First Detection and Characterization of Macrolide-Resistant Mycoplasma pneumoniae from People with Community-Acquired Pneumonia in Iran

Abstract

Objectives:

Increasing macrolide resistance of Mycoplasma pneumoniae strains is becoming a public health concern worldwide. Nevertheless, no comprehensive genomic background of circulating isolates is available in our region. We aimed to study the genetic diversity of this microorganism using the multiple-locus variable-number tandem-repeat analysis method and to investigate the relationships between MLVA types and macrolide susceptibility profiles of the isolates.

Materials and Methods:

A total of 270 patients attending Tehran general university hospitals were included in this study. One throat swab was taken from each patient. M. pneumoniae was identified using culture and PCR assay. Macrolide resistance was determined using the broth microdilution method. The MLVA was performed by amplification of four variable-number tandem-repeat loci.

Results:

Of 270 specimens, M. pneumoniae was detected in 25.2% (n = 68) and 21.8% (n = 59) samples using PCR and culture, respectively. Approximately 56.9% of isolates were resistant to macrolides. Fifty-one of 59 M. pneumoniae isolates were divided into 6 distinct MLVA types.

Conclusion:

The macrolide-resistant M. pneumoniae (MRMP) rate in this study was relatively high and most of the MRMP isolates were assigned into the type 4/5/7/2. Since a significant association between MLVA type 4/5/7/2 and macrolide resistance of M. pneumoniae isolates was observed, further monitoring of genetic diversity of MRMP isolates might facilitate better understanding of epidemiology of this microorganism. Besides surveillance of the antibiotic susceptibility might be helpful to make necessary reconsiderations on guidelines for treatment of M. pneumoniae infection.

Introduction

Mycoplasma pneumoniae is the main cause of atypical community-acquired pneumonia (CAP), ∼15–40% of cases, of which ∼18% require hospitalization.¹ M. pneumoniae causes both upper and lower respiratory diseases among all age groups, with epidemic peaks every 4–7 years.² Molecular typing of M. pneumoniae isolates facilitates access to epidemiologic data, detection of outbreaks, and analyzing the transmission of infection.³

Despite the genomic homogeneity of M. pneumoniae, the molecular typing methods of this microorganism have been developed dramatically over the past 30 years.

Previous typing methods such as P1 typing and pulsed-field gel electrophoresis (PFGE) lacked enough discriminatory power for differentiation of M. pneumoniae.⁴ Later, the multiple-locus variable-number tandem-repeat analysis (MLVA) technique was established in 2009, based on five variable number tandem repeats (VNTRs) of the M. pneumoniae genome. This method greatly improved the ability to identify M. pneumoniae types.⁵

Because of the instability of Mpn1 locus through serial passages, several studies recommended the amendment of MLVA scheme.^3,6 Finally, development of the international guideline of MLVA typing of M. pneumoniae simplified interpretation and interlaboratory comparison of MLVA data is now increasingly in use for strain characterization worldwide.⁷

MLVA typing has higher discriminatory power than previous methods as PFGE and P1 typing. Besides because of the high homogeneity of M. pneumoniae genome and a low rate of horizontal DNA transfer, VNTR loci are more prone to change over time than housekeeping genes,⁵ also MLVA is a faster method, with high-throughput and low cost in comparison with sequenci-based methods. Considering the advantages of this method in comparison with others, it was selected for this study.

Macrolide resistance among M. pneumoniae strains has been rising up since 2000 worldwide, especially in Asian countries such as Japan^8,9 and China.^10,11 The prevalence now ranging between 0% and 15% in Europe and the United States, relatively 30% in Israel, and up to 90–100% in Asian countries as China, Japan, Korea, and Iraq.^12,13 This resistance is mainly because of the point mutations in the peptidyl-transferase loop in domain V of the 23S rRNA.¹⁴

Molecular typing methods such as MLVA analysis can provide epidemiological information about common genotypes among MRMP (macrolide-resistant M. pneumoniae) strains circulating in the population.

There is no adequate information on molecular epidemiology and macrolide resistance of M. pneumoniae in our region. In addition, fast and reliable diagnostic techniques are not available. Hence, this study aims to evaluate the macrolide resistance rate and molecular characterization of M. pneumoniae isolates using MLVA method, in patients attending Tehran university hospitals in Iran.

Materials and Methods

Study design and ethics statement

A cross-sectional study was conducted on outpatients suffering from respiratory tract infection signs and symptoms attending three university hospitals in Tehran, Iran, during January to December 2018. A total of 270 patients were included in this study and 1 throat swab was taken from each patient. The study protocol was approved by the medical Ethics Committee of Tehran University of Medical Sciences (IR.TUMS.SPH.REC.1397.176), and written informed consent was obtained from all the patients.

Inclusion criteria were as follows: 1.

