Novel Variant and Known Mutation in 23S rRNA Gene of Mycoplasma pneumoniae, Northern Vietnam, 2023.

During a 2023 outbreak of Mycoplasma pneumoniae-associated community-acquired pneumonia among children in northern Vietnam, we analyzed M. pneumoniae isolated from nasopharyngeal samples. In almost half (6 of 13) of samples tested, we found known A2063G mutations (macrolide resistance) and a novel C2353T variant on the 23S rRNA gene.

P1 gene of Mycoplasma pneumoniae isolated from 2016 to 2019 and relationship between genotyping and macrolide resistance in Hokkaido, Japan.

We characterized 515 Mycoplasma pneumoniae specimens in Hokkaido. In 2013 and 2014, the p1 gene type 1 strain, mostly macrolide-resistant, was dominant and the prevalence of macrolide resistance was over 50 %. After 2017, the p1 gene type 2 lineage, mostly macrolide-sensitive, increased and the prevalence of macrolide resistance became 31.0 % in 2017, 5.3 % in 2018 and 16.3 % in 2019.

Use of different approaches for the culture-independent typing of Mycoplasma pneumoniae from two geographically distinct regions.

We used multi-locus variable-number of tandem repeat analysis (MLVA), p1, multi-locus sequence (MLS) and single nucleotide polymorphisms (SNP) typing to characterize a collection of Mycoplasma pneumoniae strains from Cuba and Germany. Among 67 strains, 5 p1, 7 MLVA, 11 MLS, and 11 SNP types were obtained.

Clonal spread of macrolide-resistant Mycoplasma pneumoniae sequence type-3 and type-17 with recombination on non-P1 adhesin among children in Taiwan.

OBJECTIVES: Mycoplasma pneumoniae is currently the most commonly detected bacterial cause of childhood community-acquired pneumonia in several countries. Of note, clonal expansion of macrolide-resistant ST3 occurred in Japan and South Korea. An alarming surge in macrolide resistance complicates the treatment of pneumonia. We aimed to evaluate the clinical manifestation and clonal relatedness of M. pneumoniae circulating among children in Taiwan. METHODS: We prospectively enrolled 626 children with radiologically confirmed pneumonia between 2017 and 2019. An M. pneumoniae infection was suspected on clinical grounds, and tested by real-time PCR and oropharyngeal swab cultures. We used multilocus sequence typing and whole-genome sequencing to characterize the genetic features of M. pneumoniae. RESULTS: A total of 226 children with M. pneumoniae pneumonia were enrolled. Macrolide resistance was found in 77% (174/226) of patients. Multi-locus sequence typing revealed that ST3 (n = 93) and its single-locus variant ST17 (n = 84) were the predominant clones among macrolide-resistant strains. ST17 presented clinical characteristics comparable to its ancestor ST3. On multivariate analysis, macrolide resistance (OR 3.5; 95% CI 1.4-8.5; p 0.007) was independently associated with fever >72 hours after macrolide treatment. By whole-genome sequencing, prediction analysis of recombination sites revealed one recombination site in ST3 and ST17 compared with M29 (a macrolide-sensitive ST3 strain isolated from China in 2005) containing cytadhesin MgpC-like protein, RepMP4 and RepMP5. ST17 had another recombination site containing an adhesin and RepMP2/3. CONCLUSIONS: In addition to macrolide resistance, ST3 and its ST17 variant might evolve through recombination between repetitive sequences and non-P1 cytadhesins for persistent circulation in Taiwan.

Diversity in Genotype Distribution of Mycoplasma pneumoniae Obtained from Children and Adults.

