Impact of donor lung pathogenic bacteria detected by next-generation sequencing on early post-transplant outcomes in lung transplant recipients

Background: The effect of donor lung pathogenic bacteria on the prognosis of lung transplantation is not clear. We used the technique of next-generation sequencing (NGS) to detect the pathogenic bacteria from the lower respiratory tract and analyzed whether the colonized bacteria of donor lung affect the outcomes of lung transplantation. Methods: All patients who underwent lung transplantation from March 2018 to June 2018 at the Wuxi People's Hospital affiliated to Nanjing Medical University were included in this study. Twelve cases of donor lung were obtained, and 17 lung transplants were performed, including 12 single lung transplantation and 5 bilateral lung transplantation. The colonized bacteria in the lower lobe tissue of donor lung were detected by NGS, and the bacteria culture method was used to detect the bacteria in the airway secretion before and after the operation. The information of extracorporeal membrane oxygenation (ECMO) support time, mechanical ventilation time, intensive care unit (ICU) stay time,duration of fever and hospital length of staywere collected for prognostic analysis. Results: Compared with bacterial culture methods, the positive rate of bacteriaby using NGS in the lungs were higher (52.9% vs 41.2%). Among the patients who had detected bacteria by NGS in donor lungs before surgery, only one patient (1/9) developed the same bacteria after lung transplantation.Either NGS or bacterial culture methods, there is no association between the colonized colonization bacteria in donor lungs and the patient outcomes of immediate posttransplant period. Conclusion: The detection of bacteria by using NGS is more sensitive than normal bacterial culture. The colonized bacteria in different parts of the lung are inconsistent. There is no association between the colonized bacteria in donor lungs and short-term outcome of lung transplantation patients.

secretions at bronchoscopy in the the donor lower airways.g. The percentage of white blood cells, neutrophils, C-reactive protein, and procalcitonin increased gradually compared with the onset of disease. h. The donor's body temperature is higher than normal. i. Blood culture is positive.

Sample collection and preservation.
According to the National Lung Transplantation Data Center for donor lung selection criteria, this study obtained 12 donor lungs. 17

Sample detection
Based on High-throughput pathogenic microbial gene detection technology (Next Generation Sequencing, NGS), the nucleic acid sequence of the pathogenic microorganism in the sample was analyzed, and the microorganism was identified by comparing with the nucleic acid sequence of the existing microorganism in the database. The detection process includes: nucleic acid extraction, library construction, sequencing, information analysis, report interpretation and etc.

Tissue sample: Sample Processing and DNA Extraction
Tissue sample from patient was collected and cut into small pieces according to standard procedures.
1.5mL microcentrifuge tube with 0.7mL lysis buffer and pieces of tissue sample and 1g 0.5mm glass bead were attached to a horizontal platform on a vortex mixer and agitated vigorously at 2800-3200RPM for 30 min. 0.3mL sample was separated into a new 1.5mL microcentrifuge tube and DNA was extracted using the TIANamp Micro DNA Kit (DP316, TIANGEN BIOTECH) according to the manufacturer's recommendation.

Construction of DNA libraries
Then, DNA libraries were constructed through DNA-fragmentation end-repair, adapter-ligation and PCR amplification. Agilent 2100 was used for quality control of the DNA libraries. Quality qualified libraries were sequenced by BGISEQ-50 platform [9].

Sequencing and bioinformatic analysis
High-quality sequencing data were generated by removing low-quality, and short (length < 35bp) reads, followed by computational substraction of human host sequences mapped to the human reference genome (hg19) using Burrows-Wheeler Alignment [10].The remaining data by removal of low-complexity reads were classified by simultaneously aligning to four Microbial Genome Databases, consisting of viruses, bacteria, fungi, and parasites.
The classification reference databases were downloaded from NCBI (ftp://ftp.ncbi.nlm.nih.gov/genomes/). RefSeq contains 4,061 whole genome sequence of viral taxa, 2,473 bacteral genomes or scaffolds, 199 fungi related to human infection, and 135 parasites associated with human diseases.

Statistical analysis
Descriptive statistics were computed for the overall sample and stratified by presence of positive bacteria detected by NGS or bacterial culture positive on donor lung samples. Mean ± standard deviation (SD) or median (interquartile range (IQR) was used for describe the continuous variables.
We used t test/ANOVA or non-parametric Wilcoxon-Mann-Whitney (for continuous variables) and chisquared or Fisher's Exact test for categorical variables to compare the difference between two groups. The significance level was set at 0.05. SPSS 19.0 for Windows (SPSS Inc, Chicago, IL, USA) was used for statistical analysis. Results:

Classification of lung colonized bacteria detected by different methods.
We also analyzed the types of lung colonized bacteria which detected by NGS, or the types of bacteria cultured from lung airway secretions from donor lungs and post-transplantation lungs. We found that the proportion of bacteria detected by NGS in donor lungs is 52.9%, the proportion of bacteria detected by bacterial culture in donor lungs is 35.3%. Only 5 (29.4%) cases of all samples, the bacteria detected by NGS in donor lungs with the tissues from the lower part of the lungs were identical to the bacteria cultured in the bronchial secretions of the lungs. This shows that in most cases, the colonized bacteria of different parts of the lungs is not consistent, and it is more sensitive to use NGS to detect bacteria than Classical bacterial culture method ( Table 2).
Of the 17 patients, 9 (52.9%) patients detected bacteria by sputum culture after operation. In 5 (29.4%) cases, the bacterial detected by NGS were compared with the sputum culture results of postoperation, and only 1 (11.1%) of the patients who detected bacteria by using NGS before operation had the same postoperative bacteria after lung transplantation. This indicates that the bacteria in lung after lung transplantation are not mainly derived from colonized bacteria in the donor lung before operation, and may be more closely related to secondary infection (Table 2).
that the donors could be infected by multi-drug resistant nosocomial bacteria in two days, and spread those bacteria to recipients [13]. Most of donor organs are donated by brain-dead patients from the ICU, it is common that the donor lungs had been infected and colonized by bacteria, leading to an increase in donor-derived infections. 2013, but those bacteria were not found during donation of organs and transplantation [15].
Therefore, the screening for colonized bacteria or infected pathogens in donor lungs is particularly important.
In this study, we currently receive donor lungs in accordance with the donor lung selection criteria of National Lung Transplantation Data Center, based on medical history, chest X-ray or chest CT, fiberoptic bronchoscopy, and blood cell counter, C-reactive protein, procalcitonin and other tests to comprehensively assess the infection of the lungs, and exclude the lungs when it was infected.
However, we can still detect pathogenic microorganisms from the lungs that meet the standards for lung utilization. We define them as colonized bacteria. Traditional methods for detecting colonized bacteria include bronchial secretions or alveolar lavage fluid. In this study, we not only used bronchial There are also some shortcomings in this research. Such as the number of study cases is not enough, the follow-up time is relatively short, the effect of lung colonization on long-term prognosis of lung transplant recipients has not been observed. Further confirmation is needed for large sample sizes and long-term follow-up studies.