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\begin{document}

\title{Molecular typing of Mycoplasma pneumoniae and its correlation with
macrolide resistance in Henan of China}



\author[1]{Pengbo Guo}%
\author[1]{Yanhong Wang}%
\author[1]{Lei Liu}%
\author[2]{Xuan Zheng}%
\author[2]{LIfeng Li}%
\author[1]{Zhi Lei}%
\author[2]{Shiyue Mei}%
\author[1]{Chongchen Zhou}%
\affil[1]{Children's Hospital Affiliated of Zhengzhou University}%
\affil[2]{Henan Children's Hospital}%


\vspace{-1em}



  
  \date{May 29, 2024}


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\selectlanguage{english}
\begin{abstract}
Background/purpose To date, molecular typing studies of Mycoplasma
pneumoniae are limited in Henan. We researched the molecular types of
Mycoplasma pneumoniae in pediatric patients in Henan in 2020. Methods M.
pneumonia was detected by SAT-MP kit. The domain V of their 23S rRNA
were sequenced for detection of macrolide-resistant point mutations.
Molecular typing with multiple locus variable-number tandem repeat
analysis (MLVA) was done for both macrolide-susceptible and macrolide
-resistance M. pneumoniae samples. Results M. pneumoniae was detected in
9.8\% (121/1237) respiratory samples in 2020. Among the M.
pneumoniae-positive samples, 96\% (116/121) had macrolide-resistant
genotypes, and all of them were A2063G mutation. 105 macrolide-resistant
strains and 4 macrolide-susceptible strains fulfill MLVA typing. MLVA
4-5-7-2 was the most frequent type (60/109, 55\%), followed by 3-4-6-2
(49/109, 45\%). There was no association between MLVA types and
macrolide resistance (p \textgreater{}0.05). Conclusion The percentage
of macrolide resistance M. pneumoniae was high (96\%) in pediatric
patients in 2020 in Henan, and A2063G was the dominant point mutation.
MLVA types was independent to macrolide resistance.%
\end{abstract}%



\sloppy


\textbf{Introduction}

\emph{Mycoplasma pneumoniae} is one of the most common causes of acute
respiratory tract infection(ARTI) and community-acquired pneumonia(CAP)
especially in children. About 20\%\textasciitilde{}40\% of CAP in
pediatric~department is infected by \emph{M.pneumoniae}
.\textsuperscript{1, 2} The infection is transmitted through close
contact with infected patients, which may leads to epidemics in family
and community. Global \emph{M.pneumoniae}epidemics occur every 3 to 7
years with various incidence rates. Studies show that an epidemic has
been spreading in many countries since 2010.\textsuperscript{3-6} Upper
and lower respiratory tract infections are often mild and self-limited;
however, sometimes occasional complications in other organs may develop
to death.

\emph{M. pneumoniae} naturally lacks a cell wall as the action position,
it is resistance to \selectlanguage{greek}β-\selectlanguage{english}lactam antibiotics such as penicillin and
cephalosporin. Macrolides are usually the first pharmaceuticals to treat
children with CAP caused by \emph{M. pneumonia} . However, long time
excessive use of macrolides provided condition for
macrolide-resistant\emph{M. pneumoniae} (MRMP) strains in the world.
Since 2000,the first case of MRMP strains was reported , MRMP has spread
rapidly worldwide, especially in East Asia.\textsuperscript{7, 8}It was
reported that the resistant rates reach to about 90\% in China and
Japan. Several point mutations on the 23s RNA account for the
macrolide-resistant of \emph{M. pneumoniae} . The most common mutation
sites were A2063 andA2064 which usually induce high-level
macrolide-resistant, the mutation site at C2617 was related to low-level
macrolide-resistant.\textsuperscript{7}

For a long time, the PCR product length analysis of the P1 gene has been
the most common molecular typing method. However, this method can only
classified

\emph{M. pneumonia} strains into two groups. In 2009, D'egrange et al.
published a new method---multiple-locus variable-number tandem repeat
(VNTR) analysis (MLVA). This method remarkably increases the typing
groups for \emph{M. pneumoniae}and provides a new powerful tool for the
epidemicology of \emph{M. pneumoniae} infections\emph{.}
\textsuperscript{9}In 2011,Dumke et al. developed the MLVA
method.\textsuperscript{10} The original project was based on VNTR at
five loci on the 23s RNA. One loci named Mpn1 was finally excluded
because of the instability problem in recent proposal project.
\textsuperscript{10-12}

The aim of this study is to explore the relation between MLVA types and
macrolide-resistant of \emph{M. pneumonia} in children in Henan
province, which might provide some interesting information for studying
the emergence of MRMP in Henan province and other regions.