The aim of this study was to explore whether there was any specific genotype responsible for the high prevalence of Mycoplasma pneumoniae infection in children. A total of 247 M. pneumoniae-DNA positive clinical specimens including 200 from children and 47 from adults, collected in Beijing, China, during the same period, were analyzed. We performed P1-restriction fragment length polymorphism analysis (RFLP), multi-locus variable number tandem repeat analysis (MLVA) and detected the macrolide resistance-associated mutations in 23S rRNA of the clinical specimens. In the present study, we observed P1 genotype 1 and MLVA type M4-5-7-2 accounted for the majority of the cases across all ages in Beijing. Macrolide resistance-associated mutants of M. pneumoniae were also at a high level with 90.5% (181/200) in children and 76.6% (36/47) in adults. However, more diverse genotypes and a higher prevalence of macrolide resistance-associated mutations were found in the pediatric specimens. Further investigations are warranted to help to explain the difference of morbidity and molecular characteristics across the demographic spectrum.

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

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.

Molecular characterization of Mycoplasma pneumoniae infections in Moscow from 2015 to 2018.

The aim of this study was to assess which Mycoplasma pneumoniae genotypes were present in Moscow during the years 2015-2018 and whether the proportion between detected genotypes changed over time. We were also interested in the presence of macrolide resistance (MR)Mycoplasma pneumoniae. We performed multilocus variable-number tandem-repeat (VNTR) analysis (MLVA), SNP typing, and mutation typing in the 23S rRNA gene from 117 M. pneumoniae clinical isolates. Our analysis suggests two major MLVA types: 4572 and 3562. In 2017-2018, MLVA type 4572 gradually became predominant. In general, the SNP type range is the same as described earlier for European countries. The analysis of MR mutations showed that 7% of the isolates had an A2063G mutation in the 23S rRNA gene with no isolates carrying an A2064G mutation. In 2017-2018, MLVA type 4572 (SNP type 1) begins to spread in Moscow, which was widespread globally, especially in Asian countries. SNP typing of our sample showed higher discriminatory power than MLVA typing.

Comparative genomics of Mycoplasma pneumoniae isolated from children with pneumonia: South Korea, 2010-2016.

BACKGROUND: Mycoplasma pneumoniae is a common cause of respiratory tract infections in children and adults. This study applied high-throughput whole genome sequencing (WGS) technologies to analyze the genomes of 30 M. pneumoniae strains isolated from children with pneumonia in South Korea during the two epidemics from 2010 to 2016 in comparison with a global collection of 48 M. pneumoniae strains which includes seven countries ranging from 1944 to 2017. RESULTS: The 30 Korean strains had approximately 40% GC content and ranged from 815,686 to 818,669 base pairs, coding for a total of 809 to 828 genes. Overall, BRIG revealed 99% to > 99% similarity among strains. The genomic similarity dropped to approximately 95% in the P1 type 2 strains when aligned to the reference M129 genome, which corresponded to the region of the p1 gene. MAUVE detected four subtype-specific insertions (three in P1 type 1 and one in P1 type 2), of which were all hypothetical proteins except one tRNA insertion in all P1 type 1 strains. The phylogenetic associations of 30 strains were generally consistent with the multilocus sequence typing results. The phylogenetic tree constructed with 78 genomes including 30 genomes from Korea formed two clusters and further divided into two sub-clusters. eBURST analysis revealed two clonal complexes according to P1 typing results showing higher diversity among P1 type 2 strains. CONCLUSIONS: The comparative whole genome approach was able to define high genetic identity, unique structural diversity, and phylogenetic associations among the 78 M. pneumoniae strains isolated worldwide.

An integrated respiratory microbial gene catalogue to better understand the microbial aetiology of Mycoplasma pneumoniae pneumonia.

