the time for cough relief between the azithromycin group (13.5 ±7.4 d) and the fluoroquinolone group (8.6 ±5.1 d) ( Table 2).

Discussion
M. pneumoniae is the main cause of CAP in humans and is usually transmitted by close contact with infected patients. Studies have found that M. pneumoniae has replaced Streptococcus pneumoniae as the major CAP pathogen, accounting for 10%-30% of cases 4,17 . Due to the lack of a cell wall in M. pneumoniae and fewer contraindications, macrolides have been the first choice for the clinical treatment of MPP 18,19 .
Recently, more than 60% of macrolide-resistant M. pneumoniae strains in pediatric patients were shown to have high resistance to 14-and 15-membered ring macrolides. In contrast to pediatric patients, the prevalence of macrolide-resistant M. pneumoniae infection in adult patients in the United States and the UK is low at 0-23% 20 and 9.3% 21 , respectively. However, several Chinese studies have reported a significantly higher percentage of macrolide-resistant M. pneumoniae strains, up to 90% 22 .
In this study, we found that the prevalence of macrolideresistant M. pneumoniae was 100% in Yantai, which is significantly higher than previous studies. This may be due to a lack of previous research into antimicrobial resistance status and the abuse of macrolides in local regions. As macrolides do not require a skin test and have few side effects, they have become commonly used antibiotics at home. Our study found that 37.5% (3/8) of patients had used macrolides before seeing a doctor. We also found that macrolide prescriptions were common in outpatient services of the Pediatric Department and the Respiratory Department in our hospital.
The mechanism of macrolide-resistance has been widely explored. Macrolide resistance in M. pneumoniae was found to be highly related to mutations in domain V of the 23S rRNA gene. The A2063G and A2064G mutations were identified as the most prevalent mutations related to macrolide resistance 23 . The A2063G mutation, which often leads to high levels of macrolide resistance, can occur in Chinese populations at a frequency of more than 95% 22 . In our study, 27 macrolide-resistant M. pneumoniae strains harbored the A2063G mutation in the 23S rRNA gene. This may be one of the reasons for 100% macrolide resistance in Yantai, China.
This study has several disadvantages. Firstly, the sample size was small. Secondly, due to concerns about macrolide resistance, MPP patients were mostly treated with fluoroquinolone and therefore, we were unable to analyze the effect of macrolide-resistance on prognosis. Thirdly, in this study we defined the diagnosis of MPP as isolation of M. pneumoniae in the pharyngeal swab. However, less than a quarter of patients had their diagnosis confirmed by paired serum antibody tests.
In summary, this study found that macrolide-resistant M. pneumoniae in adult CAP in Yantai is a major problem, with a resistance rate of 100%. Rigorous supervision of the administration of macrolides is urgently needed. Further studies on the genome of M. pneumoniae could provide us with information regarding drug resistance trends. At present, fluoroquinolone and tetracycline have good antimicrobial activity against M. pneumoniae in vitro. However, in Yantai, the abusive use of fluoroquinolones in fish and poultry industries could lead to the development of resistant M. pneumoniae strains. Management of antimicrobial drugs should be further strengthened, and drug resistance should be regularly monitored to avoid or delay the development of drug resistance.

Conflicts of interest
The authors declare no conflict of interest