\textbf{Method}

\textbf{Sample collection}

This study was proceeded from January, 2020 to December, 2020 in Henan
province of China. Samples were collected from Henan Children's
Hospital, Zhengzhou, China. A total of 1237 samples were collected from
patients 14 years old or younger preliminarily confirmed with CAP on the
clinician's decision. The exclusion criteria were as follows: (a) few
cells in the specimen or insufficient amount of nucleic acid, (b)
respiratory tract infection with a precise etiological diagnosis. This
study was approved by the Ethics Committee of the Zhengzhou Children's
Hospital (Approval number:2021-K-073). Informed consent was taken from
parents or guardians.

\textbf{Total nucleic acid extraction and detection
of\emph{M.pneumoniae}}

Total nucleic acids were extracted using a throat swab extraction kit
(Health Gene Technologies, China) according to the manufacturer's
instruction.\emph{M. pneumonia} was detected by SAT-MP kit (Shanghai
Rendu Biotechnology Co, Ltd). Primers were targeted to 16s
rRNA:MP-1(5'AATTTAATACGACTCACTATAGGGAGACACCGC

TCCACATGAAATTCCAAAACTCCC3') and MP-2(5'CGGTAATACATAGGTCGCAAGC3').The
probe is FAM-5'CGGACUAUUAAUCUAGAGUGUGUCCG3'-DABCYL. The detection limit
of this assay was 1000 DNA copies/ml regarding the respiratory
specimens.

\textbf{Detection of the macrolide resistance}

If the specimen is positive for \emph{M. pneumoniae} , macrolide
resistance was detected by amplification of the 1997-2707 nucleotides
position of 23S rRNA, followed by DNA sequencing for mutation loci
analysis (A2063 G/C, A2064 G/C,C2617 G/A).

\textbf{MLVA typing}

MLVA typing was completed based on a culture-independent method by Chao
Yan et al. with slight modifications.\textsuperscript{13} Briefly, PCR
was done using primers targeting the four loci containing tandem repeat
sequence (Table 1). The PCR was completed on a Verity thermal cycler
(Thermo Fisher Scientific Inc) following this methodology: step 1, 94\selectlanguage{ngerman}°C
for 10 min; step 2, 94°C for 1min, 58°C for 1min, and 72°C for 1min and
30 s. The second step was iterated for 35 cycles. The label PCR products
were separated by capillary electrophoresis using an 3500DX Genetic
Analyzer platform (Thermo Fisher Scientific, USA),and the data were
analyzed using GeneMapper software (version 5.0;Applied Biosystems).

\textbf{Statistical analysis}

SPSS (version 22th)was used for statistical analysis. Independence test
was used for the relationship of MLVA types and macrolide-resistant
mutation. A p value of \textless{}0.05 was considered significant.

\textbf{Results}

A total of 1237 specimens from children with CAP in the year of 2020
were tested. We detected \emph{M. pneumoniae} by real-time qPCR in 121
(9.8\%) of all specimens. Fig. 1 shows the testing results in different
months of the year. The number of \emph{M. pneumoniae} -positive
specimens fluctuated from 1 to 49 in each month. Of
all\emph{M.pneumoniae} -positive strains,116 (96\%) were macrolide
resistant by detecting point-mutations in 23S rRNA. All of 23S rRNA
mutations were A2063G, one of them was heterozygous mutation.

All of positive specimens, including 116 macrolide resistant \emph{M.
pneumoniae} (MRMP) and 5 macrolide-susceptible \emph{M.
pneumoniae}(MSMP), were further analyzed by MLVA typing. However, there
was 11 MRMP and 1 MSMP samples unable to fulfill MLVA typing, and it was
probably because the levels of \emph{M.pneumoniae} nucleic acid were too
low. Finally they were excluded in analysis.

