| The activity of New Delhi metallo-β-lactamase(NDM) is a major mechanism of resistance of Gram-negative bacteria to β-lactam antibiotics including the carbapenems. blaNDM-1 have been reported in many kinds of Gram-negative bacilli,especially in the Enterobacteriaceae and Acinetobacter spp, which could threaten the human health seriously. To explore the transmission mode of NDM-1 is of great significance to the prevention and control of prevalence of NDM-1. The epidemiological surveys as well as the genetic feature study indicate that mobile DNA elements,including transposon and plasmid play an important role in the transmission of NDM-1. Conjugation is one of the important ways to spread resistance genes among bacteria. To explore the transferability of NDM-1 among different species of bacteria through plasmid conjugation is crucially important for revealing the transmission way of NDM-1.Both Acinetobacter lwoffii(Iz4b) and Acinetobacter calcoaceticus(XM1570)carried NDM-1 plasmids, designated as pNDM-Iz4 b and pXM1. Sequencing and analysis of the plasmids have been completed. In order to investigate the patterns of the interspecific transmission of NDM-1 plasmid, several experiments were conducted as followings in this study.A. lwoffii(Iz4b) from a pet cat and A. Calcoaceticus(XM1570) from a patient were used as donor strains and different species of enterobacteriaceae bacteria were used as recipient strains. Conjugation experiment was conducted to explore the cross-species transferability of NDM-1 mediated by plasmids. Besides, the antibiotic sensitivity, growth property and genetic stabiliy of conjugants were characterized. The results indicated that these two NDM-1 plasmids were able to transfer to E. coli DH5α,but failed to transfer to other attenuated or reference strains directly. However,pNDM-Iz4 b couldn’t be stably inherited in DH5α. A relatively stable conjugants was acquired through successive passage in LB plate supplemented with imipenem. In contrast, pXM1 was relatively stable in DH5α. Then the NDM-1 plasmids were transferred to other strains(Salmonella choleraesuis C500, Salmonella typhi ATCC14028, Salmonella paratyphi A CMCC50093, Enterobacter cloacae ATCC13047) mediated by DH5α comjugant. The transfered NDM-1 plasmid endowed the host strains with resistant phenotype to many kinds of β-lactam antibiotics, and didn’t influence the growth properties of the host strains. In addtion,to explore the underlying genetic reasons for stability of pNDM-Iz4 b, gene encoding areas accounting for the replication and transfer of the plasmid were studied by walking PCR and sequencing. Sequence analysis showed that campared with original sequence, no mutations were detected in NDM-1 plasmid in unstable and stable DH5αconjugants, as well as in Salmonella choleraesuis C500 conjugants, indicating that the stability of pNDM-Iz4 b may be closely related with the adaptability of the host strains rather than the sequence mutations in NDM-1 plasmid.In order to explore the transferability of NDM-1 plasmid among Acinetobacter spp., further conjugation experiments were conducted by using different species of Acinetobacter strains isolated from different sources as recipient strains and A. lwoffii(Iz4b) as doner strain. The results showed that NDM-1 plasmid in A. lwoffii was able to cross the genetic boundaries and transfer to Acinetobacter baumannii,Acinetobacter calcoaceticus(plasmid-cured strain of XM1570) from patient and Acinetobacter baumannii/ calcoaceticus complex from pet dog. Further study of conjugants showed that NDM-1 plasmids were stably inherited in new hosts, and endowed them with resistance phenotype to most β-lactam antibiotics. The MIC of conjugants to imipenem reached up to 256μg/mL. In addtion, imipenem-added medium combined with a higher culturing temperature(41℃) were used for the first time to select suspected conjugants in our study.The effect of nano-materials on the conjugation efficiency of NDM-1 plasmid was explored. Klebsiella pneumoniae ATCC BAA-2146 was used as doner strain and E. coli J53 as recipient strain, the conjugation efficiency was calculated and compared when different concentration of nano-Al2O3(0.05/0.5/5/50 mmo L/L) were added into PBS or LB medium. The results indicated that nano-Al2O3 could promote the conjugation of NDM-1 plasmid, the conjugation efficiency was has improved about 3times with 50 mmo L/L nano-Al2O3.Taken together, the results indicated that:(1) NDM-1 plasmids were able to cross the genetic boundaries and transfer to other species of Enterobacteriaceae mediated by DH5α, but failed to transfer directly. Salmonella strains were more likely to receive NDM-1 plasmid compared with other Enterobacteriaceae strains.(2) NDM-1 plasmid in Acinetobacter lwoffii could transfer to other species of Acinetobacter spp. and was stably inherited in new host strains.(3) Nano- Al2O3 could increase the conjugation efficiency of NDM-1 plasmid. Given that nano materials were widely used in industrial manufacture, nano-materials residual in the environment could promote thewide distribution of NDM-1. This study preliminarily revealed the characteristics of horizontal cross-species transmission of NDM-1 by conjugation, which laid theoretical foundation for the prevention and control of NDM-1 transmission. |
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