| Acinetobacter baumannii is an important opportunistic pathogen which can survive in medical equipments and inside human body. It has a high mortality rate by causing nosocomial infection which are responsible for various infectious diseases. It also has high infection rate among wounded soldiers. In recent years, due to the high prevalence of multidrug-resistant(MDR) and extensively drug-resistant(XDR) isolates, A. baumannii has been identified as one of the top seven pathogens threatening our healthcare-delivery system by the Infectious Diseases Society of America. In China, clinical isolation rate of A. baumannii is listed on the top 5. The resistance situation is also increasingly serious, which has brought great challenge to the prevention and treatment of related infection. Close monitoring and analysis of drug resistance and genetic polymorphism of A. buamannii would help to provide more targeted treatment strategy, and improve the efficiency of treatment.In addition to the strong drug resistance, A. baumannii is also one of the most common bacterial causes of biofilm-related contamination of medical devices. Biofilms are assemblages of microorganisms encased in a matrix that function as a cooperative consortium. Biofilm-specific resistance has been reported to be significantly higher than antibiotic resistance of planktonic bacteria. Also, bacteria in biofilm could get a long-term survival and responsible for chronic infections. Therefore biofilm-related infections are more threatening to our health. Since biofilm is the main exist mode for bacteria both in vivo and in vitro, biofilm-specific resistance is more important in practice. Biofilm-specific resistance was reported to be 10-1000 times higher than antibiotic resistance, greatly enhancing the difficulty in treatment. Biofilm formation and drug resistance are two important abilities for bacteria to survive, hence investigation of the relationship between these two factors, and clear the strength and influencing factors of biofilm-specific resistance would greatly help understanding biofilm-related infections and exploring more effective ways in prevention and treatment, which is now the burning issue in practice. These problems though have been involved in several researches, are still inconclusive. Based on the above issues, this study mainly carried out the following research.In the first part, we evaluated antibiotic resistance and genetic polymorphism of 272 A. baumannii isolated from Beijing and Xiamen during 2010-2013. The antibiotic resistance profiles to 21 recommended antibiotics in nine antimicrobial categories showed the resistance situation was quite serious. Resistant rate to cephems was the highest(98.2%), followed by penicillins(90.8%), tetracyclines(68.4%), fluoroquinolones(66.9%), penicillins + β-lactamase inhibitors(65.4%), aminoglycosides(62.1%), carbopenems(59.9%), folate pathway inhibitors(59.9%) and lipopeptides(3.7%). Among the 272 isolates, 72.4% were either MDR or XDR. Isolates from Beijing have higher resistance rates to most antibiotics except for levofloxacin and polymyxin B than isolates from Xiamen.PFGE analysis indicated a high genetic diversity among the A. baumannii strains. Meanwhile MDR and XDR isolates built up the 8 predominant clusters, some of which have been prevalent in both Beijing and Xiamen. We also found that the resistance rate of the isolates in the predominant clusters were relatively low to one or several of five antibiotics including amikacin, tobramycin, minocycline, levofloxacin, and piperacillin/tazobactam, suggesting that we could select more targeted antibiotics in infection treatment. MLST analysis revealed that the majority of main STs were MDR and XDR isolates. We found 55 isolates belong to 53 new STs, most of which were non-MDR isolates, indicating a higher mutation rate among non-MDR isolates.In the second part, we evaluated the biofilm forming capacity of all the isolates, and investigated the resistance before and after biofilm formation. Then we analysed the relationship between antibiotic resistance, biofilm formation and biofilm-specific resistance. Biofilm forming capacity was evaluated using the crystal violet staining method. The results were also verified by confocal laser scanning microscopy. There was a great difference in biofilm forming capacities between the isolates. The OD550 values ranged from 0.078 ± 0.003 to 2.556 ± 0.137. Additionally,249(91%) isolates exhibited positive biofilm formation, and 63(23%) were stronger biofilm formers than type strain ATCC19606. Since no significant difference in growth rate was observed between isolates with strong and weak biofilm forming capacity, we believe that the great difference in biofilm formation was not due to the difference in growth rate. A negative correlation was revealed between biofilm forming capacity and antibiotic resistance, which means isolates with higher level of resistance often form weaker biofilm while non-MDR isolates tended to form stronger biofilms. This result indicated a complementary relationship between biofilm formation and antibiotic resistance, the two ways that promote A. baumannii survival.Then we quantitatively measured the minimal inhibitory concentration(MIC) and minimal biofilm eliminating capacity(MBEC) of 31 isolates to cefotaxime, imipenem and ciprofloxacin before and after biofilm formation. The isolates were selected using a systematic sampling method according to their biofilm formation capacities. Results showed that the enhancement in resistance to the three antibiotics differed significantly from each other. For cefotaxime, MIC ranged