Acinetobacter baumannii as an oro-dental pathogen: a red alert!!

OBJECTIVES: This review highlights the existence and association of Acinetobacter baumannii with the oro-dental diseases, transforming this systemic pathogen into an oral pathogen. The review also hypothesizes possible reasons for the categorization of this pathogen as code blue due to its stealthy entry into the oral cavity. METHODOLOGY: Study data were retrieved from various search engines reporting specifically on the association of A. baumannii in dental diseases and tray set-ups. Articles were also examined regarding obtained outcomes on A. baumannii biofilm formation, iron acquisitions, magnitude of antimicrobial resistance, and its role in the oral cancers. RESULTS: A. baumannii is associated with the oro-dental diseases and various virulence factors attribute for the establishment and progression of oro-mucosal infections. Its presence in the oral cavity is frequent in oral microbiomes, conditions of impaired host immunity, age related illnesses, and hospitalized individuals. Many sources also contribute for its prevalence in the dental health care environment and the presence of drug resistant traits is also observed. Its association with oral cancers and oral squamous cell carcinoma is also evident. CONCLUSIONS: The review calls for awareness on the emergence of A. baumannii in dental clinics and for the need for educational programs to monitor and control the sudden outbreaks of such virulent and resistant traits in the dental health care settings.

Resistance to bacteriophage incurs a cost to virulence in drug-resistant Acinetobacter baumannii.

Introduction . Acinetobacter baumannii is a critical priority pathogen for novel antimicrobials (World Health Organization) because of the rise in nosocomial infections and its ability to evolve resistance to last resort antibiotics. A. baumannii is thus a priority target for phage therapeutics. Two strains of a novel, virulent bacteriophage (LemonAid and Tonic) able to infect carbapenem-resistant A. baumannii (strain NCTC 13420), were isolated from environmental water samples collected through a citizen science programme.Gap statement. Phage-host coevolution can lead to emergence of host resistance, with a concomitant reduction in the virulence of host bacteria; a potential benefit to phage therapy applications.Methodology. In vitro and in vivo assays, genomics and microscopy techniques were used to characterize the phages; determine mechanisms and impact of phage resistance on host virulence, and the efficacy of the phages against A. baumannii.Results. A. baumannii developed resistance to both viruses, LemonAid and Tonic. Resistance came at a cost to virulence, with the resistant variants causing significantly reduced mortality in a Galleria mellonella larval in vivo model. A replicated 8 bp insertion increased in frequency (~40 % higher frequency than in the wild-type) within phage-resistant A. baumannii mutants, putatively resulting in early truncation of a protein of unknown function. Evidence from comparative genomics and an adsorption assay suggests this protein acts as a novel phage receptor site in A. baumannii. We find no evidence linking resistance to changes in capsule structure, a known virulence factor. LemonAid efficiently suppressed growth of A. baumanni in vitro across a wide range of titres. However, in vivo, while survival of A. baumannii infected larvae significantly increased with both remedial and prophylactic treatment with LemonAid (107 p.f.u. ml-1), the effect was weak and not sufficient to save larvae from morbidity and mortality.Conclusion. While LemonAid and Tonic did not prove effective as a treatment in a Galleria larvae model, there is potential to harness their ability to attenuate virulence in drug-resistant A. baumannii.

[Analysis of the virulence and genetic differences of carbapenem-resistant Acinetobacter baumannii epidemic clones].