BACKGROUND: The imbalanced respiratory microbiota observed in pneumonia causes high morbidity and mortality in childhood. Respiratory metagenomic analysis demands a comprehensive microbial gene catalogue, which will significantly advance our understanding of host-microorganism interactions. RESULTS: We collected 334 respiratory microbial samples from 171 healthy children and 76 children with pneumonia. The respiratory microbial gene catalogue we established comprised 2.25 million non-redundant microbial genes, covering 90.52% of prevalent genes. The major oropharyngeal microbial species found in healthy children were Prevotella and Streptococcus. In children with Mycoplasma pneumoniae pneumonia (MPP), oropharyngeal microbial diversity and associated gene numbers decreased compared with those of healthy children. The concurrence network of oropharyngeal microorganisms in patients predominantly featured Staphylococcus spp. and M. pneumoniae. Functional orthologues, which are associated with the metabolism of various lipids, membrane transport, and signal transduction, accumulated in the oropharyngeal microbiome of children with pneumonia. Several antibiotic resistance genes and virulence factor genes were identified in the genomes of M. pneumoniae and 13 other microorganisms reconstructed via metagenomic data. Although the common macrolide/ß-lactam resistance genes were not identified in the assembled M. pneumoniae genome, a single-nucleotide polymorphism (A2063G) related to macrolide resistance was identified in a 23S ribosomal RNA gene. CONCLUSIONS: The results of this study will facilitate exploration of unknown microbial components and host-microorganism interactions in studies of the respiratory microbiome. They will also yield further insights into the microbial aetiology of MPP.

Mycoplasma pneumoniae outbreak, Southeastern Finland, 2017-2018: molecular epidemiology and laboratory diagnostic lessons.

This study characterizes a large Mycoplasma pneumoniae outbreak observed in Kymenlaakso in Southeastern Finland during August 2017-January 2018. The first part of the investigation included 327 patients, who sought healthcare consultation at local GPs or hospitals due to clinical symptoms, and were tested for M. pneumoniae antibodies (Patient cohort). The second part of the investigation, conducted approximately 4 weeks after the peak of the outbreak, consisted of school screening of pupils (N = 239) in three different school buildings by PCR on respiratory specimens and questionnaires (Screening cohort). PCR positive respiratory specimens were subsequently utilized for molecular typing. The outbreak peaked in late October 2017. Of the Patient cohort, 9/106 (8.5%) respiratory specimens were PCR positive. In contrast, 3/182 (1.6%) of the Screening cohort were PCR positive. Asymptomatic carriage was observed. Multiple-locus variable-number tandem-repeat analysis (MLVA) identified two distinct MLVA types. All typed M. pneumoniae strains belonged to P1 type 1. No mutations leading to macrolide resistance were observed. In total, 61/327 (19%) of the Patient cohort had a serological indication of recent infection. The IgM test reactivity at the time of a negative PCR test result varied from a completely non-reactive value up to very strong reactivity, highlighting the difficulty in a single specimen serodiagnosis.

Evaluation of the ELITe InGenius PCR Platform for Detection of Mycoplasma pneumoniae.

Mycoplasma pneumoniae is the leading cause of bacterial community-acquired pneumonia in persons of all ages. Due to the fastidious nature of this bacterium and the necessary specialized growth media, nucleic acid amplification testing is currently the most reliable means for patient diagnostics. Analytical sensitivity, specificity, reproducibility, and clinical performance of the ELITe InGenius automated PCR platform with its MGB Alert M. pneumoniae real-time PCR research use only reagents (ELITechGroup, Inc., Bothell, WA) were compared with those of a laboratory-developed real-time PCR assay targeting repMp1 for detection of M. pneumoniae The ELITe InGenius PCR assay successfully detected 31 distinct M. pneumoniae clinical isolates and reference strains, and there was no cross-reactivity with other mollicutes, Gram-positive bacteria, or Gram-negative bacteria. In testing 223 clinical samples, the ELITe InGenius PCR showed 95.79% and 99.22% positive and negative agreement with the repMp1 assay, respectively. Additionally, the ELITech platform showed 98.91% positive and 96.95% negative predictive values, and there was no significant difference detected between the two assays (McNemar's test, P = 0.375). The ELITe InGenius PCR assay limit of detection was 0.16 CFU/PCR test or 4.16 genome copies (GCs)/test. Accuracy, instrument ease-of-use, and decreased hands-on time make the ELITe InGenius platform suitable for detection of M. pneumoniae directly from clinical specimens.

Polyclonal spread of multiple genotypes of Mycoplasma pneumoniae in semi-closed settings in Yamagata, Japan.

