Bacterial strains and Clinical characteristic
Samples isolated from the feces, sputa, and blood were collected from the neonates infected with K. pneumoniae. All the sputum samples were collected from the neonates who were diagnosed with neonatal pneumonia, acute bronchopneumonia, and bronchitis. Diagnoses were made based on both clinical and radiologic findings. The strains isolated from the same patient were paired. 43 isolates of K. pneumoniae were collected from feces, sputa, and blood of 21 neonates. All the neonates were admitted to the Second Xiangya Hospital of Central South University, China, from July 2014 to April 2015. All the data of the neonates were collected by chart review from the hospital’s unified electronic database. These isolates were identified by using the BD Phoenix 100 Automated Microbiology System (BD Diagnostic Systems, MD, USA). Escherichia coli ATCC 25922 and K. pneumoniae ATCC 700603 were used as quality control strains.
Antibiotic susceptibility testing
All bacterial isolates were subjected to antibiotic sensitivity tests using the agar dilution method following the standard antibiotic susceptibility test chart from the CLSI guidelines . The results were interpreted by measuring the minimum inhibitory concentrations (MICs) which were determined as the lowest concentration of antibiotics at which the strains showed no visible growth after overnight incubation at 37oC. The isolates resistant to carbapenems were verified with the Kirby-Bauer/disk diffusion method following the CLSI guidelines [ 46].
PCR and sequencing for resistant genes
Genomic DNA from the isolates was prepared for PCR and genetic analyses using the TIAN amp Bacterial DNA Kit (Tian Gen Biotech, Beijing, Co., Ltd.). The β-lactamase antibiotic resistance genes which were prevalent in K. pneumoniae were mainly detected (including NDM-1, KPC-2, OXA-1, OXA-2, OXA-9, OXA-48, OXA-181, CTX-M-1, CTX-M-2, CTX-M-8, CTX-M-14, CTX-M-15, CMY-4, CMY-8, TEM-1, and SHV; Table 3). These resistance genes were screened through PCR assays, and the PCR products were sent to Sangon Biotech (Shanghai)Co., Ltd. for sequencing analysis. The entire sequence of each gene was compared to the sequences in the Gen-Bank nucleotide database at http://www.ncbi.nlm.nih.gov/blast/.
Multiple Locus Sequence Typing
The MLST assay was performed as previously described . Briefly, seven K. pneumoniae housekeeping genes (infB, tonB, pgi, gapA, phoE, rpoB, and mdh) were amplified and sequenced. Alleles and STs were assigned using the K. pneumoniae MLST database (http://bigsdb.web.pasteur.fr/klebsiella/klebsiella.html).
The products of the housekeeping genes were compared and analyzed by utilizing the program BLAST. To explore the phylogenetic relationship among the isolates, the seven loci (rpoB, gapA, mdh, pgi, infB, phoE, and tonB) of each isolate were concatenated and aligned using the Clustal X program. An evolutionary tree for the data set was formed by the UMPGA tree using the software MEGA X. The stability of the phylogenetic relationship was evaluated by bootstrap analysis based on 1000 replicates . The tree was drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree .
We performed PFGE analysis using Bio-Rad system . First, bacterial suspension was prepared, and then the restriction enzyme XbaI was used. Second, the electrophoretic gel was imprinted, and stained with ethidium bromide. Finally, electrophoretic images were analyzed with the software BioNumerics (Applied Maths, Inc.). A similarity coefficient >80% was selected to define a major cluster.
All data were analyzed with SPSS 19.0 statistical software. Categorical variables were evaluated by the Fisher's exact test. Values were presented as percentages of the group from which they were derived (categorical variables). A p value of <0.05 was considered statistically significant. Bio Numerics 5.10 software was used for PFGE.