BackgroundThe emergence of multidrug- or extensively drug-resistant Acinetobacter baumannii has made it difficult to treat and control infections caused by this bacterium. It is urgently necessary to search for alternatives to conventional antibiotics for control of severe A. baumannii infections. In recent years, bacteriophages and their derivatives, such as depolymerases, showed great potential as antibacterial or antivirulence agents against bacterial infections. Nonetheless, unlike broad-spectrum bactericidal antibiotics, phage-encoded depolymerase targets only a limited number of bacterial strains. Therefore, identification of novel depolymerases and evaluation of their ability to control A. baumannii infections is important.MethodsA bacteriophage was isolated from hospital sewage using an extensively drug-resistant A. baumannii strain as the host bacterium, and the phage’s plaque morphology and genomic composition were studied. A polysaccharide depolymerase (Dpo48) was expressed and identified, and the effects of pH and temperature on its activity were determined. Besides, a serum killing assay was conducted, and amino acid sequences homologous to those of putative polysaccharide depolymerases were compared.ResultsPhage IME200 yielded clear plaques surrounded by enlarged halos, with polysaccharide depolymerase activity against the host bacterium. A tail fiber protein with a Pectate_lyase_3 domain was identified as Dpo48 and characterized . Dpo48 was found to degrade the capsule polysaccharide of the bacterial surface, as revealed by Alcian blue staining. Dpo48 manifested stable activity over a broad range of pH (5.0–9.0) and temperatures (20–70 °C). Results from in vitro serum killing assays indicated that 50% serum was sufficient to cause a five log reduction of overnight enzyme-treated bacteria, with serum complement playing an important role in these killing assays. Moreover, Dpo48 had a spectrum of activity exactly the same as its parental phage IME200, which was active against 10 out of 41 A. baumannii strains. Amino acid sequence alignment showed that the putative tail fiber proteins had a relatively short, highly conserved domain in their N-terminal sequences, but their amino acid sequences containing pectate lyase domains, found in the C-terminal regions, were highly diverse.ConclusionsPhage-encoded capsule depolymerases may become promising antivirulence agents for preventing and controlling A. baumannii infections.

Acinetobacter baumannii is an important opportunistic pathogen associated with a variety of nosocomial infections. A rapid and sensitive molecular detection in clinical isolates is quite needed for the appropriate therapy and outbreak control of A. baumannii. Group 2 carbapenems have been considered the agents of choice for the treatment of multiple drug-resistant A. baumannii. But the prevalence of carbapenem-resistant A. baumannii (CRAB) has been steadily increasing in recent years. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of A. baumannii in clinical samples by using high-specificity primers of the blaOXA-51 gene. Then we investigated the OXA-carbapenemases molecular epidemiology of A. baumannii isolates in two comprehensive hospitals in Beijing. The results showed that the LAMP assay could detect target DNA within 60 min at 65°C. The detection limit was 50 pg/μl, which was about 10-fold greater than that of PCR. Furthermore, this method could distinguish A. baumannii from the homologous A. nosocomialis and A. pittii. A total of 228 positive isolates were identified by this LAMP-based method for A. baumannii from 335 intensive care unit patients with clinically suspected multi-resistant infections in two hospitals in Beijing. The rates of CRAB are on the rise and are slowly becoming a routine phenotype for A. baumannii. Among the CRABs, 92.3% harbored both the blaOXA-23 and blaOXA-51 genes. Thirty-three pulsotypes were identified by pulsed-field gel electrophoresis, and the majority belonged to clone C. In conclusion, the LAMP method developed for detecting A. baumannii was faster and simpler than conventional PCR and has great potential for both point-of-care testing and basic research. We further demonstrated a high distribution of class D carbapenemase-encoding genes, mainly OXA-23, which presents an emerging threat in hospitals in China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) pose a significant threat to global public health. In present research, a total of 80 CRKP strains belonging to ST11 were collected with 70% (56 of 80 isolates) expressing a K47 capsular type. Thus, it is significant to prevent and control infections caused by these bacteria. Capsule depolymerases could degrade bacterial surface polysaccharides to reduce their virulence and expose bacteria to host immune attack. Previous studies have demonstrated the potential of phage-encoded depolymerases as antivirulent agents in treating CRKP infections in vitro and in vivo. Here, two capsule depolymerases (Dpo42 and Dpo43) derived from phage IME205 were expressed and characterized. Although both depolymerases act on strains with a capsular serotype K47, they are active against different subsets of strains, indicating subtle differences in capsule composition that exist within this serotype. The host range of phage IME205 matched to the sum of specificity range of Dpo42 and Dpo43. These two enzymes maintained stable activity in a relatively broad range of pH levels (pH 5.0-8.0 for Dpo42 and pH 4.0-8.0 for Dpo43) and temperatures (20-70 • C). Besides, both Dpo42 and Dpo43 could make host bacteria fully susceptible to the killing effect of serum complement and display no hemolytic activity to erythrocytes. In summary, capsule depolymerases are promising antivirulent agents to combat CRKP infections.