PURPOSE: To clarify the spread of Mycoplasma pneumoniae infections in semi-closed settings such as schools and family homes using molecular typing methods. METHODOLOGY: We retrospectively searched for school- and family-based clusters of M. pneumoniae infections based on information regarding patients from whom M. pneumoniae strains had been isolated between 2011 and 2013 in Yamagata, Japan. The molecular typing profile, including the P1 type and the four-locus (Mpn13, 14, 15 and 16) multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) type, was obtained from our previous study. RESULTS: We identified 11 school-based clusters involving 71 patients and 16 family-based clusters involving 38 patients, including 14 duplications between these types of clusters. A total of 95M. pneumoniae strains isolated from those patients were divided into 4 genotypes: 33 strains of type 4-5-7-2, 1; 31 of type 4-5-7-3, 1; 24 of type 3-5-6-2, 2c; and 7 of type 3-5-6-2, 2a. Of the 11 school-based clusters, 6 clusters (54.5%) consisted of multiple genotypes, and the remaining 5 clusters consisted of a single genotype. Moreover, the presence of multiple genotypes was identified in three classrooms of a school. On the other hand, in 14 (87.5%) of the 16 family-based clusters, the genotypes of the M. pneumoniae strains isolated from each family member were identical. CONCLUSION: The spread of M. pneumoniae infection in schools is likely polyclonal, since M. pneumoniae strains are brought into schools from various sites, such as family homes, which are important sites of disease transmission.

Molecular Typing of Mycoplasma pneumoniae Strains in Sweden from 1996 to 2017 and the Emergence of a New P1 Cytadhesin Gene, Variant 2e.

Mycoplasma pneumoniae causes respiratory infections, such as community-acquired pneumonia (CAP), with epidemics recurring every 3 to 7 years. In 2010 and 2011, many countries experienced an extraordinary epidemic peak. The cause of these recurring epidemics is not understood, but decreasing herd immunity and shifts in the strains' antigenic properties have been suggested as contributing factors. M. pneumoniae PCR-positive samples were collected between 1996 and 2017 from four neighboring counties inhabited by 12% of Sweden's population. A total of 578 isolates were characterized directly from 624 clinical samples using P1 typing by sequencing and multilocus variable number tandem repeat analysis (MLVA). A fluorescence resonance energy transfer (FRET)-PCR approach was also used to detect mutations associated with macrolide resistance in the 23S rRNA gene. Through P1 typing, the strains were classified into type 1 and type 2, as well as variants 2a, 2b, 2c, and a new variant found in nine of the strains, denoted variant 2e. Twelve MLVA types were distinguished, and 3-5-6-2 (42.4%), 4-5-7-2 (37.4%), and 3-6-6-2 (14.9%) predominated. Several P1 and MLVA types cocirculated each year, but type 2/variant 2 strains and MLVA types 3-5-6-2 and 4-5-7-2 predominated during the epidemic period comprising the peak of 2010 and 2011. In 2016 and 2017, type 1 became more common, and MLVA type 4-5-7-2 predominated. We also found that 0.2% (1/578) of the strains carried a macrolide resistance-associated mutation, indicating a very low prevalence of macrolide resistance in this region of Sweden.

Multilocus Sequence Typing of Mycoplasma pneumoniae, Japan, 2002-2016.

In Japan, Mycoplasma pneumoniae resistance to macrolides is high. To compare sequence types (STs) of susceptible and resistant isolates, we performed multilocus sequence typing for 417 isolates obtained in Japan during 2002-2016. The most prevalent ST overall was ST3, for macrolide-resistant was ST19, and for macrolide-susceptible were ST14 and ST7.

Mycoplasma pneumoniae DNA detection and specific antibody class response in patients from two tertiary care hospitals in tropical Sri Lanka.