Objective: To evaluate the virulence levels of carbapenem-resistant Acinetobacter baumannii ST191, ST195, and ST208, and to analyze the differences in virulence factors among these epidemic clones. Methods: The study involved the genomic sequencing of 233 Acinetobacter baumannii strains that were isolated from the Fifth Medical Center of the Chinese People's Liberation Army General Hospital (North Hospital) between 2011 and 2019. The genomic data was cross-referenced with the Virulence Factor Database (VFDB) to examine the presence of virulence genes in the strains. Furthermore, a Galleria mellonella infection survival model was used to evaluate the virulence levels of the strains, and the association between virulence levels and virulence genes was analyzed. Results: The study included 38 strains of the ST191 clone, 104 strains of the ST195 clone, and 91 strains of the ST208 clone. In the Galleria mellonella infection survival experiment, the average mortality rate for ST191 was 23.0%, with 3 (7.9%) highly virulent strains. For ST195, the average mortality rate was 53.0%, with 34 (32.7%) highly virulent strains. For ST208, the average mortality rate was 47.0%, with 20 (21.9%) highly virulent strains. There was a significant statistical difference in mortality rates between ST191 and ST195 (χ2=13.9, P<0.001) as well as between ST191 and ST208 (χ2=15.2, P<0.001). A comparison of the strains with the VFDB revealed significant differences in the virulence genes carried by the clones. Specifically, the type Ⅵ secretion system-related genes (clpV/tssH, hcp/tssD, tagX, tssA, tssB, tssC, tssE, tssF, tssG, tssK, ssL, tssM) and the sugar transferase gene ACICU_RS00475 were found to be universally absent in ST191 strains (0%) while being prevalent in ST195 (100.0%) and ST208 (>82.0%) strains. Statistical analysis revealed an association between the mortality rate of the clones and the presence of virulence genes(clpV/tssH P<0.001, hcp/tssD P=0.001, tagX P<0.001, tssA P<0.001, tssB P=0.001, tssC P=0.001, tssE P=0.001, tssF P=0.001, tssG P<0.001, tssK P<0.001, tssL P<0.001, tssM P=0.001, ACICU_RS00475 P=0.001). Conclusion: Among the carbapenem-resistant epidemic clones of Acinetobacter baumannii, the ST191 clone shows lower mortality rates in Galleria mellonella, possibly because of the lack of type Ⅵ secretion system and sugar transferase genes.

The tRNA methyltransferase TrmB is critical for Acinetobacter baumannii stress responses and pulmonary infection.

IMPORTANCE: As deficiencies in tRNA modifications have been linked to human diseases such as cancer and diabetes, much research has focused on the modifications' impacts on translational regulation in eukaryotes. However, the significance of tRNA modifications in bacterial physiology remains largely unexplored. In this paper, we demonstrate that the m7G tRNA methyltransferase TrmB is crucial for a top-priority pathogen, Acinetobacter baumannii, to respond to stressors encountered during infection, including oxidative stress, low pH, and iron deprivation. We show that loss of TrmB dramatically attenuates a murine pulmonary infection. Given the current efforts to use another tRNA methyltransferase, TrmD, as an antimicrobial therapeutic target, we propose that TrmB, and other tRNA methyltransferases, may also be viable options for drug development to combat multidrug-resistant A. baumannii.

Use of the waxworm Galleria mellonella larvae as an infection model to study Acinetobacter baumannii.

Galleria mellonella larvae have been increasingly used in research, including microbial infection studies. They act as suitable preliminary infection models to study host-pathogen interactions due to their advantages, such as the ability to survive at 37°C mimicking human body temperature, their immune system shares similarities with mammalian immune systems, and their short life cycle allowing large-scale studies. Here, we present a protocol for simple rearing and maintenance of G. mellonella without requiring special instruments and specialized training. This allows the continuous supply of healthy G. mellonella for research purposes. Besides, this protocol also provides detailed procedures on the (i) G. mellonella infection assays (killing assay and bacterial burden assay) for virulence studies and (ii) bacterial cell harvesting from infected larvae and RNA extraction for bacterial gene expression studies during infection. Our protocol could not only be used in the studies of A. baumannii virulence but can also be modified according to different bacterial strains.

Effect of Hcp Iron Ion Regulation on the Interaction Between Acinetobacter baumannii With Human Pulmonary Alveolar Epithelial Cells and Biofilm Formation.

