Significance of the determination of DNA load of drug-resistant mycoplasma pneumoniae and 23sRNA gene mutation locus in children

Purpose: To determine the significance of the detection of Mycoplasma pneumoniae (MP)-DNA load and 23sRNA gene mutation locus in children with drug-resistant MP pneumonia. Methods: A total of 158 children with MP pneumonia received drug sensitivity tests. The patients were divided into resistance group and non-resistance group. The MP-DNA load index (MPLI) and mutation rate of 23sRNA gene at 2063 locus were assessed and compared between the two groups: the MPLI-negative group and the MPLI-positive group, based on whether MPLI was greater than 6.12. The association of MPLI of all the patients and the 23sRNA gene mutation at 2063 locus in the resistance group, as well as clinical indicators were analyzed. Results: The MPLI of the resistance group was lower than that of the non-resistance group. In the MPLI-positive group, the duration of disease, defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity, and length of stay were all longer than those of the MPLI-negative group, while the proportion of cases with extrapulmonary complications and the white blood cell (WBC) count were higher than those of the MPLI-negative group. The mutation rate of 23sRNA gene at 2063 locus in the resistance group was higher than that in non-resistance group (p < 0.05). The defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were longer in the mutation-positive group than those in the mutation-negative group (p < 0.05). Conclusion: The mutation rate of 23sRNA gene at 2063 locus is higher in children with drug-resistant MP pneumonia. Furthermore, low MPLI and 23sRNA gene mutations at 2063 locus are associated with the duration of disease, disappearance time of clinical symptoms and other clinical indicators.


INTRODUCTION
Mycoplasma pneumonias (MP) mainly causes respiratory tract infection, which leads to up to 40 % of community-acquired pneumonia (CAP) in all age groups, and is also the cause of regional epidemics.MP is generally susceptible to all groups, but the harm to children is more serious [1].At present, MP pneumonia is often treated with macrolide antibiotics, and the 23sRNA gene at 2063 locus has been verified to be their target [2,3].MP quantitative detection is an important indicator for diagnosing MP pneumonia and evaluating antibiotic resistance, which is mainly regulated by MP-DNA.Therefore, it is of great significance to investigate the MP-DNA load used for evaluating antibiotic resistance.In this study, the MP-DNA load and genotype of 23sRNA gene at 2063 locus in children with drug-resistant MP pneumonia were analyzed to determine their associations with the clinical characteristics of pediatric patients.

Clinical profile of patients
Children (158) with MP pneumonia treated in our hospital from December 2017 to December 2019 were selected, including 86 males and 72 females aged 2 -14 years, with a mean of (7.27 ± 1.51) years.This study was approved by the ethical committee of Affiliated Hospital 2 of Nantong University (approval no.2021KT112).The study was conducted by following the Declaration of Helsinki.

Inclusion criteria
1) Pediatric patients meeting the diagnostic criteria for MP pneumonia [4]; 2) those approved by the Ethics Committee of Children's Hospital of Soochow University [5], and 3) those aged ≤ 14 years.

Exclusion criteria
1) Pediatric patients with severe heart, liver or kidney dysfunction; 2) those accompanied by other types of lung diseases such as bronchial asthma; 3) those infected with viruses, bacteria or other pathogens; 4) those with severe immune diseases; or 5) those treated with glucocorticoids within 1 week before the start of this study.

Drug sensitivity test
Pharyngeal swab specimens were collected within 2 h after admission, added into the drugsensitive plate with MP medium in the negative wells, and cultured in an incubator at 37 °C for 24 h, followed by analysis of drug resistance.If the medium in the control well turned yellow compared with that in the negative control well, the specimens were resistant to antibiotics.In contrast, if the medium in the control well had no change in color compared with that in the negative control well, the specimens were sensitive and non-resistant to antibiotics.

DNA extraction
Pharyngeal swab specimens were rinsed with normal saline, and centrifuged using a TGL-16GB high-speed centrifuge (Shanghai Anting Scientific Instrument Factory) at 13,000 r/min for 10 min.Then the lower-layer precipitate was harvested, added with 50 μL of DNA lysis buffer, placed in a metal bath and centrifuged again.The supernatant was harvested for later use.

Determination of DNA load
The content of MP-DNA was detected by fluorescence quantitative polymerase chain reaction (PCR).PCR amplification (PE5700 gene detection system for data processing) was performed with the kit provided by Anlong Gene Technology Co. Ltd and the following conditions: pre-denaturation at 94 °C for 5 min, followed by 40 cycles of denaturation at 94 °C for 30 s, annealing at 57 °C for 30 s and extension at 72 °C for 1 min; final extension at 72 °C for 5 min.Besides, with H actin-DNA content as an internal control, PCR was conducted as follows: predenaturation at 94 °C for 4 min, followed by 40 cycles of denaturation at 93 °C for 30 s and annealing at 60 °C for 30 s.The primers and probe sequences are shown in Table 1.

Calculation of MP-DNA load index (MPLI)
MPLI was computed as in Eq 1.

