| Pseudomonas aeruginosa, which belongs to the genus of non-fermentative bacteria, is widely distributed in natural and clinical environments, and also one of most important opportunistic pathogens causing nosocomial infections. Due to the wide use of antibiotics, P. aeruginosa is increasingly resistant to various classes of antibiotics, especially those key antibiotics such as β-lactamases, quinolones and aminoglycosides. This has pose a real threat to the public health as it has frequently led to the clinical failure in the treatment of such resistant organisms. P. aeruginosa possessed very complex resistant mechanisms, which can be achieved through many ways. Firstly, it can show natural resistance to certain antibiotics duo to its low outer membrane permeability, chromosomally encoded AmpC enzyme and active efflux system. Secondly, it can further develop adaptive resistance through frequent genome mutations. Finally, it can rapidly develop resistance by acquiring resistance genes mediated by gene horizontal transfer elements, which could further lead to the expansion and wide dissemination of resistance. In fact, these mechanisms mention above act together, leading to resistance eventually. Therefore, the investigation of resistance evolution and dissemination is of great importance for both the control measures and the reduction of resistant strain development. Our study was focused on the imported molecular resistance mechanisms of clinical P. aeruginosa which had been performed on the resistance profiles before, aiming at illustrating the resistance evolution of P. aeruginosa. Our results may provide a good foundation for clinical therapy and infection control.Methods1. A total of260non-repetitive clinical isolates of P. aeruginosa were obtained in three hospitals from Yantai, Hefei, and Zhenjiang regions. All the strains were identified using BioMerieux company microbiological analysis instruments or API kits. Antimicrobial susceptibility testing was performed by K-B disk diffusion methods. Statistical analysis was performed by WHONET5.0software. Integrons were detected by PCR. 2. The variable region of intll and intI2genes positive isolates were further characterized by PCR-RFLP (Restriction fragments length polymorphism analysis). ISCR1genes were detected by PCR, and their variable regions were further characterized by PCR-RFLP. Complex integrons were determined by PCR mapping. The PCR products were purified and then sent to the life technology company for both strands sequencing, with parts of products cloned into pTG-18T vectors for sequencing. The genetic contents of variable regions were determined by online BLAST analysis of the sequences assembled. The transferability of integrons was confirmed by electrotransformation of extracted plasmids into competent cell.Bioinformatics analysis of the nucleotides and the deduced protein sequence of the novel blaoxA-251gene was used by a series of bioinformatics software including DNAstar, clustal1.8and mega4.0and followed by creation of phylogenetic trees. The complete CDs of blaOXA-10and blaOXA-251were cloned into pET-28a expression vectors, then transformed into BL21cell and finally selected on plates containing antibiotics. Both the wide type of BL21and the transformants were performed on the MIC testing to various antibiotics using agar dilution methods. The crude enzyme was extracted from transformants by sonication methods and the pI was determined by isoelectric focusing.Results1. The clinical isolates of P. aeruginosa in this surveillance were mainly obtained from inpatients’ sputum specimens (75%), and were distributed mostly in departments including ICU (25.4%), Respiration (24.2%), Neurology (11.5%) and so on. The resistant results showed P. aeruginosa exhibited high resistance rates to both CTX and STX(>60%), low resistance rates to IPM and AK, but full susceptibility to PB, resistance rates to other antibiotics from low to high including FEP(20.30%), TZP(21.5%), CAZ(22.6%), ATM(31.9%), TOB(31.9%), CFP(38.1%), GN(38.8%), PRL(41.9%) and CIP(49.2%). A total of123(47%) multi-drug resistant isolates were identified,89(34.2%) of which were simultaneous resistant to three key antibiotics including β-lactamase, quinolones and aminoglycosides. Class1integrons were presented in109isolates; Class2integrons were