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We collected respiratory specimens from 128 pediatric patients diagnosed with pneumonia in Beijing in late 2023. Mycoplasma pneumoniae was detected in 77.3% (99/128) patients, with 36.4% (4/11), 82.9% (34/41), 80.3% (61/76) in children aged less than 3 years, 3-6 years, over 7 years, respectively. Mycoplasma pneumoniae ( M. pneumoniae ) was characterized using P1 gene typing, MLVA typing and sequencing of domain V of the 23S rRNA gene. P1 gene type 1 (P1-1; 76.1%, 54/71) and MLVA type 4-5-7-2 (73.7%, 73/99) were predominant. MLVA identified a new genotype: 3-4-6-2. Macrolide resistance-associated mutations were detected in 100% of samples, with A2063G accounting for 99% and A2064G for 1%. The positive rate of M. pneumoniae was higher compared to previous reports, especially in children less than 3 years, suggesting a M. pneumoniae epidemic showing a younger age trend occurred in late 2023 in Beijing, China. Higher proportions of macrolide-resistant M. pneumoniae , P1-1 and 4-5-7-2 genotype M. pneumoniae indicated increased macrolide resistance rate and genotyping shift phenomenon, which might be attributable to this epidemic. Additionally, complete clinical information from 73 M. pneumoniae pneumonia inpatients were analyzed. The incidence of severe M. pneumoniae pneumonia was 56.2% (41/73). Mycoplasma pneumoniae pneumonia patients exhibited longer duration of fever, with a median value of 10.0 days (IQR, 8.0-13.0), and higher incidence of complications (74.0%, 54/73). However, in this cohort, we found that the severity of M. pneumoniae pneumonia, co-infection, or complications were not associated with M. pneumoniae P1 gene or MLVA types. Clinicians should be aware that patients infected with macrolide-resistant M. pneumoniae exhibited more severe clinical presentations., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Chen, Jia, Gao, Ren, Du, Zhao, Feng, Xue, Cui, Gan, Feng, Fan, Fu, Xu, Yu, Yang, Zhao, Huang, Ke, Cao, Yan and Yuan.)

Mycoplasma pneumoniae is a common causative pathogen of community-acquired pneumonia. An accurate and sensitive detection method is important for evaluating disease severity and treatment efficacy. Digital droplet PCR (ddPCR) is a competent method enabling the absolute quantification of DNA copy number with high precision and sensitivity. We established ddPCR for M. pneumoniae detection, using clinical specimens for validation, and this showed excellent specificity for M. pneumoniae . The limit of detection of ddPCR was 2.9 copies/reaction, while that for real-time PCR was 10.8 copies/reaction. In total, 178 clinical samples were used to evaluate the ddPCR assay, which correctly identified and differentiated 80 positive samples, whereas the real-time PCR tested 79 samples as positive. One sample that tested negative in real-time PCR was positive in ddPCR, with a bacterial load of three copies/test. For samples that tested positive in both methods, the cycle threshold of real-time PCR was highly correlated with the copy number of ddPCR. Bacterial loads in patients with severe M. pneumoniae pneumonia were significantly higher than those in patients with general M. pneumoniae pneumonia. The ddPCR showed that bacterial loads were significantly decreased after macrolide treatment, which could have reflected the treatment efficacy. The proposed ddPCR assay was sensitive and specific for the detection of M. pneumoniae . Quantitative monitoring of bacterial load in clinical samples could help clinicians to evaluate treatment efficacy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zhao, Yan, Feng, Du, Feng, Cui, Cui, Gan, Fan, Xu, Fu, Yu, Yuan and Xue.)

Staphylococcus aureus is an opportunistic pathogen that shows a unique ability to quickly respond to a variety of antibiotics. The Crp/Fnr family transcriptional regulator ArcR controls expression of arginine deiminase pathway genes arcABDC , which enable the utilization of arginine as an energy source for cell growth under anaerobic conditions. However, ArcR shares low overall similarity with other Crp/Fnr family proteins, suggesting that they differ in the response to environmental stress. In this study, MIC and survival assays were performed to determine the role of ArcR in antibiotic resistance and tolerance. The results showed that deletion of arcR reduced tolerance of S.aureus to fluoroquinolone antibiotics, mainly through a defect in the response to oxidative stress. In ΔarcR mutant, the expression of the major catalase gene katA was downregulated, and katA overexpression restored bacterial resistance to oxidative stress and antibiotics. We showed that ArcR directly regulated katA transcription by binding to the promoter region of katA . Therefore, our results revealed the contribution of ArcR in bacterial tolerance to oxidative stress and subsequently to fluoroquinolones antibiotics. This study added our understanding on the role of Crp/Fnr family in bacterial susceptibility to antibiotics., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Fu, Fan, Li, Li, Du, Liu, Cui, Zhang, Zhao, Feng, Xue, Cui, Yan, Gan, Feng, Xu, Yu, Tian, Ding, Chen, Chen and Yuan.)

