Utility of Conventional Culture and MALDI-TOF MS for Identification of Microbial Communities in Bronchoalveolar Lavage Fluid in Comparison with the GS Junior Next Generation Sequencing System

BACKGROUND: Diverse microbiota exist in the lower respiratory tract. Although next generation sequencing (NGS) is the most widely used microbiome analysis technique, it is difficult to implement NGS in clinical microbiology laboratories. Therefore, we evaluated the performance of conventional culture methods together with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) in identifying microbiota in bronchoalveolar lavage (BAL) fluid. METHODS: BAL fluid samples (n=27) were obtained from patients undergoing diagnostic bronchoscopy for lung mass evaluation. Bacterial and fungal culture was performed with conventional media used in clinical microbiology laboratories. On an average, 20 isolated colonies were picked from each agar plate and identified by MALDI-TOF MS. Microbiome analysis using 16S rRNA NGS was conducted for comparison. RESULTS: Streptococcus spp. and Neisseria spp. were most frequently cultured from the BAL fluid samples. In two samples, Enterobacteriaceae grew predominantly on MacConkey agar. Actinomyces and Veillonella spp. were commonly identified anaerobes; gut bacteria, such as Lactobacillus, Bifidobacterium, and Clostridium, and fungi were also isolated. NGS revealed more diverse bacterial communities than culture, and Prevotella spp. were mainly identified solely by NGS. Some bacteria, such as Staphylococcus spp., Clostridium spp., and Bifidobacterium spp., were identified solely by culture, indicating that culture may be more sensitive for detecting certain bacteria. CONCLUSIONS: Culture and NGS of BAL fluid samples revealed common bacteria with some different microbial communities. Despite some limitations, culture combined with MALDI-TOF MS might play a complementary role in microbiome analysis using 16S rRNA NGS.

Molecular epidemiology and resistome analysis of multidrug-resistant ST11 Klebsiella pneumoniae strain containing multiple copies of extended-spectrum ß-lactamase genes using whole-genome sequencing.

The aim of this work was to investigate the mechanism responsible for multidrug resistance in ST11 Klebsiella pneumoniae YMC 2013/7/B3993 containing multiple copies of ESBL genes using multiple parallel sequencing technology. In-depth analysis of the strain revealed multiple copies of ESBL genes, 2 copies of blaSHV-12 and 1 copy of blaCTX-M-15. Furthermore, 1 copy of blaOXA-9 and 3 copies of blaTEM-1 were found. The insertion of Tn1331 was detected, which consisted of blaOXA-9, blaTEM-1, aac(6')-lb-cr, and aadA1 genes. The acquisition of multiple copies of resistance genes was due to the insertion of transposons in the bacterial genome and plasmid. The genotypic analysis revealed that the isolates belonging to ST11 showed severe resistance phenotypes and greater dissemination potential. To the best of our knowledge, this is the first report demonstrating multiple copies of same ESBL genes in K. pneumoniae ST11 isolate. Furthermore, massive parallel sequencing studies of genetic factors to enhance the fitness of this type strain would be warranted to determine whether ST11 K. pneumoniae can spread the KPC-type gene.

Molecular Epidemiology and Characterization of Carbapenemase-Producing Enterobacteriaceae Isolated at a University Hospital in Korea during 4-Year Period / 대한임상미생물학회지

BACKGROUND: Carbapenemase-producing Enterobacteriaceae (CPE) has been increasingly reported worldwide in the past 10 years, which is an important infection control concern. Since the epidemiology and characteristics of these CPEs vary according to institutes, we aimed to characterize CPEs in a university hospital during the recent 4 years. METHODS: From October 2011 to September 2015, CPE isolates from clinical specimens and hospital surveillance cultures were collected. Carbapenem resistance was confirmed by disk diffusion method and Minimal Inhibitory Concentration (MIC) was determined by agar dilution method. Carbapenemase production was tested by double disk test using aminophenylboronic acid and dipicolic acid. PCR and sequence analysis were performed to detect bla(KPC), bla(IMP-1), bla(VIM-2), bla(NDM-1)-like genes and bla(OXA-48) gene. Pulsed-field gel electrophoresis (PFGE) and Multilocus sequence typing (MLST) were conducted for KPC-producing Klebsiella pneumoniae isolates. RESULTS: Twenty-five isolates (11%) of CPE were identified among 222 carbapenem-resistant Enterobacteriacae isolates during the study period. The most prevalent CPE was KPC-producing K. pneumonia and others were IMP-1, VIM-2, NDM-1 type and OXA-48 producing CPEs. Most of these CPEs showed resistance to carbapenems with variable MICs. The sequence types (STs) of KPC-producing K. pneumoniae were ST307 and ST11. The PFGE of ST11 and ST307 showed clonality in each group suggesting the possibility of in-hospital outbreak. CONCLUSION: The prevalence of CPE has been increasing. In our institute, KPC-producing K. pneumoniae was the most frequently isolated CPE in the recent 4 years. CPE including KPC producers can easily transfer their resistance. Therefore continuous monitoring and more intensified infection control for CPE should be considered.