The physician's decision according to:

- A new or progressive infiltrate on a chest X-ray

- Auscultator findings consistent with pneumonia

- Following signs and symptoms: nonproductive cough, headache, fever or hypothermia, dyspnea, sore throat, cervical adenopathy, and fatigue.

Exclusion criteria were as follows:

Hospital-acquired pneumonia

History of lung transplantation

Chronic diseases such as cystic fibrosis, cancer, tuberculosis, asthma, bronchitis.

The obtained throat swabs were placed into PPLO broth (MicroMedia, Australia), and then transferred to the Mycoplasma Laboratory, Department of Pathobiology, School of Public Health, Tehran University of Medical Sciences, for immediate processing.¹⁵

Culture

PPLO broth containing tubes were vortexed, the swab was discarded, and the contents passed through 0.45 μm filters and inoculated in sterile PPLO broth and incubated under microaerophilic conditions (5–10% CO₂) at 37°C until signs of Mycoplasma growth are observed. The growth of Mycoplasma spp. is indicated by a color change to yellow or orange, because of acid fermentation of glucose. Then, as soon as this color changed, 500 μL of the broth medium was subcultured on PPLO agar plate and incubated within 4 weeks. After 14 days of incubation, agar plates were observed using an inverted microscope, at 400 × magnification for the observation of Mycoplasma colonies. Further biochemical characterization was performed using hemadsorption and hemolysis tests.¹⁶

Molecular identification

Genomic DNA was directly extracted from specimens, using the FavorPrep™ Tissue Genomic DNA Extraction Mini Kit (Favorgen Biotech Corporation, Taiwan) according to the manufacturer's instruction. To identify the M. pneumoniae, a 450 bp fragment of the P1 gene was amplified by PCR assay as previously described.¹⁷

Molecular typing

Genomic DNA was extracted from broth culture of M. pneumoniae isolates. MLVA typing was applied to M. pneumoniae isolates using fluorescently labeled primers targeting four VNTR loci (Mpn13, Mpn14, Mpn15, and Mpn16), followed by capillary electrophoresis as described by Dégrange et al.⁵ The data were analyzed and interpreted according to the four-loci nomenclature and the international typing protocol of M. pneumoniae as previously described.^3,7 Fifty-one isolates were divided into six distinct types, other eight isolates were excluded because one or more VNTRs were not amplified.

Antibiotic susceptibility testing

Minimum inhibitory concentration (MIC) broth microdilution method was used to determine the susceptibility of isolates to erythromycin and azithromycin. The procedures were performed according to the guidelines of the Clinical and Laboratory Standards Institute (CLSI M43a). Strain M129 (ATCC 29342) was chosen as the sensitive reference strain. Ninety-six-well microdilution plate was used for the assay. Each well contained a serial dilution of the antimicrobial agent and 175 μL of the desired dilution of the bacteria inoculum (10⁴–10⁵ CFU/mL). The MIC was recorded as the lowest concentration of antimicrobial agent without a visible color change in broth medium wells because of the growth of organisms, at the time when the organism control well first shows color change to yellow.

Data analysis

Statistical analysis of demographic data was performed using Statistical Package for the Social Sciences (SPSS) (V24; IBM Corp.). Categorical variables were compared using Pearson or Fisher's exact tests as appropriate. All statistic tests were two-sided, and statistical significance was defined as p < 0.05.

The data of the calculated number of repeats were imported into Bionumerics software package (V7.6.3; Applied Maths) and a minimum spanning tree (MST) was generated based on macrolide susceptibility profile and MLVA types of isolates for visualization of the relationship between isolates.

Results

Frequency of M. pneumoniae

Of the 270 specimens (97 males, 173 females) M. pneumoniae was detected in 25.2% (n = 68) and 21.8% (n = 59) of the samples using PCR and culture, respectively. Approximately 3.33% (n = 9) of the negative culture specimens were identified by PCR. The median age was 53.9 ± 16.7 (interquartile range = 41–67). The incidence of M. pneumoniae infection did not differ significantly among the four age groups (p = 0.27). However, the frequency among elderly patients (29.5%) was higher than young adults (23.6%) and middle-aged patients (20.2%).

The clinical signs and symptoms are given in Table 1. Dyspnea and chest pain were the most prevalent symptoms among patients and only dyspnea was statistically significant for M. pneumoniae infection (p = 0.03).

Table 1.