With the increasingly severe problem of bacterial resistance, colistin, as the last line of defense, has attracted attention again. Mobile colistin resistance ( mcr -1) gene is involved in the horizontal transmission of colistin resistance in Gram-negative bacteria (GNB), which is a serious threat to human health. Therefore, rapid detection of mcr -1 gene presence in clinical samples is crucial. In this study, a Recombinase-aided amplification(RAA) method for mcr -1 was successfully constructed, with sensitivity of 20 copies/reaction. In addition, amplification signal could only be detected in the strain containing mcr -1 gene among 14 different bacterial species. The method was then used to test a total of 672 clinical samples from a pediatric hospital in Beijing. Five strains harbored mcr -1 genes were isolated from mcr -1-positive clinical samples and identified as Escherichia coli . Multi-locus sequence typing (MLST) analysis showed that the five E. coli belonged to different ST types. Notably, the mcr -1 gene from the isolates could be transferred conjugately to the recipient strain E. coli J53, with highest transfer efficiency up to 57-58%, suggesting that the mcr -1 gene was located on the plasmid. These findings showed that the RAA assay has potential to be a rapid and sensitive mcr -1 gene screening test for clinical samples, and mcr -1 could be transmitted vertically and horizontally between and within bacterial species in a plasmid-mediated manner., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fan, Feng, Xu, Feng, Yan, Fu, Zhao, Cui, Gan, Liu, Du, Zhang, Xu, Li, Xue and Yuan.)

Streptococcus pneumoniae ( S. pneumoniae ) is a common major human pathogen associated with community-acquired pneumonia, septicemia, meningitis, and otitis media. It is difficult to isolate and identify S. pneumoniae form clinical samples. To evaluate a novel, rapid, sensitive, and specific loop-mediated isothermal amplification (LAMP) assay to detect S. pneumoniae pneumonia in children, we designed specific LAMP primers targeting lytA and psaA genes. We optimized the reaction time and reaction system, and evaluated its sensitivity and specificity of detection using real-time turbidity monitoring and visual observation. We also analyzed the molecular characteristics of the isolates obtained from the positive samples. The primer sets LytA-1 and PsaA-2 amplified the genes in the shortest times, and 63°C was confirmed as the optimum reaction temperature. The detection sensitivity of each reaction was 10 and 100 copies/μL with primer sets LytA-1 and PsaA-2, respectively. This LAMP assay showed no cross-reactivity with other 27 pathogens. To describe the availability of this method, we collected 748 clinical samples from children with pneumonia. Among them, 135 were confirmed to be S. pneumoniae positive by LAMP. The sensitivity was 100% (95% CI 96.4-100%), specificity 99.0% (95% CI 97.8-99.6%). Including them, 50 were co-infected with Mycoplasma pneumoniae . This LAMP assay detected S. pneumoniae in 1 h and the results can be identified with visual naked eyes. Thus, it will be a powerful tool for S. pneumoniae early diagnosis and effective antibiotic therapy., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Du, Yan, Du, Zhao, Xue, Zheng, Feng, Cui, Gan, Feng, Fan, Xu, Fu, Fu, Zhang, Li, Zhang, Liu, Li, Cui, Zhou, Zhang, Chen and Yuan.)

New Delhi metallo-β-lactamase, a metallo-β-lactamase carbapenemase type, mediates resistance to most β-lactam antibiotics including penicillins, cephalosporins, and carbapenems. Therefore, it is important to detect bla NDM genes in children's clinical samples as quickly as possible and analyze their characteristics. Here, a recombinase-aided amplification (RAA) assay, which operates in a single one-step reaction tube at 39°C in 5-15 min, was established to target bla NDM genes in children's clinical samples. The analytical sensitivity of the RAA assay was 20 copies, and the various bacterial types without bla NDM genes did not amplify. This method was used to detect bla NDM genes in 112 children's stool samples, 10 of which were tested positive by both RAA and standard PCR. To further investigate the characteristics of carbapenem-resistant bacteria carrying bla NDM in children, 15 carbapenem-resistant bacteria ( Escherichia coli , Klebsiella pneumoniae , Acinetobacter baumannii , Citrobacter freundii , Klebsiella oxytoca , Acinetobacter junii , and Proteus mirabilis ) were isolated from the 10 samples. Notably, more than one bacterial type was isolated from three samples. Most of these isolates were resistant to cephalosporins, cefoperazone-sulbactam, piperacillin-tazobactam, ticarcillin-clavulanic acid, aztreonam, co-trimoxazole, and carbapenems. bla NDM - 1 and bla NDM - 5 were the two main types in these samples. These data show that the RAA assay has potential to be a sensitive and rapid bla NDM gene screening test for clinical samples. The common existence of bla NDM and multi-drug resistance genes presents major challenges for pediatric treatment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Feng, Xue, Feng, Yan, Cui, Gan, Zhang, Zhao, Xu, Li, Liu, Du, Zhang, Yao, Tai, Ma, Zhang, Qu, Wei and Yuan.)