Acinetobacter baumannii is a type of bacterial nosocomial infection with severe drug resistance. Hemolysin co-regulated protein (Hcp) is a marker of activated type VI secretion system (T6SS), a key secretory system that promotes Gram-negative bacteria colonization, adhesion, and invasion of host cells. Hcp is also regulated by iron ions (Fe). In this study, an ATCC17978 hcp deletion strain (ATCC17978Δhcp), an hcp complement strain (ATCC17978Δhcp+ ), and an A. baumannii-green fluorescent protein (GFP) strain were constructed and used to investigate the role of hcp in bacterial adhesion to cells (human pulmonary alveolar epithelial cells (HPAEpiC)) and biofilm formation. Our results indicate that the inhibitory concentrations of the three A. baumannii strains (ATCC17978 wild type, ATCC17978Δhcp, and ATCC17978Δhcp+) were drug-sensitive strains. A. baumannii hcp gene and iron ions might be involved in promoting the formation of a biofilm and host-bacteria interaction. Iron ions affected the ability of A. baumannii to adhere to cells, as there was no significant difference in the bacterial numbers when assessing the adhesion of the three strains to HPAEpiC in the presence of iron ion concentrations of 0 µM (F = 3.1800, p = 0.1144), 25 µM (F = 2.067, p = 0.2075), 100 µM (F = 30.52, p = 0.0007), and 400 µM (F = 17.57, p = 0.0031). The three strains showed significant differences in their ability to adhere to HPAEpiC. The numbers of bacteria adhesion to HPAEpiC were ATCC17978Δhcp>ATCC17978Δhcp+>ATCC17978 in descending order. Hcp gene was positively regulated by iron ions in the bacteria-cells' co-culture. It is speculated that the effect of iron ions on the interaction between A. baumannii and HPAEpiC might be related to the transport function of hcp and bacterial immune escape mechanisms.

Genomic and Phenotypic Evolution of Tigecycline-Resistant Acinetobacter baumannii in Critically Ill Patients.

Acinetobacter baumannii is an important opportunistic pathogen of nosocomial infections. A. baumannii presently exhibits increasing antibiotic resistance, which poses great challenges to public health. The occurrence of tigecycline-resistant A. baumannii is related to tigecycline treatment and the within-host evolution of bacteria. We analyzed isogenic A. baumannii isolates from two critically ill patients who underwent tigecycline treatment. Whole-genome sequencing and comparative analyses were performed to determine the characteristics of genomic evolution. We conducted phenotypic studies, including in vitro antibiotic sensitivity tests, biofilm formation tests, growth curve determination, serum bactericidal determination, and Galleria mellonella lethality assays. In vivo emergent tigecycline resistance was observed after tigecycline treatment. After the withdrawal of tigecycline pressure, tigecycline-resistant isolates were not isolated from one patient. Four tigecycline-resistant isolates exhibited lower growth rates. The biofilm formation and virulence characteristics of tigecycline-resistant isolates were reasonably different between the two patients. A special phenotype appeared after tigecycline treatment in both patients, accompanied by reduced serum tolerance, enhanced biofilm formation ability, and reduced virulence of Galleria mellonella. Most of the genomic variation occurred after the tigecycline treatment, primarily involving transcription-, signal transduction-, translation-, ribosomal biogenesis-, and cell wall biogenesis-related genes. We determined that the genomic variations in baeR, wzc, aroQ, rluC, and adeS and acquisition of ISAba1 were associated with tigecycline resistance in vivo. Capsular polysaccharide-related genes, wzc, and itrA2, and aroQ, were the key genes related to the virulence evolution of A. baumannii within the host. IMPORTANCE Multidrug-resistant Acinetobacter baumannii poses a huge challenge to clinical treatment, and tigecycline is considered a last-line drug for the treatment of multidrug-resistant A. baumannii. However, the mechanism of tigecycline resistance in vivo has not been elucidated. This study analyzed the genomic and phenotypic evolution of tigecycline-resistant A. baumannii in two critically ill patients. In this study, after treatment with tigecycline, tigecycline-resistant A. baumannii emerged with higher fitness costs. After the withdrawal of tigecycline pressure, tigecycline-resistant isolates were not isolated from one patient. The in vivo and in vitro virulence of the isolates exhibited diametrically opposite results in the two patients. Genomic variations in baeR, wzc, aroQ, rluC, and adeS and acquisition of ISAba1 were associated with tigecycline resistance in vivo. The capsular polysaccharide-related genes, wzc, itrA2, and aroQ, were the key genes related to the virulence of A. baumannii in hosts. Our research provides a theoretical basis for elucidating the mechanism of tigecycline resistance and presents new clues for future surveillance and treatment of multidrug-resistant A. baumannii.