This research demonstrates that intranasal application of bacteriophage is viable, and could provide complete protection from pneumonia caused by A. baumannii.

The emergence of multidrug- and extensively drug-resistant Acinetobacter baumannii has made it difficult to treat and control infections caused by this bacterium. Thus, alternatives to conventional antibiotics for management of severe A. baumannii infections is urgently needed. In our previous study, we found that a capsule depolymerase Dpo48 could strip bacterial capsules, and the non-capsuled A. baumannii were significantly decreased in the presence of serum complement in vitro. Here, we further explored its potential as a therapeutic agent for controlling systemic infections caused by extensively drug-resistant A. baumannii. Prior to mammalian studies, the anti-virulence efficacy of Dpo48 was first tested in a Galleria mellonella infection model. Survival rate of Dpo48-pretreated bacteria or Dpo48 treatment group was significantly increased compared to the infective G. mellonella without treatment. Furthermore, the safety and therapeutic efficacy of Dpo48 to mice were evaluated. The mice treated with Dpo48 displayed normal serum levels of TBIL, AST, ALT, ALP, Cr, BUN and LDH, while no significant histopathology changes were observed in tissues of liver, spleen, lung, and kidney. Treatment with Dpo48 could rescue normal and immunocompromised mice from lethal peritoneal sepsis, with the bacterial counts in blood, liver, spleen, lung, and kidney significantly reduced by 1.4–3.3 log colony-forming units at 4 h posttreatment. Besides, the hemolysis and cytotoxicity assays showed that Dpo48 was non-homolytic to human red blood cells and non-toxic to human lung, liver and kidney cell lines. Overall, the present study demonstrated the promising potential of capsule depolymerases as therapeutic agents to prevent antibiotic-resistant A. baumannii infections.

Heat shock proteins (HSPs) display adjuvant functions when given as fusion proteins to enhance vaccination efficiency. To evaluate enhanced potency of Hantaan virus (HTNV) glycoprotein (GP) and nucleocapsid protein (NP) immunogenicity by heat shock protein 70 (HSP70), a recombinant adenovirus rAd-GnS0.7-pCAG-HSP70C expression vector was developed by genetically linking the HSP70 C-terminal gene (HSP70 359–610 aa, HSP70C) to the Gn and 0.7 kb fragment of the NP (aa1–274-S0.7). C57BL/6 mice were immunized with these recombinant adenoviral vectors. A series of immunological assays determined the immunogenicity of the recombinant adenoviral vectors. The results showed that rAd-GnS0.7-pCAG-HSP70C induced a stronger humoral and cellular immune response than other recombinant adenoviruses (rAd-GnS0.7-pCAG and rAd-GnS0.7) and the HFRS vaccine control. Animal protection experiments showed that rAd-GnS0.7-pCAG-HSP70C was effective at protecting C57BL/6 mice from HTNV infection. The results of the immunological experiments showed that HSP70C lead to enhanced vaccine potency, and suggested significant potential in the development of genetically engineered vaccines against HTNV.

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The rapid expansion of Acinetobacter baumannii clinical isolates exhibiting resistance to most or all available antibiotics is a global concern. Current treatments for infections caused by this bacterium have become less effective, and the need to explore new alternative therapies is urgent. Depolymerases derived from phages are emerging as attractive anti-virulence agents. In this study, a previously isolated A. baumannii phage (designated as vB_AbaM_IME285) was characterized, and genomic study was carried out using various bioinformatics tools. A gene predicted as encoding for the depolymerase was cloned and expressed, and the depolymerase activity of the recombinant enzyme (Dp49) was identified both in vitro and in experimental mice. The results showed that phage IME285 formed translucent halos around the plaques when inoculated onto a lawn of the host bacteria, exibiting depolymerase activity against this strain. On the basis of complete genome sequencing and bioinformatics analysis, ORF49 was speculated to be a gene encoding for the putative capsule depolymerase. The expressed recombinant Dp49 displayed an effective depolymerase activity and had a spectrum of activity similar to its parental phage IME285, which was active against 25 out of 49 A. baumannii strains. It was found that Dp49 greatly improved the inhibitory effect of serum on bacterial growth in vitro , and the administration of this enzyme significantly increased the survival rates of A. baumannii -infected mice in the animal experiment. In conclusion, the phage-encoded depolymerase Dp49 might be a promising alternative means of controlling infections mediated by multidrug-resistant A. baumannii .