Most clinical isolates of Acinetobacter baumannii, a nosocomial pathogen, are multidrug-resistant (MDR), fueling the search for alternative therapies. Bacteriophage-derived endolysins have potent antibacterial activities and are considered as alternatives to antibiotics against A. baumannii infection. Gram-negative bacteria possess outer lipid membrane that prevents direct contact between the endolysins and the cell wall. We hypothesized that the fusion of antimicrobial peptide (AMP) with endolysin could help to reduce bacterial endolysin resistance and increase antimicrobial activity by membrane permeability action. Accordingly, we fused cecropin A, a commonly used AMP, with the N-terminus of AbEndolysin, which enhances the bactericidal activity of the chimeric endolysin. The bactericidal activity of cecropin A-fused AbEndolysin increased by at least 2–8 fold for various MDR A. baumannii clinical isolates. The in vitro bactericidal activity results also showed higher bacterial lysis by the chimeric endolysin than that by the parental lysin. The engineered AbEndolysin (eAbEndolysin) showed synergistic effects with the beta-lactam antibiotics cefotaxime, ceftazidime, and aztreonam, and an additive effect with meropenem and imipenem. eAbEndolysin had no cytotoxic effect on A549 cell line and rescued mice (40% survival rate) from systemic A. baumannii infection. Together, these findings suggest the potential of lysin therapy and may prompt its use as an alternative to antibiotics.

Treatment of infections caused by Acinetobacter baumannii nosocomial strains has become increasingly problematic owing to their resistance to antibiotics. ppGpp is a secondary messenger involved in growth control and various stress responses in bacteria. The mechanism for inhibition of antibiotic resistance via ppGpp is still unidentified in various pathogenic bacteria including A. baumannii. Here, we investigated the effects of ppGpp on efflux pump (EP)-related genes in A. baumannii. ppGpp-deficient and -complementary strains were constructed by conjugation and we confirmed (p)ppGpp measurements by thin-layer chromatography. We observed that the ppGpp-deficient strain (ΔA1S_0579) showed abnormal stretching patterns by transmission electron microscopy analysis. The MICs of antimicrobial agents for the WT A. baumannii (ATCC 17978), ppGpp-deficient and complementary strains were determined by the Etest and broth dilution assay methods. The expression levels of EP-related genes were determined by quantitative RT–PCR. We observed morphological differences between a ppGpp-deficient strain (ΔA1S_0579) and the WT strain. Dramatic reductions of MICs in the ppGpp-deficient strain compared with the WT were observed for gentamicin (2.6-fold), tetracycline (3.9-fold), erythromycin (4-fold) and trimethoprim (>4-fold). Expression of the EP-related genes abeB (2.8-fold), tet(A) (2.3-fold), adeB (10.0-fold), adeI (9.9-fold), adeJ (11.8-fold) and adeK (14.4-fold) was also decreased in the ppGpp-deficient strain. This study demonstrates that ppGpp regulates EP-related gene expression in A. baumannii, affecting antibiotic susceptibility. To date, treatment for MDR A. baumannii has had no new antimicrobial agents, so the A1S_0579 gene could be a novel therapeutic target for rational drug design by affecting ppGpp production.

Acinetobacter baumannii, a major nosocomial pathogen, survives in diverse hospital environments, and its multidrug resistance is a major concern. The ppGpp-dependent stringent response mediates the reprogramming of genes with diverse functions in several bacteria. We investigated whether ppGpp is involved in A. baumannii’s pathogenesis by examining biofilm formation, surface motility, adhesion, invasion, and mouse infection studies. Transcriptome analysis of early stationary phase cultures revealed 498 differentially-expressed genes (≥ 2-fold change) in a ppGpp-deficient A. baumannii strain; 220 and 278 genes were up and downregulated, respectively. Csu operon expression, important in pilus biosynthesis during early biofilm formation, was significantly reduced in the ppGpp-deficient strain. Our findings suggest that ppGpp signaling influences A. baumannii biofilm formation, surface motility, adherence, and virulence. We showed the association between ppGpp and pathogenicity in A. baumannii for the first time; ppGpp may be a novel antivirulence target in A. baumannii.

Acinetobacter baumannii is an important nosocomial pathogen that can survive in different environmental conditions and poses a severe threat to public health due to its multidrug resistance properties. Research on transcriptional regulators, which play an essential role in adjusting to new environments, could provide new insights into A. baumannii pathogenesis. LysR-type transcriptional regulators (LTTRs) are structurally conserved among bacterial species and regulate virulence in many pathogens. We identified a novel LTTR, designated as LeuO encoded in the A. baumannii genome. After construction of LeuO mutant strain, transcriptome analysis showed that LeuO regulates the expression of 194 upregulated genes and 108 downregulated genes responsible for various functions and our qPCR validation of several differentially expressed genes support transcriptome data. Our results demonstrated that disruption of LeuO led to increased biofilm formation and increased pathogenicity in an animal model. However, the adherence and surface motility of the LeuO mutant were reduced compared with those of the wild-type strain. We observed some mutations on amino acids sequence of LeuO in clinical isolates. These mutations in the A. baumannii biofilm regulator LeuO may cause hyper-biofilm in the tested clinical isolates. This study is the first to demonstrate the association between the LTTR member LeuO and virulence traits of A. baumannii.

Acinetobacter baumannii is known for its virulence in severely ill, hospitalized patients and for exhibiting multidrug resistance. A. baumannii infection treatment poses a serious problem in clinical environments. The outer membrane protein A (OmpA) of the Acinetobacter genus is involved in bacterial virulence. Regulatory factors of OmpA in the post-transcriptional stage have been previously identified. However, the regulatory factors that act before the transcriptional stage remain unclear. We investigated the A1S_0316 gene that encodes a putative transcription factor for OmpA expression in A. baumannii. A1S_0316 was purified and examined using size-exclusion chromatography, which revealed that it forms an oligomer. The binding affinity of A1S_0316 to the OmpA promoter region was also examined. We compared the binding affinity to the OmpA promotor region between A1S_0316 and the AbH-NS protein. A1S_0316 showed higher binding affinity to the OmpA promotor region than did H-NS. We examined the regulatory effect of these proteins on OmpA expression in A. baumannii using real-time qPCR and various in vitro tools. Our results indicated that A1S_0316 acts as an anti-repressor on the promotor region of the OmpA gene by inhibiting the binding of the AbH-NS protein. This study was the first demonstration of the transcriptional regulation of OmpA expression.

Acinetobacter baumannii is a nosocomial pathogen, which is a problem worldwide due to the emergence of a difficult-to-treat multidrug-resistant A. baumannii (MDRAB). Endolysins are hydrolytic enzymes produced by a bacteriophage that can be used as a potential therapeutic agent for multidrug-resistant bacterial infection in replacing antibiotics. Here, we isolated a novel bacteriophage through prophage induction using mitomycin C from clinical A. baumannii 1656-2. Morphologically, ΦAb1656-2 was identified as a Siphoviridae family bacteriophage, which can infect MDRAB. The whole genome of ΦAb1656-2 was sequenced, and it showed that it is 50.9 kb with a G + C content of 38.6% and 68 putative open reading frames (ORFs). A novel endolysin named AbEndolysin with an N-acetylmuramidase-containing catalytic domain was identified, expressed, and purified from ΦAb1656-2. Recombinant AbEndolysin showed significant antibacterial activity against MDRAB clinical strains without any outer membrane permeabilizer. These results suggest that AbEndolysin could represent a potential antimicrobial agent for treating MDRAB clinical isolates.