Thioredoxin-mediated alteration of protein content and cytotoxicity of Acinetobacter baumannii outer membrane vesicles.

Acinetobacter baumannii is a Gram-negative bacterium responsible for many hospital-acquired infections including ventilator-associated pneumonia and sepsis. We have previously identified A. baumannii thioredoxin A protein (TrxA) as a virulence factor with a multitude of functions including reduction of protein disulfides. TrxA plays an important role in resistance to oxidative stress facilitating host immune evasion in part by alteration of type IV pili and cell surface hydrophobicity. Other virulence factors such as outer membrane vesicles (OMV) shed by bacteria have been shown to mediate bacterial intercellular communication and modulate host immune response. To investigate whether OMVs can be modulated by TrxA, we isolated OMVs from wild type (WT) and TrxA-deficient (ΔtrxA) A. baumannii clinical isolate Ci79 and carried out a functional and proteomic comparison. Despite attenuation of ΔtrxA in a mouse challenge model, pulmonary inoculation of ΔtrxA OMVs resulted in increased lung permeability compared to WT OMVs. Furthermore, ΔtrxA OMVs induced more J774 macrophage-like cell death than WT OMVs. This ΔtrxA OMV-mediated cell death was abrogated when cells were incubated with protease-K-treated OMVs suggesting OMV proteins were responsible for cytotoxicity. We therefore compared WT and mutant OMV proteins using proteomic analysis. We observed that up-regulated and unique ΔtrxA OMV proteins consisted of many membrane bound proteins involved in small molecule transport as well as proteolytic activity. Bacterial OmpA, metalloprotease, and fimbrial protein have been shown to enhance mammalian cell apoptosis through various mechanisms. Differential packaging of these proteins in ΔtrxA OMVs may contribute to the increased cytotoxicity observed in this study.

Acinetobacter baumannii Regulates Its Stress Responses via the BfmRS Two-Component Regulatory System.

Acinetobacter baumannii is a common nosocomial pathogen that utilizes numerous mechanisms to aid its survival in both the environment and the host. Coordination of such mechanisms requires an intricate regulatory network. We report here that A. baumannii can directly regulate several stress-related pathways via the two-component regulatory system BfmRS. Similar to previous studies, results from transcriptomic analysis showed that mutation of the BfmR response regulator causes dysregulation of genes required for the oxidative stress response, the osmotic stress response, the misfolded protein/heat shock response, Csu pilus/fimbria production, and capsular polysaccharide biosynthesis. We also found that the BfmRS system is involved in controlling siderophore biosynthesis and transport, and type IV pili production. We provide evidence that BfmR binds to various stress-related promoter regions and show that BfmR alone can directly activate transcription of some stress-related genes. Additionally, we show that the BfmS sensor kinase acts as a BfmR phosphatase to negatively regulate BfmR activity. This work highlights the importance of the BfmRS system in promoting survival of A. baumannii. IMPORTANCE Acinetobacter baumannii is a nosocomial pathogen that has extremely high rates of multidrug resistance. This organism's ability to endure stressful conditions is a key part of its ability to spread in the hospital environment and cause infections. Unlike other members of the gammaproteobacteria, A. baumannii does not encode a homolog of the RpoS sigma factor to coordinate its stress response. Here, we demonstrate that the BfmRS two-component system directly controls the expression of multiple stress resistance genes. Our findings suggest that BfmRS is central to a unique scheme of general stress response regulation by A. baumannii.

Association of capsular polysaccharide locus 2 with prognosis of Acinetobacter baumannii bacteraemia.

