Increased Constituent Ratios of Klebsiella sp., Acinetobacter sp., and Streptococcus sp. and a Decrease in Microflora Diversity May Be Indicators of Ventilator-Associated Pneumonia: A Prospective Study in the Respiratory Tracts of Neonates

Abstract

Ventilator-associated pneumonia (VAP) is a common complication and cause of death in neonates on mechanical ventilation. However, it is difficult to define the causes of VAP. To understand the causes of VAP, we undertook a prospective study based on the diversity of the microflora in VAP. The experimental group consisted of newborns who suffered from respiratory distress syndrome (RDS) and VAP, while the control group suffered from RDS without VAP. Sputa were collected within 1, 3, and 5 days of ventilation and were divided into six groups. DNA was extracted from the samples, and the 16S rDNA was PCR amplified, separated using denaturing gradient gel electrophoresis (DGGE), cloned and sequenced. The resulting sequences were compared using BLAST. The DGGE pictures were measured, and the richness, Shannon-Wiener index, and cluster maps were analyzed. No differences were found regarding the constituent ratio of any genus between the Non-VAP and VAP group within 1 day after intubation. After 1 to 3 days, the constituent ratios of Klebsiella sp., Acinetobacter sp., and Streptococcus sp. in the VAP group were higher than those in the Non-VAP group, and the ratios of Serratia sp. and Achromobacter sp. were lower. After 3 to 5 days, the ratios of Klebsiella sp., Acinetobacter sp., Serratia sp., and Achromobacter sp. were lower than those in the Non-VAP group. The richness and Shannon-Wiener index of the Non-VAP group were higher than those of the VAP group from 1 to 3 days after intubation, while no differences were found within 1 day and from 3 to 5 days. We conclude that during the first three days of intubation, the microflora diversity in the lower respiratory tract was reduced due to VAP, and the greater constituent ratios of Klebsiella sp., Acinetobacter sp., and Streptococcus sp. in the sputum may be indicators of VAP.

Microbial investigations in throat swab and tracheal aspirate specimens are beneficial to predict the corresponding endotracheal tube biofilm flora among intubated neonates with ventilator-associated pneumonia

“…A previous study has demonstrated that decreased microbial diversity may be associated with VAP among intubated neonates (21), indicating that it is an important measurement for VAP surveillance. In the present study, the findings of similar microbial diversity shared by tracheal aspirate and ET biofilm samples suggests that the microbial diversity profile of ET biofilms may be evaluated using the corresponding tracheal aspirate samples instead.…”

“…The PCR was conducted as previously described (21). The reaction included 6 µl microbial DNA as template DNA, 25 µl Premix Taq Version 2.0 (TaKaRa Biotechnology Co., Ltd., Dalian, China), 0.5 µl each primer and 18 µl sterile ddH 2 O. Template DNA of sterile ddH 2 O was regarded as the negative control.…”

“…A second PCR targeting 16S rRNA gene V3 region was conducted using primers 357f without the 'GC' clamp (5'-CCTACGGGAGGCAGCAG-3') and 518r as previously described (21). The PCR products were purified using Agarose Gel DNA Purification Kit Version 3.0 (Takara Bio, Inc., Otsu, Japan).…”

“…The purified DNA was cloned using the PMD18-T Vector system (Takara Bio, Inc.) and transformed into Escherichia coli DH5α competent cells (Takara Biotechnology Co., Ltd.). The resulting cells were cultured overnight at 37˚C on Luria-Bertani (LB) medium with ampicillin and positive clones were selected for sequencing (Biotech Corporation, Shanghai, China) as described previously (21). Sequences were assigned and identified to phylogenetic species using BLAST in NCBI Genbank database (https://blast.ncbi.nlm.…”

Determining the Possible Etiology of Hospital-Acquired Pneumonia Using a Clone Library Analysis in Japan

“…Sequence-based molecular methods are useful for bacteriological identification, and there are several reports showing the etiological data of nosocomial pneumonia patients, mostly VAP patients (Bahrani-Mougeot et al 2007;Bousbia et al 2012;Lu et al 2014). Using 16S rRNA gene sequencing, we have previously reported the microbiota in community-acquired pneumonia (CAP) (Yamasaki et al 2013), healthcare-associated pneumonia (HCAP) in bronchoalveolar lavage fluid (BALF), and bacterial pleurisy using pleural effusion (Kawanami et al 2011), and these results indicated the important roles of oral streptococci and anaerobes.…”