presented in4isolates; No class3 integrons were presented in any isolates. The resistance rates to antibiotics examined in intll positive isolates were significantly higher than those in intll negative isolates (P<0.01). In addition, the multi-resistance rates in intll positive isolates were also significantly higher than that in intll negative isolates.2. Among the109intll positive isolates,95were positive for the variable region amplification,9of which represented empty integrons and3represented non-specific amplification.12different resistant gene cassettes were identified, including aminoglycosides resistance genes (aadAl, aadA2, aadA3, aadA3C, aacA7, aadB, aadA13), sulfonamides resistance gene(dfrA12), chloramphenicol resistance gene(cmlA8) and β-lactamase resistance genes(blaP1, blaOXA-251, blaOXA-10,blaIMP-4). Two novel resistant gene cassettes were found, including an aadA3C gene and a blaOXA-251gene. The aacA3C gene differed from the aacA3reference gene (GenBank: M29695) by three nucleotides which lead to an Asp85Gly amino acid mutation while the blaOXA-251showed Gly128Asp, Lys137Asn and Ile187amino acid mutations compared to its nearest match blaOXA-10(GenBank:U37105). The sequence of blaOXA-251has been submitted to the web site of http://www.lahey.org/Studies, named OXA-251. A total of seven arrays formed by gene cassettes were found, including blaPl, aacA7, aadB-aadA1, blaVIM-4-blaP1, drfA12-orf-aadA2, aacA3C-aadA13-blaoxA-251and aacA3-aadA13-clmA8-blaOXA-10.Two new arrays including aadB-aadAl and aacA3C-aadA13-blaOXA-251were firstly identified in P. aeruginosa, with assigned accession number JN157817and JN118546, respectively. The aacA3C-aadA13-blaOXA-251has never been found in any species before, and thus named In713by the web site of http://integrall.bio.ua.pt. Four intI2gene positive isolates carrying the same dfrAl-satl-aadAl array were found. In addition, these isolates contained both the class1integrons and class2integrons, with the type of blaP1as well as dfrAl-sat1-aadAl coexisted. ISCR1genes were present in15isolates. PCR-RFLP analysis showed the variable regions of ISCRl were the same. DNA sequencing revealed the genetic contents of the variable region contained blaPER-1, ABC transporter, gst, and qacEΔ genes. PCR mapping identified three types of complex class1integron structures, including intl1-aacA7-qacEΔ-sul-ISCR1-blaPER-1-gst-ABC transporter-qacEΔ, intll-aacA3C-aadA13-blaOXA-251-ISCR1-blaPER-1-gst-ABC transporter-qacEΔ and intI-aacA3-aadA13-clmA8-blaOXA-10-ISCR1-blaPER-1-gst-ABC transporter-qacEΔ. The blaPl and drfA12-orf-aadA2were successfully transferred into competent E.coli.3. The complete sequence of blaOXA-251was obtained, which possessed a nucleotide of804bp coding for a protein of266aa. Results from bioinformatics analysis revealed the OXA-251was derived from the mutations of OXA-10. The transformants harbouring blaOXA-10and blaOXA-251genes were successfully constructed and the pI of7.1and7.5were also determined, respectively. The susceptibility results showed the MIC of the blaOXA-251transformants to PRL, CTX, ATM, CAZ, CFP, FEP and FOX were obviously higher than those of the blaOXA-10transformant, among which the MIC to PRL, CTX and ATM were in the resistant ranges, but the MIC to IPM and MEM had no changes.Conclusions1. Class1integrons were the most common types of integrons and were clinically relevant to bacterial resistance among clinical isolates of P. aeruginosa. Class2integrons were less common, and no class3integrons were detected in any P. aeruginosa isolats.2. Resistance to anbiotics including β-lactamases, aminoglycosides, chloramphenicol and trimethoprim can be mediated by integrons and also be horizontally transferred through plasmids.3. Novel resistant genes including aacA3C and blaOXA-251as well as novel integrons including aadB-aadAl and aacA3C-aadAI3-blaOXA-251were firstly found in P. aeruginosa, which suggested integrons had a strong adaptive capability to external enviroments through their genomic diversity and flexibility.4. The complex integron structure containing the ISCR1gene was described in P. aeruginosa, and its role in the evolution of drug resistance was also illustrated in the present study.5. blaOXA-251was successfully cloned and expressed. OXA-251was proved to be a new Ex-OXA derived from OXA-10mutations. |