Acinetobacter baumannii causes healthcare-associated infections worldwide. Capsular polysaccharide (CPS) is shown an important virulence factor of A. baumannii both in vitro and in vivo. Capsule locus 2 (KL2) for CPS is the most common KL type and is associated with carbapenem resistance. It is unclear whether KL2 is related to the clinical outcome of invasive A. baumannii infection. Here we had followed patients with A. baumannii bacteraemia prospectively between 2009 and 2014. One-third of the unduplicated blood isolates were randomly selected each year for microbiological and clinical studies. The KL2 gene cluster was identified using polymerase chain reaction. A total of 148 patients were enrolled randomly. Eighteen isolates (12.2%) carried KL2, and 130 isolates (87.8%) didn't. Compared with non-KL2 isolates, KL2 isolates had significantly higher resistance to imipenem, sulbactam, and tigecycline. Compared with the non-KL group, in the KL2 group, the hospital stay before development of bacteraemia was longer (P < 0.001), a higher percentage had pneumonia (P = 0.004), and the white blood cell count was lower (P = 0.03). Infection with KL2 A. baumannii predicted mortality (adjusted hazard ratio [aHR], 2.03; 95% confidence interval [CI], 1.09-3.78; P = 0.03), independently of the Pitt bacteraemia score (aHR, 1.34; 95% CI, 1.23-1.46; P < 0.001) and leucopenia (aHR, 2.16; 95% CI, 1.30-3.57; P = 0.003). Thrombocytopenia contributed to the effect of KL2 on mortality in bacteraemia (Sobel test P = 0.01). Large-scale studies are warranted to confirm these findings and the underlying mechanisms deserve further investigation.

Antiseptic 9-Meric Peptide with Potency against Carbapenem-Resistant Acinetobacter baumannii Infection.

Carbapenem-resistant A. baumannii (CRAB) infection can cause acute host reactions that lead to high-fatality sepsis, making it important to develop new therapeutic options. Previously, we developed a short 9-meric peptide, Pro9-3D, with significant antibacterial and cytotoxic effects. In this study, we attempted to produce safer peptide antibiotics against CRAB by reversing the parent sequence to generate R-Pro9-3 and R-Pro9-3D. Among the tested peptides, R-Pro9-3D had the most rapid and effective antibacterial activity against Gram-negative bacteria, particularly clinical CRAB isolates. Analyses of antimicrobial mechanisms based on lipopolysaccharide (LPS)-neutralization, LPS binding, and membrane depolarization, as well as SEM ultrastructural investigations, revealed that R-Pro9-3D binds strongly to LPS and impairs the membrane integrity of CRAB by effectively permeabilizing its outer membrane. R-Pro9-3D was also less cytotoxic and had better proteolytic stability than Pro9-3D and killed biofilm forming CRAB. As an LPS-neutralizing peptide, R-Pro9-3D effectively reduced LPS-induced pro-inflammatory cytokine levels in RAW 264.7 cells. The antiseptic abilities of R-Pro9-3D were also investigated using a mouse model of CRAB-induced sepsis, which revealed that R-Pro9-3D reduced multiple organ damage and attenuated systemic infection by acting as an antibacterial and immunosuppressive agent. Thus, R-Pro9-3D displays potential as a novel antiseptic peptide for treating Gram-negative CRAB infections.

Deciphering Multidrug-Resistant Acinetobacter baumannii from a Pediatric Cancer Hospital in Egypt.

