A hypervirulent Acinetobacter baumannii strain has robust anti-phagocytosis ability

Abstract

Background: Acinetobacter baumannii (A. baumannii) is associated with both hospital-acquired infections (HAP) and community-acquired pneumonia (CAP). In this study, we present a novel CAP-associated A. baumannii (CAP-AB) strain causing severe pneumonia in an afore healthy male patient without underlying conditions. Subsequently, we investigated the pathogenicity and immunogenicity of this CAP-AB strain using a mice pneumonia model.

Results: A 58-year-old male patient with no underlying conditions experienced worsening symptoms of a productive cough, sputum, and fever that developed acutely, in just 24 h. The diagnosis was severe community-acquired pneumonia (CAP) and type-1 respiratory failure. An A. baumannii strain was isolated from his sputum and blood cultures. To gain a deeper understanding of the rapid progression of its pathology, we utilized the CAP-associated A. baumannii strain YC128, a previously obtained hospital-acquired pneumonia A. baumannii (HAP-AB) strain YC156, and a highly virulent A. baumannii control strain LAC-4 to construct a mouse pneumonia model, and subsequently compared the mortality rate of the three groups. Following inoculation with 10⁷ CFU of A. baumannii, the mortality rate for the YC128, LAC-4, and YC156 groups was 60% (6/10), 30% (3/10), and 0%, respectively. The bacterial burden within the pulmonary, liver, and spleen tissues of mice in the YC128 group was significantly higher than that of the YC156 group, and slightly higher than that of the LAC-4 group. Pathological analysis of lung tissue using HE-staining revealed that the inflammatory pathological changes in mice from the YC128 group were significantly more severe than those in the YC156 group. Additionally, CT scan images displayed more pronounced inflammation in the lungs of mice from the YC128 group compared to the YC156 group. Local levels of cytokines/chemokines such as IL-1β, IL-6, TNF-α, and CXCL1 were assessed via RT-qPCR in lung tissues. In comparison with the YC156 strain, the highly virulent YC128 strain induced the expression of proinflammatory cytokines more rapidly and severely. Furthermore, we examined the in vitro anti-phagocytosis ability of YC128 and YC156 strains against mice peritoneal macrophages, revealing that the highly virulent YC128 isolate displayed greater resistance to macrophage uptake in contrast to YC156. Results from Whole Genome Sequencing (WGS) indicated that YC128 harbored a complete type VI secretion system (T6SS) gene cluster, while YC156 lacked the majority of genes within the T6SS gene cluster. The other virulence-related genes exhibited minimal differences between YC128 and YC156. Drawing from previous studies, we postulated that the T6SS is linked to the hypervirulence and robust anti-phagocytic ability of YC128.

Conclusions: This article reports on the isolation of a novel hypervirulent CAP-AB strain, YC128, from a severe CAP patient. The results demonstrate that this CAP-AB strain, YC128, is capable of inducing fatal pneumonia and extrapulmonary dissemination in a mouse pneumonia model. Moreover, this highly virulent CAP-AB strain exhibits significantly stronger anti-phagocytic abilities compared to the HAP-AB YC156 strain. Genome sequencing comparisons reveal that the heightened hypervirulence and enhanced anti-phagocytosis abilities observed in YC128 may be attributed to the presence of the T6SS.

Keywords: Acinetobacter baumannii; Anti-phagocytosis; Community-acquired pneumonia; Hypervirulent; Type VI secretion system.

Fig. 1

YC128 induced significantly increased mortality rate in mice. Sixty mice were intranasally inoculated with A. baumannii in 50 μl of saline, with each group 10 mice, in addition, five mice was intranasally inoculated with 50 μl saline as negative control. a Every mouse in group YC128(H), LAC-4(H) and YC156(H) was inoculated with 10⁸ CFU A. baumannii respectively. b Every mouse in group YC128(L), LAC-4(L) and YC156(L) was inoculated with 10⁷ CFU A. baumannii respectively. (H: high A. baumannii suspension, 10⁸ CFU; L: low A. baumannii suspension, 10⁷ CFU)

Fig. 2

YC128 induced significantly higher bacterial burdens in tissues. The bacterial burden of YC128, LAC-4 and YC156 in mice pneumoniae model. Twenty-four mice were intranasally inoculated with 10⁷ CFU A. baumannii in 50 μl of saline, with each group 8 mice. Four mice were sacrificed at 24 h and 48 h after inoculation in each group. The lungs, liver and spleen from three mice were conducted quantitative culture. a Bacterial counting of lungs. b Bacterial counting of liver. c Bacterial counting of spleen

Fig. 3

The inflammatory pathological changes of the CAP-AB infected mice were significantly more severe than the HAP-AB group. Pulmonary pathology of CAP-AB YC128 and HAP-AB YC156 group mice. a 24 h after YC156 inoculation. Moderate edema in pulmonary interval (red arrow), some lymphocytes and neutrophils invasion (black arrow). b 48 h after YC156 inoculation. Focal infiltration of lymphocytes and neutrophils in bronchial epithelium (black arrow), protein effusion with inflammatory cell infiltration in some alveolus (red arrow). c 72 h after YC156 inoculation, some of the alveolus atrophy and lung consolidation (black arrow), focal intra alveolar hemorrhage (red arrow). d 24 h after YC128 inoculation. Most alveoli atrophy, lung consolidation (black arrow), the alveoli fused into large alveoli (red arrow). e 48 h after YC128 inoculation. Lung consolidation, destruction of alveolus, mononuclear and neutrophils invasion (black arrow), some alveolis fused into large alveoli (red arrow). f 72 h after YC128 inoculation. Lung consolidation, destruction of alveolus (black arrow) with a large number of mononuclear, lymphocytes and neutrophils invasion (red arrow)

Fig. 4

YC128 induced more severe imaging changes shown in chest CT scan. a 24 h after YC156 inoculation. Slight patchy opacities in both lungs. b 48 h after YC156 inoculation. Moderate patchy opacities in both lungs. c 72 h after YC156 inoculation. Patchy opacities alleviation in both lungs. d 24 h after YC128 inoculation. Slight patchy opacities in both lungs. e 48 h after YC128 inoculation. Complete consolidation was present in the whole left lung and local patchy opacities in the right lung. f 72 h after YC128 inoculation. consolidation appeared in bilateral lungs like “white lung”

Fig. 5

YC128 induced more rapid cytokines expressions in the lung. The local level in lung of cytokines/chemokine were detected by RT-qPCR. a IL-1β. b IL-6. c TNF-α. d CXCL1

Fig. 6

YC128 showed significant stronger in vitro anti-phagocytosis ability than that of HAP-AB strain. Peritoneal macrophages isolated from 5 mice and each mouse macrophages were distributed into 6 wells. Then, 20 μl per well of YC156 and YC128 bacterial suspension were added into the cell culture. After two hours later, the cell culture medium was collected to quantify the extracellular bacterial number. Then the wells were washed using ice-cold PBS and sterile water added into wells to lysis macrophage. Two hours later, the suspension was cultured to quantify the intracellular bacterial number. a Intracellular bacterial number. b Extracellular bacterial number

Fig. 7

The Comparisons of Whole Genome Sequencing between YC128 and YC156. a Virulence related genes. b Antimicrobial resistance genes