Conceived and designed the experiments: Contributed to the writing of the manuscript: The authors confirm that all data underlying the findings are fully available without restriction. All relevant data are within the paper and its Supporting Information files. Due to the limited information of the contribution of various antibiotic resistance mechanisms in clinical Burkholderia cepacia complex isolates, Antibiotic resistance mechanisms, including integron analysis, identification of quinolone resistance-determining region mutations, measurement of efflux pump activity, and sequence analysis of efflux pump regulators, were investigated in 66 clinical B.
Clonal relatedness determined by PFGE analysis revealed 30 pulsotypes, including two major pulsotypes that comprised Among six levofloxacin-resistant isolates, five had single-base substitutions in the gyrA gene and three demonstrated efflux pump activities.
Among the 42 isolates exhibiting resistance to at least one antimicrobial agent, Quantitation of efflux pump RNA level and sequence analysis revealed that over-expression of the RND-3 efflux pump was attributable to specific mutations in the RND-3 efflux pump regulator gene. In conclusion, high-level expression of
Cheung po tsai homosexual discrimination pumps is prevalent in B. Mutations in the RND-3 efflux pump regulator gene are the major cause of efflux pump activity, resulting in the resistance to antibiotics in clinical B.
The Burkholderia cepacia complex is comprised of Gram-negative, non-fermenting bacilli that are commonly found in natural and hospital environments . The difficulty in treating B. The causes of multiple antibiotic resistance in the B.
Because the contribution of various antibiotic resistance mechanisms in clinical B.
We also differentiated the B. A total of 66 B. Antibiotic resistant mechanisms of bacterial isolates were analysed in this study whereas the human specimens or patient's information were not included.
The study subjects were bacterial isolates and the informed consents were waived. The genomovar typing of B. Sample preparation and crystallisation were performed as described by Degand et al. Antimicrobial susceptibility testing was performed by the standard broth microdilution method according to the guidelines of the Clinical and Laboratory Standards Institute CLSI .
The following antimicrobial agents were tested: To evaluate the Cheung po tsai homosexual discrimination pump activity of B. Louis, MO by the broth microdilution method . The gels were then stained with ethidium bromide and photographed under UV light. Dice Cheung po tsai homosexual discrimination indices were employed to construct a dendrogram of pulsotype relationships via the unweighted pair group method using arithmetic averages UPGMA with BioNumerics software version 6.
PCR assays were performed to amplify the full sequences of the class 1 and 2 integron cassettes int 1 and int 2 Table 1 .
Primers used for amplification of the sequences are listed in Table 1 . Reactions were carried out in a ABI machine following the manufacturer's protocol.
The primers are listed in Table 1 . The non-induced strain No. The BCAM gene, which was stably expressed under antibiotics, was evaluated as an internal control .
Categorical variables Cheung po tsai homosexual discrimination analysed with the Chi-square test. The Mann-Whitney U test was used to determine if there were differences between isolates in terms of the expression level of efflux pumps, both for isolates that did and did not exhibit resistance to antimicrobial agents.
In addition, four B. Except for ceftazidime, no difference in the other antimicrobial resistance rates was observed between isolates of the two major pulsotypes Table S2.
Antimicrobial susceptibility results are presented in Table 2 and the MIC data for each isolate was supplied in file S1. The antimicrobial susceptible rates for the tested antimicrobial agents of three species are revealed in Table S1. No significant differences in those rates between species are found. Class 1 integron was identified in 18 B. Six isolates exhibited the In0 structure GenBank accession number: Two isolates with integron containing catB3 gene, which encodes chloramphenicol acetyltransferase were resistant to chloramphenicol.
The result revealed integron's role in the resistance to sulfamethoxazole, chloramphenicol, and aminoglycoside.
Genes are shown as arrows with the direction of transcription indicated by the arrowheads. The proportions of strains exhibiting efflux pump activity for various antimicrobial agents are revealed in Fig. The presence of efflux pump activity was significantly correlated with resistance to any of the following antimicrobial agents: Resistant mechanism of levofloxacin has been known via accumulation of mutations in the QRDR of topoisomerase genes and efflux pump activation .
Among the 66 B. To understand whether these efflux pumps were expressed in clinical isolates, some representative isolates with efflux pump activity no. Based on previous studies that determined that the over-expression of multidrug resistance efflux pumps was usually Cheung po tsai homosexual discrimination with mutations in regulator genes in clinical Pseudomonas aeruginosa isolates two regulator genes RND-3, and RND-9 including their promoter regions were sequenced in the five B.
The regulators of RND-9 demonstrated no amino acid changes when compared to B. For three of isolates with efflux pump activity no. The deletion of a guanine nucleotide at position could result in the translational frameshifting in B.
The five nucleotide deletions from position to caused protein frameshifting.