Genotype detection of 23sRNA gene at 2063 locus
The 23sRNA gene amplification was carried out using the primer sequences shown in Table 1.The polymerase chain reaction (PCR) conditions were as follows: pre-denaturation at 94 °C for 5 min, followed by 35 cycles of denaturation at 94 °C for 15 s, annealing at 52 °C for 30 s and extension at 72 °C for 30 s; final extension at 72 °C for 10 min.The PCR product was stored at 4 °C and detected with 1 % agarose gel electrophoresis.Then, the single product band was recovered, purified and sequenced.The sequencing result was compared with the reference sequence in the gene bank [7], and the genotype at 2063 locus was determined: A as wild type, and C, G and T as mutant type.

MPLI data
According to the results of the drug sensitivity test, the patients were divided into a resistance group (n = 112) and a non-resistance group (n = 46).The MPLI in the resistance group (5.03 ± 1.27) was lower than that of the non-resistance group (6.03 ± 1.32); (t = 4.373, p = 0.000).

Association between MPLI and clinical indicators
In the MPLI-positive group, the duration of disease, defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were all longer than those in the MPLI-negative group, and the proportion of cases with extrapulmonary complications and the white blood cell (WBC) count were higher than those in the MPLInegative group (p < 0.05; Table 2).

Genotype of 23sRNA gene at 2063 locus in the two groups
The mutation rate of 23sRNA gene at 2063 locus in the resistance group was higher than that in the non-resistance group (p < 0.05) (Table 3).

Association between 23sRNA gene mutation at 2063 locus and clinical indicators in resistance group
The defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were longer in the mutation-positive group than in the mutationnegative group (p < 0.05; Table 4).

DISCUSSION
With the wide use of macrolides in clinical practice, the drug resistance rate has been increasing.The mechanism of MP resistance to macrolides is mainly related to the mutation of the target locus.The base point mutation in 23sRNA domain directly binding to macrolides can lead to a decrease in affinity between antibiotics and ribosomes, resulting in drug resistance [8].With the use of antibiotics, the proliferation of resistant strains is inhibited, and the DNA copy number declines, so their MPLI is different from that of the sensitive strains.In this study, the MPLI in the resistance group was lower than that in the non-resistance group, suggesting that the MP-DNA copy number in children with drug-resistant MP pneumonia is high, which is consistent with the result of the relevant study [9].It can be seen that the antibiotic resistance of MP is related to the MP-DNA load, and antibiotic resistance in child patients can be judged by detecting the MP-DNA load, and whether to give antibiotic therapy can then be determined based on the detection results.
The decrease in MPLI is primarily related to the fact that the division and proliferation of resistant strains are not restricted by therapeutic drugs during treatment.The higher the MP-DNA load, the longer the retention time of MP in the respiratory tract.As a result, persistent damage will be caused to the bronchial mucosa, and the patients become less sensitive to drugs, weakening the efficacy and delaying rehabilitation [10,11].In this study, it was found that in MPLI-positive group, the duration of disease, defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were all longer than those in MPLI-negative group, and the proportion of cases with extrapulmonary complications and the WBC count were higher than those in MPLI-negative group.The above results demonstrated that the efficacy can be judged by determining the MP-DNA load.
Resistant locus mutation is the main cause of drug-resistant MP pneumonia in children.Zhou et al. [12,13] determined the genotype of 23sRNA gene at 2063 locus in MP-resistant and MP-sensitive child patients and found that the probability of genotype C, G and T of 23sRNA gene at 2063 locus in MP-resistant group is far higher than that in MP-sensitive group.In this study, it was further confirmed that the mutation rate of 23sRNA gene at 2063 locus in resistance group was higher than that in non-resistance group.The main reason is that 23sRNA gene at 2063 locus is the target of macrolide antibiotics, and its mutation will lead to MP resistance.MP is the major pathogen causing communityacquired respiratory tract infections which frequently occur in preschool children.Recently, MP infections have been increasing, but the incidence of MP resistance has gradually emerged due to the wide use of antibiotics.MP resistance is the main cause of refractory conditions, which will affect the normal life and growth and development of children [14].MP resistance in children is mainly attributed to the locus change of 23sRNA domain of the 50S ribosomal subunit [15].Due to the nucleotide sequence change of 23sRNA domain of the 50S ribosomal subunit, the affinity between antibiotics and ribosomes is reduced, thus leading to antibiotic resistance.It has been found that 23sRNA gene mutation at 2063 locus corresponds to a higher probability of MP resistance [16].In this study, the defervescence time, disappearance time of cough and expectoration, disappearance time of chest opacity and length of stay were longer in mutation-positive group than those in mutationnegative group, suggesting that the efficacy on child patients with mutant 23sRNA gene at 2063 locus is less significant than those with wild-type gene.The main reason is that 23sRNA gene mutation at 2063 locus may lead to MP resistance, thus weakening the treatment effect.

Table 1 :
Gene primer sequences

Table 2 :
Association between MPLI and clinical indicators

Table 3 :
Genotype of 23sRNA gene at 2063 locus in the two groups [n (%)]

Table 4 :
Association between 23sRNA gene mutation at 2063 locus and clinical indicators in resistance group