PURPOSE: Respiratory tract infections are a major cause of global morbidity and mortality. Pneumonia is the ninth leading cause of mortality in Sri Lanka. Atypical pathogens cause about one-fifth of community-acquired pneumonia, while Mycoplasma pneumoniae accounts for about 50 %. This study aimed to determine the seroprevalence of M. pneumoniae respiratory tract infections in Sri Lanka while attempting to understand the relationships between the serology and PCR. METHODOLOGY: Paired sera from 418 adult patients (pneumonia, n=97; bronchitis, n=183; pharyngitis, n=138) and 87 healthy controls were studied. IgM, IgG and IgA antibodies were tested by M. pneumoniae enzyme-linked immunosorbent assay (ELISA). Positive IgM and or IgG seroconversion was considered to be seropositive. M. pneumoniae DNA were tested by PCR in age and gender-matched seropositives and seronegatives. RESULTS: M. pneumoniae IgG was in 14.4 % (14/97), 6.0 % (11/183) and 1.5 % (2/138) of pneumonia, bronchitis and pharyngitis patients, respectively, whilst IgM was in 6.2 % (6/97), 1.1 % (2/183) and 0 % (0/138), respectively. Amongst the pneumonia seropositives, 64.7 % (11/17) showed IgG alone, 17.5 % (3/17) showed IgM alone and 17.5 % (3/17) showed IgM and IgG. Amongst the bronchitis seropositives, 84.6 % (11/13) had IgG alone and 15.4 % (2/13) had IgM alone. In the pharyngitis seropositives, only IgG was detected 100 % (2/2). M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. In pneumonia or bronchitis patients, specific DNA was in 77.8 % (7/10) and 50 % (6/12) of patients, respectively. M. pneumoniae DNA was not found in pharyngitis patients. Of the seropositive PCR-negative pneumonia patients, 66.7 % (2/3) showed IgG alone and 33.3 % (1/3)showed IgM alone. In bronchitis patients, 83.3 % (5/6) showed IgG alone and 16.7 % (1/6) showed IgM alone. Of the seronegative PCR-positive patients, 16.7 % (2/12) had pneumonia and 18.2 % (2/11) had bronchitis. CONCLUSION: The serological evidence for M. pneumoniae infection in Sri Lanka comprised the following prevalences: 17.5 % (17/97), 7.1 % (13/183) and 1.4 % (2/138) in adults with pneumonia, bronchitis or pharyngitis, respectively. M. pneumoniae DNA was in 52.2 % (12/23) of seropositives and 15.4 % (4/26) of seronegatives. IgG was predominant in PCR positives and negatives.

Clinical characteristics of infections caused by Mycoplasma pneumoniae P1 genotypes in children.

Mycoplasma pneumoniae (M. pneumoniae) isolates can be classified into two major genetic groups, P1 type 1 (MP1) and P1 type 2 (MP2), based on the DNA sequence of the P1 adhesion protein gene. The aim of our study was to determine if M. pneumoniae P1 genotype is associated with disease manifestation and severity of acute M. pneumoniae infection. We compared epidemiological and clinical data of children infected with either MP1 or MP2. In addition, we separately analysed data of patients presenting with individual manifestations of M. pneumoniae infection. Data of 356 patients infected with MP1 were compared with those of 126 patients infected with MP2. MP2-infected children presented with higher median baseline C-reactive protein levels and were admitted to the hospital more often. The distribution of P1 genotype varied among groups of patients with different manifestations of M. pneumoniae infection. MP2 was more common than MP1 among patients with neurological and cardiovascular manifestations, whereas MP1 was more prevalent in other manifestations. The results from our large cohort indicate that the two P1 subtypes may have different pathogenic potential and that infections with MP2 strains could be more virulent than those with MP1 strains.

Re-evaluation of the etiology and clinical and radiological features of community-acquired lobar pneumonia in adults.