Demographic Data and Clinical Features Among Mycoplasma pneumoniae Positive and Mycoplasma pneumoniae-Negative Patients in Tehran University Hospitals

	M. pneumoniae positive, N = 68, n (%)	M. pneumoniae negative, N = 202, n (%)	Total, N = 270, n (%)	p
Age, years
0–18	2 (2.9)	2 (1)	4 (1.5)	0.27
19–44	17 (25)	55 (27.2)	72 (26.7)
45–59	18 (26.5)	71 (35.1)	89 (33)
60<	31 (45.6)	74 (36.6)	105 (38.9)
Total	68 (100)	202 (100)	270
Sex
Female	45 (66.2)	128 (63.4)	173 (64.1)	0.77
Male	23 (33.8)	74 (36.6)	97 (35.9)	0.77
Fever	11 (16.2)	25 (12.4)	36 (13.3)	0.53
Headache	7 (10.3)	30 (14.9)	37 (13.7)	0.41
Vomit	2 (2.9)	1 (0.5)	3 (1.1)	0.15
Sore throat	1 (1.5)	13 (6.4)	14 (5.2)	0.2
Dyspnea	55 (80.9)	135 (66.8)	190 (70.4)	0.03
Chest pain	36 (52.9)	96 (47.5)	132 (48.9)	0.48
Sputum production	30 (44.1)	109 (54)	139 (51.5)	0.16
Nonproductive cough	35 (51.5)	89 (44.1)	124 (45.9)	0.32
Fatigue	19 (27.9)	67 (33.2)	86 (31.9)	0.45

Macrolide susceptibility testing

Of 51 M. pneumoniae isolates, 29 isolates (56.9%) had MICs of 32, 64, or >64 μg/mL for both macrolides tested (azithromycin and erythromycin) and categorized as macrolide resistant. The other 22 isolates had MICs of ≤0.5 μg/mL and were considered macrolide susceptible (Table 2).

Table 2.

MIC Ranges of Mycoplasma pneumoniae Isolates by Broth Microdilution Method According to Clinical and Laboratory Standards Institute M43a

Antimicrobial agent	Range tested, μg/mL	No. of strains, MIC, μg/mL									Susceptible strains, MIC ≤0.5 μg/mL, n (%)	Resistant strains, MIC ≥1 μg/mL, n (%)
Antimicrobial agent	Range tested, μg/mL	≤0.5	1	2	4	8	16	32	64	>64	Susceptible strains, MIC ≤0.5 μg/mL, n (%)	Resistant strains, MIC ≥1 μg/mL, n (%)
Azithromycin	0.5–64	22	0	0	0	0	0	3	1	25	22 (43.1)	29 (56.9)
Erythromycin	0.5–64	22	0	0	0	0	0	0	3	26	22 (43.1)	29 (56.9)

MIC, minimum inhibitory concentration.

Genotyping

The MLVA assay was applied to all 59 M. pneumoniae isolates. Fifty-one isolates were divided into six distinct MLVA types by fragment analysis of PCR products, other eight isolates were excluded because one or more VNTRs were not amplified.

MLVA types 3/5/6/2 (21/51; 41.2%) and 4/5/7/2 (18/51; 35.5%) were the two predominant types; furthermore, four other less frequent MLVA types were observed (Table 3; Fig. 1).

FIG. 1.

Minimum Spanning Tree of the results obtained by MLVA typing for the 51 Mycoplasma pneumoniae isolates. Each circle indicates a genotype. The size of the circle indicates the number of isolates with the same type. All lines linking the MLVA genotypes are thick, denoting a single locus difference between the types. MLVA, multiple-locus variable-number tandem-repeat analysis.

Table 3.

The Relationships Between MLVA Types and Macrolide Resistance

MLVA type	Total, n (%)	MRMP, N = 29	MSMP, N = 22, n (%)	p
3/5/6/2	21 (41.2)	7 (24.1)	14 (63.6)	0.09
4/5/7/2	18 (35.3)	17 (58.6)	1 (4.5)	0.00
3/5/7/2	4 (7.8)	1 (3.4)	3 (13.6)	0.30
3/6/6/2	5 (9.8)	1 (3.4)	4 (18.2)	0.15
4/5/6/2	2 (3.9)	2 (6.9)	0 (0)	0.50
4/5/7/3	1 (2)	1 (3.4)	0 (0)	1.00

Bold indicates P < 0.05.

MLVA, multiple-locus variable-number tandem-repeat analysis; MRMP, macrolide-resistant M. pneumoniae; MSMP, macrolide-susceptible M. pneumoniae.

The most prevalent type among macrolide-susceptible M. pneumoniae isolates was 3/5/6/2 (63.6%), whereas 4/5/7/2 was the most common among MRMP isolates (58.6%). The association between MLVA types and macrolide resistance was determined. The only type that had a significant association with macrolide resistance was 4/5/7/2 (p = 0.00) (Table 3; Fig. 1).

Discussion

To our knowledge, this is the first report of MLVA typing and antibiotic susceptibility testing of M. pneumoniae isolates from Iran. Such studies are too limited in the Middle East.