from 0.5-1,024 μg/m L, MBEC from 256-524,288 μg/m L, so the enhancement could reach 8-2048 times. For imipenem, MIC ranged from 0.25-128 μg/m L, MBEC from 8-4,096 μg/m L, so the resistance could enhance 32-512 times. As for ciprofloxacin, MIC ranged from 0.25-256 μg/m L, MBEC from 8-8,192 μg/m L, so the resistance could enhance 16-512 times. No significant correlation was found between the enhancement and biofilm biomass. For each of the antibiotics, biofilm-specific resistance(MBEC) was positively related with antibiotic resistance(MIC). This means that for MDR and PDR isolates, even though their biofilms are always weak, they could still get a great enhancement in resistance once formed into biofilms. This could greatly increased the difficulty of anti-infection treatment.Based on the above findings, in the third part, we preliminarily explored the mechanisms from five aspects: biofilm-formation-related genes, plasmid profiles, CRISPR, persisters, and transcriptome analysis. The detection of seven previously reported genes related to biofilm formation(including bap, bfs, aba I, bfm R, bfm S, csu A, and csu AB) revealed that three of them had a regular distribution pattern among strains with different antibiotic resistance phenotypes. Strains with higher level of resistance were more likely harbouring bap, while bfm R and bfm S were more likely detected in more susceptible strains. Previous studies have reported that Bap encoded by bap gene could improve the level of biofilm-specific resistance which could explain that even though MDR and PDR isolates only formed weak biofilms, they could still get a great enhancement in resistance. As for bfm R and bfm S, their distribution pattern was consistent with our finding that there’s a negative correlation between biofilm formation and antibiotic resistance. Also, bfm S was reported to be related with imipenem and ciprofloxacin resistance. Our results further supported that biofilm related genes are of great possibility to be involved in the process of antibiotic resistance.Plasmids were detected in 255 of the 272 isolates. The isolates harboured one to seven plasmids and the sizes ranged from 5.28 kbp to 207.6kbp. Plasmid profile analysis revealed that resistant isolates often harbored more plasmids, while there’s no obvious relationship between biofilm forming capacity and plasmid profiles. Although plasmids were reported to be involved in both the antibiotic resistance and biofilm formation, we failed to explain the relationship between these two factors only by the plasmid profile. Further sequence analyses are required.CRISPR system detection found that only 14 of the 272 isolates harboured a complete CRISPR structure, and there was a large fragment replacement in another isolate. The biofilms formed by strains harbouring CRISPR were significantly stronger than those of CRISPR negative strains. Moreover, the cas1 and cas3 genes were upregulated in biofilm, suggesting that the CRISPR system may be more active in biofilm. Because the exchange channels in biofilm are conducive to horizontal gene transfer, the role of CRISPR in the regulation of resistance in biofilm might be even stronger than in planktonic mode.Seven isolates covering resistant/susceptible, strong/weak biofilm formation, strong/weak biofilm-specific resistance, and huge/small enhancement in resistance after biofilm formation were used to analyze the persister rate in their biofilms, and found the rate was between 0.0001%- 0.01%. Strains with stronger antibiotic resistance tended to have more persisters in their biofilms. This could be explaining that although resistant isolates formed weak biofilms, the enhancement in resistance could still be as high as susceptible isolates.We chose two isolates, resistant and suspectible to imipenem respectively, to analyze the transcriptome sequence in four growing conditions: planktonic, biofilm, planktonic under imipenem challenge and biofilm under imipenem challenge. There’s a higher mutation rate and plenty changes in material metabolisms changed and in the process of biofilm formation and under antibiotic stimulation. Moreover, genes related to flagella assembly, ABC transporter, type I and type II secretion system ifferentially expressed in different growth mode, which meant that these pathways were most likely to participating in both biofilm formation and antibiotic resistance process in A. baumannii.In summary, this study proposes a high MDR and XDR rate, and a high genetic polymorphism in A. baumannii. The predominant clusters, the majority of which were MDR and XDR isolates, have relatively low resistance rates to one or several of five antibiotics: amikacin, tobramycin, minocycline, levofloxacin, and piperacillin/tazobactam. There’s a negative correlation between biofilm formation and antibiotic resistance. Biofilm-specific resistance was positively related with antibiotic resistance. The enhancement in resistance after biofilm formation is not directly related with biofilm biomass. This means weak biofilms could also significantly improve the resistance level. Persisters and bap gene might be associated with this phenomenon. CRISPR system, flagella, ABC transporter, type I and type II secretion system might also be involved in the regulation of biofilm-specific resistance. These findings suggested that we could use more targeted antibiotics in treatment, strengthened our understanding of A. baumannii biofilm infection, and provided new insights for putting forward more effective strategies in the prevention and treatment in related infection. |
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