As a result, the majority (60/105, 57\%) of strains in the MRMP group
could be matched to MLVA type 4-5-7-2 by the 4-loci typing scheme,45
strains in the MRMP group were 3-4-6-2. On the other hand, all of
strains in the MSMP group were 3-4-6-2(Table 2). The independence test
showed that MLVA types had no relationship with macrolide-resistant
mutation (P\textgreater{}0.05).The distribution of MLVA types among MRMP
and MSMP is showed in Fig 2.

\textbf{Discussion}

MLVA type 4-5-7-2 was a dominant stain of \emph{M. pneumoniae} in many
countries in Asia, Europe, America .\textsuperscript{14-17} Our results
showed a similar result in Henan province of China. The relationship
between MLVA types and macrolide-resistant mutation was also shown in
our study. MLVA types have no relationship with macrolide-resistant
mutation.

The relationship between MLVA types and macrolide resistance mutation
have been reported before. In 2015, Xue et al. reported that increasing
MRMP in China was linked to the increase of \emph{M. pneumoniae} strains
typed 4-5-7-2,\textsuperscript{18} and the similar finding was also
reported in Hongkong and Japan.\textsuperscript{19, 20} However, MLVA
type 4-5-7-2 is not always related to macrolide-resistance mutation. For
example, 4-5-7-2 was also the predominant type in the United States of
America, European, Australia, and Thailand,\textsuperscript{21-24} where
the prevalence rate of MRMP was low. The percentage of MRMP among MLVA
typed 4-5-7-2 may change with time. In Japan, the percentage of A2063G
mutation in M. pneumonia strains typed 4-5-7-2 was only 0.9\% between
2004 and 2010, but increased rapidly to 83.8\% between 2011 and
2014.\textsuperscript{19}

In this study, the A2063G mutation was the primary mutation, accounting
for 100\%, no other mutations such as 2064 or 2617 were found. Beside
this, our study found mixed infection of wild strain and mutation strain
in one sample. Several mixed infections were reported as case reports
before.\textsuperscript{25-27} In these reports, patients were first
infected by the macrolide-sensitive \emph{M. pneumoniae} . After a
period of treatment (usually longer than a week), the
macrolide-resistance mutation on 23s rRNA was found. However, whether
these patients had received macrolide antibiotics before specimen
collection was difficult to confirm. So, these patients might have
developed macrolide resistance during treatment, or mixed strains might
have infected them before specimen collection. More research is needed
to do in this area.

Macrolides were the first line pharmaceutics for children with \emph{M.
pneumoniae} infection. Some mutations\selectlanguage{english}(A2063 and A2064\selectlanguage{english})are associated
with high-level resistance to macrolides,\textsuperscript{7} widespread
use of macrolides for \emph{M. pneumoniae} with these mutations is not
only useless but also dangerous to induce more MRMP. Delayed usage of
appropriate antibiotics against MRMP is responsible for poor clinical
response and prolonged clinical course.\textsuperscript{28}As a result,
severe CAP caused by MRMP will increase. To break this vicious circle,
reasonable usage of antibody is important not only in treating the
patient but also in preventing further increase of MRMP.

There was a limitation in our study. All samples were from one center in
one year. The results of this study should be interpreted cautiously due
to possible selection bias.

In conclusion, this is the first study about MLVA profile and its
correlation with macrolide resistance in pediatric patients in Henan
province. In 2020, \emph{M. pneumoniae} was responsible for 9.8\%
pediatric patients with CAP. Macrolide resistance genes was detected in
96\% strains in Henan province in 2020. All the MRMP strains carried
A2063G mutation (100\%). The most common type was 4-5-7-2 ,followed by
3-4-6-2. MRMP was independent to MLVA types.

\textbf{Funding}

This work was supported by grants from the Medical Science and
Technology Projects of Henan Province
(LHGJ20190955\selectlanguage{english},LHGJ20200638\selectlanguage{english},LHGJ20190925\selectlanguage{english},SB201903028).

\textbf{Declarations of competing interest}None.

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