In this research, M. pneumoniae was detected in a quarter of the samples, which is in agreement with the recent study conducted by Arfaatabar et al. in Tehran¹⁵ and with other countries in our region, such as Saudi Arabia,¹⁸ Iraq,¹⁹ and also in other Asian countries, including China²⁰ and India.²¹

However, several studies have demonstrated that M. pneumoniae frequency varies between 1% and 10% in different parts of Iran. The lowest rate was reported by Oskooee et al.,²² following by higher rates in East Azerbaijan, Ahvaz, and Shiraz, 6.1%,²³ 8%,²⁴ and 10%,²⁵ respectively. However, these differences may be because of different sample types, transportation methods, diagnostic methods, and patient age groups.

In this study, we observed higher frequency of M. pneumoniae among elderly patients than younger adults, which might be in association with declining immune responses, as it has also been stated in other researches.²⁶ Although the overall mortality of M. pneumoniae is low,²⁷ up to 30% has been reported among the elderly people.²⁸ Therefore, rapid diagnosis and treatment of this pathogen is very important in elderly patients.

In this study, more than half (56.9%) of the isolates were resistant to macrolides. Similarly, recent reports have documented the high levels of macrolide resistance of M. pneumoniae isolates worldwide, which is reported to be up to 100% in Asian countries.¹⁴ Because atypical CAP such as M. pneumoniae infections is mostly treated empirically, antimicrobial susceptibility surveillance is necessary for current choices especially macrolides, that are the first-line antibiotics. Among the factors that can affect antimicrobial susceptibility, different geographic regions and responses to antimicrobial selection pressure are mentioned.²⁹ Antimicrobial selection pressure can lead to the clonal expansion of the resistant strains.³⁰ Thus, publication of new guidelines to treat M. pneumonia with alternative antibiotics may help to reduce the prevalence of MRMP. Because of the high macrolide resistance of M. pneumoniae isolates and teeth damage arising from tetracycline use, new guidelines were published for treatment of M. pneumoniae in Japan in 2011. Recommending respiratory fluoroquinolone/tosufloxacin as a second-line drug for use in cases where M. pneumoniae responded poorly to macrolides resulted in decreased prevalence of MRMP during 2013–2015.³¹

Molecular characterization of M. pneumoniae seemed to be impossible because of the highly homogenous nature of this microorganism, but today a variety of methods have been developed for genotyping of M. pneumoniae, and MLVA is the most widely used technique. MST modeling demonstrates the relationship of subtypes based on the number of VNTR loci that differ between MLVA genotypes. In our study, 51 M. pneumoniae isolates were classified into 6 distinct MLVA types, that the MST shows they did not differ by more than one locus. Although the spread of MLVA strains in our area was polyclonal, our findings showed that M. pneumoniae isolates circulating in Tehran are not highly heterogeneous. The two MLVA types 3/5/6/2 and 4/5/7/2 constitute the majority (76%) of all detected MLVA types, which is consistent with previous studies.^26,32,33

In our study, 94.4% (17/18) of the isolates with MLVA type 4/5/7/2 were resistant to macrolides. This finding is in concordance with previous studies. In China, high MRMP prevalence was related to increasing macrolide resistance in the MLVA type 4/5/7/2 from 25% in 2011 to 100% in 2014.³² In this study, macrolide-resistant isolates were distributed across all MLVA types but most of these isolates were assigned into the MLVA type 4/5/7/2 (58.6%), as this MLVA type represents >90% of MRMP isolates in Beijing.^34,35 In studies conducted by Dégrange et al.⁵ and Benitez et al.,⁶ 33.3% (4/12) and 90% (9/10), respectively, of MRMP isolates belonged to MLVA type 4/5/7/2. In another study in the United States, 68.4% (13/19) of MRMP isolates were MLVA type 4/5/7/2.³⁶ Therefore, high MRMP rate might have resulted from the clonal expansion of MLVA type 4/5/7/2, as stated in previous studies.

The small number of samples was one of our limitations in this study. A larger number of samples can help to have a better understanding of the epidemiology of this infection. In this study, only MLVA typing was used for molecular characterization of this microorganism. Using other typing methods in combination with MLVA might reveal more molecular characteristics of M. pneumoniae, especially MRMP isolates.

Conclusion

MRMP rate in our study is relatively high but not as high as in other Asian countries. Most of the MRMP isolates were assigned into the MLVA type 4/5/7/2. Because a significant association between MLVA type 4/5/7/2 and macrolide resistance of M. pneumoniae isolates was observed, further monitoring of genetic diversity of MRMP isolates might facilitate better comprehension of epidemiology of infection. Besides surveillance of the frequency and antibiotic resistance might be helpful to make necessary reconsiderations on guidelines for treatment of M. pneumoniae infection.

Footnotes

Acknowledgment

All authors read and approved the final version of the article.

Disclosure Statement

No competing financial interests exist.

Funding Information

This study was supported by Tehran University of Medical Sciences, Tehran, Iran (Grant No. 39778).

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