OBJECTIVE: The aims of this study were to elucidate the frequency and etiology of community-acquired lobar pneumonia (CALP) and the clinical and radiological differences between CALP and tuberculous lobar pneumonia (TLP). PATIENTS AND METHODS: We retrospectively reviewed medical records of patients with community-acquired pneumonia (CAP) (n = 1032) and tuberculosis (n = 1101) admitted to our hospital. RESULTS: Sixty-nine (6.7%) patients with CAP and 23 (2.1%) with pulmonary tuberculosis developed CALP. Legionella species were the most common pathogen (27 patients, 39.1%), followed by Streptococcus pneumoniae (19 patients, 27.5%) and Mycoplasma pneumoniae (18 patients, 26.1%). Symptom duration was longer in the patients with TLP than in those with CALP. On chest radiographs, cavitation in the area of lobar pneumonia and nodular shadows were radiological findings predictive of TLP. High-resolution computed tomography showed cavitation in the area of lobar pneumonia, well-defined centrilobular nodules, and tree-in-bud sign to be the radiological findings predictive of TLP by multivariate logistic regression models. CONCLUSION: Common causes of CALP are Legionella species, S. pneumoniae, and M. pneumoniae. TLP should be considered in patients with lobar pneumonia, particularly in patients with long symptom duration, cavitation, and nodular shadows on chest radiographs, and cavitation, well-defined centrilobular nodules, and tree-in-bud sign on CT.

Epidemiology and Molecular Identification and Characterization of Mycoplasma pneumoniae, South Africa, 2012-2015.

During 2012-2015, we tested respiratory specimens from patients with severe respiratory illness (SRI), patients with influenza-like illness (ILI), and controls in South Africa by real-time PCR for Mycoplasma pneumoniae, followed by culture and molecular characterization of positive samples. M. pneumoniae prevalence was 1.6% among SRI patients, 0.7% among ILI patients, and 0.2% among controls (p<0.001). Age <5 years (adjusted odd ratio 7.1; 95% CI 1.7-28.7) and HIV infection (adjusted odds ratio 23.8; 95% CI 4.1-138.2) among M. pneumonia-positive persons were associated with severe disease. The detection rate attributable to illness was 93.9% (95% CI 74.4%-98.5%) in SRI patients and 80.7% (95% CI 16.7%-95.6%) in ILI patients. The hospitalization rate was 28 cases/100,000 population. We observed the macrolide-susceptible M. pneumoniae genotype in all cases and found P1 types 1, 2, and a type 2 variant with multilocus variable number tandem repeat types 3/6/6/2, 3/5/6/2, and 4/5/7/2.

Molecular characterization of genomic DNA in mycoplasma pneumoniae strains isolated from serious mycoplasma pneumonia cases in 2016, Yunnan, China.

Mycoplasma pneumoniae (MP) is particularly prevalent in low-immunity school-age children. Few data have been reported on MP prevalence in Yunnan, China. This study was designed to investigate the prevalence and characterize genomic DNA of MP in a small outbreak in 2016, Southwest China. RepMP4 and RepMP2/3 genes of MP positive samples were amplified for molecular typing through sequence alignment and PCR-RFLP assay. Phylogenetic trees were constructed by MEGA5.0. The results showed that two distinct P1 types (type I and type II) were prevalent in this MP outbreak. Type I was the most prevalent type, and clustered in the same evolutionary branch of C26 (China, 2012). Only 1 MP isolate belonged to type II, and clustered in the branch of KCH405 (Japan, 2016). Fifty-nine nucleotide mutations were observed in P1 genes of type I isolates (51 in RepMP4, 8 in RepMP2/3). Ninety-five nucleotide mutations were observed in P1 genes of the type II isolates (33 in RepMP4, 62 in RepMP2/3). It is noteworthy that 31 mutation sites were clustered in an 84-bp fragment in the RepMP4 gene of type II isolates. One new fragment that appeared in two of the type I strains was not found in NCBI. Nucleotide diversity analyze results showed that RepMP4 was more likely to be genetically diverse than RepMP2/3. Two-tailed Z-test result of RepMP4 suggested positive selection between 6 P1 type I isolates and M29 (China, 2005). According to secondary structure prediction, 36 new possible protein binding sites were found and another 9 sites were lost, 2 helices were missed and 1 new helix appeared in type I isolates. As for type II isolates, 16 protein binding regions were gained and 31 were lost. This study may help to understand the intrinsic geographical relatedness and contributes further to the research of MP.