[Diversity analysis of biofilm bacteria on tracheal tubes removed from intubated neonates].
OBJECTIVE
The catheter-related infections caused by mechanical ventilation have become a intractable clinical problem, and it is related to the formation of bacterial biofilm (BF) on the surface of the implanted material. The majority of natural biofilms are formed by multiple bacterial species. However, there always only one or limited species were detected on tracheal tubes removed from intubated neonates by using traditional methods including bacterium culture and antigen detection. The aims of this study were to observe the bacterial communities diversity of BF on endotracheal tube (ETT), and discuss the difference between traditional bacterium culture methods and the use of molecular biology techniques on the basis of denatured gradient gel electrophoresis (DGGE), to provide new ideas for clinical prevention, diagnosis and treatment of bacterial infections.


METHODS
Thirty-five ETTs were obtained from 26 neonates on mechanical ventilator (from October 2012 to March 2013) in Department of Neonatology of Children's Hospital. Among the patients, 18 were boys and 8 girls, and 19 patients were < 37 weeks gestational age and 7 patients ≥ 37 weeks. DGGE profiling of 16S rDNA gene amplicons was used to assess the diversity of the bacterial population by using the software of quantity one. TA Cloning Kit and sequencing were used to investigate the distribution of bacteria and common dominant bacteria in ETT-BF.


RESULTS
The mean bands of 35 ETTs cases were 13.8 ± 5.4 from 16S rDNA PCR-DGGE, and the mean Shanon-Wiener indexes was 2.42 ± 0.38. The 16 ETTs were collected in different stages of diseases from the 7 patients. The indwelling days of 6/7 patients' ETTs increased, the Shanon-siener indexes were decreased. Among the 6 cases from different basic illnesses, and there were different Shanon-siener indexes. The result of molecular cloning and sequencing for 24 dominant bands showed that 35 cases (100%) contained Klebsiella SP·, 28 cases (80%) had Pseudomonas SP·, 27 cases (77%) had Streptococcus SP·, and 32 cases (91%) had Uncultured bacterium, while more than 2 bacterial species were found in 34 cases (97%). 28/35 (80%) Klebsiella SP· and 22/27(82%) Streptococcus SP· were accompanied by Pseudomonas SP·. There were 22 positive results of sputum culture from 26 newborns, including 10 strains (45%) of Klebsiella pneumoniae, 2 strains (9%) of Acinetobacter baumannii, Enterobacter cloacae and non-cultured bacterium in each patient (5%), but only one bacterium isolated from every sputum. Eight sputum samples had normal flora only, corresponding to the ETTs on which Klebsiella and other bacterial genuses were found.


CONCLUSIONS
The diversity of microbiota in BF on ETT was confirmed. 16S rDNA PCR-DGGE could produce a more complete picture of bacterial community than traditional bacterium culture method. Klebsiella, Pseudomonas and Streptococcus were common dominant bacteria in ETT-BF, and there might be interactions among them in the formation of BF.
Department of Neonatology, Children's Hospital of Chongqing Medical University; Ministry of Education Key Laboratory of Child Development and Disorders; Key Laboratory of Pediatrics in Chongqing; Chongqing International Science and Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China.
Chao
C
Song
Jia-lin
JL
Yu
Qing
Q
Ai
Dong
D
Liu
Wei
W
Lu
Qi
Q
Lu
Ning-ning
NN
Peng
D001419:Bacteria / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D044822:Biodiversity; D018441:Biofilms; D055499:Catheter-Related Infections / Q000382:microbiology*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D058645:Denaturing Gradient Gel Electrophoresis; D005260:Female; D006801:Humans; D007231:Infant, Newborn; D007442:Intubation, Intratracheal / Q000009:adverse effects*; D007709:Klebsiella / Q000235:genetics / Q000302:isolation & purification; D008297:Male; D016133:Polymerase Chain Reaction; D011549:Pseudomonas / Q000235:genetics / Q000302:isolation & purification; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012121:Respiration, Artificial / Q000009:adverse effects*; D013183:Sputum / Q000382:microbiology; D013291:Streptococcus / Q000235:genetics / Q000302:isolation & purification; D012122:Ventilators, Mechanical / Q000382:microbiology
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S