Infection by multidrug-resistant (MDR) Acinetobacter baumannii is one of the major causes of hospital-acquired infections worldwide. The ability of A. baumannii to survive in adverse conditions as well as its extensive antimicrobial resistance make it one of the most difficult to treat pathogens associated with high mortality rates. The aim of this study was to investigate MDR A. baumannii that has spread among pediatric cancer patients in the Children's Cancer Hospital Egypt 57357. Whole-genome sequencing was used to characterize 31 MDR A. baumannii clinical isolates. Phenotypically, the isolates were MDR, with four isolates showing resistance to the last-resort antibiotic colistin. Multilocus sequence typing showed the presence of eight clonal groups, two of which were previously reported to cause outbreaks in Egypt, and one novel sequence type (ST), Oxf-ST2246. Identification of the circulating plasmids showed the presence of two plasmid lineages in the isolates, strongly governed by sequence type. A large number of antimicrobial genes with a range of resistance mechanisms were detected in the isolates, including ß-lactamases and antibiotic efflux pumps. Analysis of insertion sequences (ISs) revealed the presence of ISAba1 and ISAba125 in all the samples, which amplify ß-lactamase expression, causing extensive carbapenem resistance. Mutation analysis was used to decipher underlying mutations responsible for colistin resistance and revealed novel mutations in several outer membrane proteins, in addition to previously reported mutations in pmrB. Altogether, understanding the transmissibility of A. baumannii as well as its resistance and virulence mechanisms will help develop novel treatment options for better management of hospital-acquired infections. IMPORTANCE Acinetobacter baumannii represents a major health threat, in particular among immunocompromised cancer patients. The rise in carbapenem-resistant A. baumannii, and the development of resistance to the last-resort antimicrobial agent colistin, complicates the management of A. baumannii outbreaks and increases mortality rates. Here, we investigate 31 multidrug resistant A. baumannii isolates from pediatric cancer patients in Children's Cancer Hospital Egypt (CCHE) 57357 via whole-genome sequencing. Multilocus sequence typing (MLST) showed the presence of eight clonal groups including a novel sequence type. In silico detection of antimicrobial-resistant genes and virulence factors revealed a strong correlation between certain virulence genes and mortality as well as several point mutations in outer membrane proteins contributing to colistin resistance. Detection of CRISPR/Cas sequences in the majority of the samples was strongly correlated with the presence of prophage sequences and associated with failure of bacteriophage therapy. Altogether, understanding the genetic makeup of circulating A. baumannii is essential for better management of outbreaks.

Importance of twitching and surface-associated motility in the virulence of Acinetobacter baumannii.

Acinetobacter baumannii is a pathogen of increasing clinical importance worldwide, especially given its ability to readily acquire resistance determinants. Motile strains of this bacterium can move by either or both of two types of motility: (i) twitching, driven by type IV pili, and (ii) surface-associated motility, an appendage-independent form of movement. A. baumannii strain MAR002 possesses both twitching and surface-associated motility. In this study, we isolated spontaneous rifampin-resistant mutants of strain MAR002 in which point mutations in the rpoB gene were identified that resulted in an altered motility pattern. Transcriptomic analysis of mutants lacking twitching, surface-associated motility, or both led to the identification of deregulated genes within each motility phenotype, based on their level of expression and their biological function. Investigations of the corresponding knockout mutants revealed several genes involved in the motility of A. baumannii strain MAR002, including two involved in twitching (encoding a minor pilin subunit and an RND [resistance nodulation division] component), one in surface-associated motility (encoding an amino acid permease), and eight in both (encoding RND and ABC components, the energy transducer TonB, the porin OprD, the T6SS component TagF, an IclR transcriptional regulator, a PQQ-dependent sugar dehydrogenase, and a putative pectate lyase). Virulence assays showed the reduced pathogenicity of mutants with impairments in both types of motility or in surface-associated motility alone. By contrast, the virulence of twitching-affected mutants was not affected. These results shed light on the key role of surface-associated motility and the limited role of twitching in the pathogenicity of A. baumannii.

Mechanical ventilation-associated pneumonia caused by Acinetobacter baumannii in Northeast China region: analysis of genotype and drug resistance of bacteria and patients' clinical features over 7 years.

OBJECTIVE: To investigate the clinical features and outcomes of patients with mechanical ventilation-associated pneumonia (VAP) caused by Acinetobacter baumannii (Ab), and to characterize the drug resistance of pathogenic strains and carbapenem resistance-associated genes. METHODS: Clinical data were collected from the PICU of Shengjing Hospital. Patients who met the diagnostic criteria of VAP and for whom Ab was a pathogen were selected as study participants. The patients were divided into carbapenem-resistant A. baumannii (CRAB) and carbapenem-sensitive A. baumannii (CSAB) groups. The genes closely associated with Ab resistance to carbapenems and the efflux pump-related genes were detected by real-time polymerase chain reaction, and results compared between the two groups. RESULTS: The total mechanical ventilation time and the administration time of antibiotics after a diagnosis of Ab infection were significantly higher in the CRAB group. And the CRAB group strains were only sensitive to amikacin, cephazolin, compound sulfamethoxazole, and tigecycline. Genetic test results indicated that IPM expression was not significantly different between two groups. The OXA-51 and OXA-23 in the CRAB group was markedly higher than that in the CSAB group, while OXA-24 expression was markedly lower. The expression of AdeABC and AdeFGH was significantly greater in the CRAB compared to CSAB group. CONCLUSION: In pediatric patients with VAP caused by Ab infection, the detection rate of CRAB strains is far higher than that of CSAB strains; The abnormal expression of ß-lactamase-producing genes (OXA-23, OXA-24, and OXA-51) and efflux pump-related genes (AdeABC and AdeFGH) is closely related to the production of CRAB.

Role of Iron-Containing Alcohol Dehydrogenases in Acinetobacter baumannii ATCC 19606 Stress Resistance and Virulence.

Most bacteria possess alcohol dehydrogenase (ADH) genes (Adh genes) to mitigate alcohol toxicity, but these genes have functions beyond alcohol degradation. Previous research has shown that ADH can modulate quorum sensing in Acinetobacter baumannii, a rising opportunistic pathogen. However, the number and nature of Adh genes in A. baumannii have not yet been fully characterized. We identified seven alcohol dehydrogenases (NAD+-ADHs) from A. baumannii ATCC 19606, and examined the roles of three iron-containing ADHs, ADH3, ADH4, and ADH6. Marker-less mutation was used to generate Adh3, Adh4, and Adh6 single, double, and triple mutants. Disrupted Adh4 mutants failed to grow in ethanol-, 1-butanol-, or 1-propanol-containing mediums, and recombinant ADH4 exhibited strongest activity against ethanol. Stress resistance assays with inorganic and organic hydroperoxides showed that Adh3 and Adh6 were key to oxidative stress resistance. Virulence assays performed on the Galleria mellonella model organism revealed that Adh4 mutants had comparable virulence to wild-type, while Adh3 and Adh6 mutants had reduced virulence. The results suggest that ADH4 is primarily involved in alcohol metabolism, while ADH3 and ADH6 are key to stress resistance and virulence. Further investigation into the roles of other ADHs in A. baumannii is warranted.

Trehalose-deficient Acinetobacter baumannii exhibits reduced virulence by losing capsular polysaccharide and altering membrane integrity.

Acinetobacter baumannii has become a leading cause of bacterial nosocomial infections, in part, due to its ability to resist desiccation, disinfection and antibiotics. Several factors contribute to the tenacity and virulence of this pathogen, including production of a broad range of surface glycoconjugates, secretory systems and efflux pumps. We became interested in examining the importance of trehalose in A. baumannii after comparing intact bacterial cells by high-resolution magic angle spinning nuclear magnetic resonance and by noting high levels of this disaccharide, obscuring all other resonances in the spectrum. Since this was observed under normal growth conditions, we speculated that trehalose must serve additional functions beyond osmolyte homeostasis. Using the virulent isolate A. baumannii AB5075 and mutants in the trehalose synthesis pathway, osmoregulatory trehalose synthesis proteins A and B (△otsA and △otsB), we found that the trehalose-deficient △otsA showed increased sensitivity to desiccation, colistin, serum complement and peripheral blood mononuclear cells, while trehalose-6-phosphate producing △otsB behaved similar to the wild-type. The △otsA mutant also demonstrated increased membrane permeability and loss of capsular polysaccharide. These findings demonstrate that trehalose deficiency leads to loss of virulence in A. baumannii AB5075.

ppGpp signaling plays a critical role in virulence of Acinetobacter baumannii.

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.

Screening of small molecules attenuating biofilm formation of Acinetobacter baumannii by inhibition of ompA promoter activity.

Anti-virulence therapeutic strategies are promising alternatives against drug-resistant pathogens. Outer membrane protein A (OmpA) plays a versatile role in the pathogenesis and antimicrobial resistance of Acinetobacter baumannii. Therefore, OmpA is an innovative target for anti-virulence therapy against A. baumannii. This study aimed to develop a high-throughput screening (HTS) system to discover small molecules inhibiting the ompA promoter activity of A. baumannii and screen chemical compounds using the bacterial growth-based HTS system. The ompA promoter and open reading frame of nptI fusion plasmids that controlled the expression of nptI encoding resistance to kanamycin by the ompA promoter were constructed and then transformed into A. baumannii ATCC 17978. This reporter strain was applied to screen small molecules inhibiting the ompA promoter activity in a chemical library. Of the 7,520 chemical compounds, 15 exhibited ≥ 70% growth inhibition of the report strain cultured in media containing kanamycin. Three compounds inhibited the expression of ompA and OmpA in the outer membrane of A. baumannii ATCC 17978, which subsequently reduced biofilm formation. In conclusion, our reporter strain is useful for large-scale screening of small molecules inhibiting the ompA expression in A. baumannii. Hit compounds identified by the HTS system are promising scaffolds to develop novel therapeutics against A. baumannii.

Multidrug-resistant Acinetobacter baumannii as an emerging concern in hospitals.

Acinetobacter baumannii has become a major concern for scientific attention due to extensive antimicrobial resistance. This resistance causes an increase in mortality rate because strains resistant to antimicrobial agents are a major challenge for physicians and healthcare workers regarding the eradication of either hospital or community-based infections. These strains with emerging resistance are a serious issue for patients in the intensive care unit (ICU). Antibiotic resistance has increased because of the acquirement of mobile genetic elements such as transposons, plasmids, and integrons and causes the prevalence of multidrug resistance strains (MDR). In addition, an increase in carbapenem resistance, which is used as last line antibiotic treatment to eliminate infections with multidrug-resistant Gram-negative bacteria, is a major concern. Carbapenems resistant A. baumannii (CR-Ab) is a worldwide problem. Because these strains are often resistant to all other commonly used antibiotics. Therefore, pathogenic multi-drug resistance A. baumannii (MDR-Ab) associated infections become hard to eradicate. Plasmid-mediated resistance causes outbreaks of extensive drug-resistant. A. baumannii (XDR-Ab). In addition, recent outbreaks relating to livestock and community settings illustrate the existence of large MDR-Ab strain reservoirs within and outside hospital settings. The purpose of this review, proper monitoring, prevention, and treatment are required to control (XDR-Ab) infections. Attachment, the formation of biofilms and the secretion of toxins, and low activation of inflammatory responses are mechanisms used by pathogenic A. baumannii strain. This review will discuss some aspects associated with antibiotics resistance in A. baumannii as well as cover briefly phage therapy as an alternative therapeutic treatment.

Risk Factors Associated With 30-Day Mortality Among Neonates With A. baumannii Sepsis.

BACKGROUND: Acinetobacter baumannii sepsis constitutes an extreme threat with a poor prognosis and is a difficult infection to control, especially in Asia. Moreover, a knowledge gap in the risk of mortality in neonatal A. baumannii sepsis still exists. METHODS: This study aimed to identify the risk factors of mortality in neonates with A. baumannii sepsis in Thailand from 1996 to 2019. A multivariable logistic regression model was analyzed for nonsurvivors and survivors of neonatal A. baumannii sepsis. RESULTS: In a 24-year period, 91 neonates with A. baumannii sepsis were reviewed. The median (interquartile range) gestational age and birth weight were 33 (28.5, 37.5) weeks and 1740 (987.5, 2730.0) g, respectively. The 30-day case fatality rate was 36.3% (33/91). In univariable analysis, nonsurvivors of neonatal A. baumannii sepsis was associated with smaller neonates, lower Apgar scores, septic shock, mechanical ventilation, umbilical catheterization, neutropenia, severe thrombocytopenia, carbapenem-resistant A. baumannii sepsis, inadequate empiric antimicrobial therapy, and acute kidney injury. In multivariable analysis, nonsurvivors of neonatal A. baumannii sepsis were associated with septic shock (adjusted odds ratio [OR] = 41.38; 95% confidence intervals [CI]: 3.42-501.13; P = 0.003), severe thrombocytopenia (adjusted OR = 33.70; 95% CI: 3.44-330.55; P = 0.002), and inadequate empiric antimicrobial therapy (adjusted OR = 10.05; 95% CI: 1.40-71.98; P = 0.02). CONCLUSION: In high multidrug-resistant areas, empiric treatment with broader spectrum antimicrobials should be considered in neonates with sepsis shock or severe thrombocytopenia.