Background
Although influenza viruses by itself can cause highly fatal primary influenza pneumonia, the excess mortality rates during influenza pandemics is mainly caused by secondary bacterial pneumonia [
1]. Epidemiologic evidence suggests that 95% and 70% of deaths during the influenza pandemic of 1918–19 and 1957–58, respectively, were due to bacterial pneumonia [
2]. During the first season (between May 2009 and August 2009) of 2009 pandemic H1N1 influenza, 29% of fatal cases in the United States were associated with a secondary bacterial infection, which was also correlated with the severity of the pneumonia [
3]. In hospitalized patients with seasonal influenza, sepsis with or without bacteremia were one of the severe complications, and associated with increased duration of hospitalization days and the requirement of intensive care [
4]. The secondary bacterial pneumonia was also observed in patients infected by avian influenza viruses including H5N1 and H7N9 [
5]. However, the frequency of secondary bacterial respiratory infection and its effect on the disease severity of avian influenza virus-infected human cases were still lacking.
Acinetobacter baumannii is one of the major opportunistic pathogens that have been implicated in various nosocomial infections [
6]. The most frequent clinical manifestation of nosocomial
A. baumannii infection is ventilator-associated pneumonia [
7]. Although the clinical impact of nosocomial
A. baumannii infection has been a matter of continuing debate as many patients with severe influenza had major underlying diseases [
8], it has been concluded that
A. baumannii infection was associated with an increase in attributable mortality, ranging from 7.8 to 23% [
9].
A. baumannii is attracting much attention owing to the increase in antimicrobial resistance and occurrence of strains that are resistant to virtually all available drugs [
10]. The rapid global emergence of multidrug-resistant
A. baumannii (MDR-AB) strains resistant to all β-lactams, including carbapenems, illustrated the potential of this organism to respond swiftly to changes in selective environmental pressure [
11]. Though it is still very rare, recently, resistance to polymyxins has also been described [
12], which leads to a pandrug-resistant
A. baumannii (PDR-AB) that can be fully refractory to the currently available antimicrobial armamentarium.
From March 2013, hundreds of human cases infected by avian influenza A (H7N9) virus with a mortality of 40% were reported and the epidemic situation seems more severe currently with over 100 new cases within December 2016 (
http://www.who.int/influenza). Among the H7N9 patients complicated by bacterial infections, MDR-AB was the most common etiology for secondary bacterial pneumonia revealed by independent descriptive studies. Herein, we studied the occurrence of secondary infection by
A. Baumannii in these severe H7N9 patients. We sought to investigate the role of dysfunctional immunity in the pathogenesis of severe pneumonia in H7N9 patients nosocomially co-infected with
A. baumannii. Our study may improve the understanding of the high pathogenicity of H7N9 in humans and provide useful recommendations for the clinical diagnosis and treatment.
Discussion
It is known that secondary bacterial pneumonia during influenza virus infection is a major cause for high mortality of severe or fatal cases. Compared with the dominant bacterial infection of
Streptococcus pneumoniae and
Staphylococcus aureus in other influenza virus pandemics [
30],
A. baumannii is the most commonly encountered pathogen in sputum or endotracheal samples in the H7N9 virus infected clinical cases [
31]. Here, we presented a typical cohort of the H7N9 patients confected by
A. baumannii and elucidated their dysfunctional immune responses. Furthermore, the genome features of the
A. baumannii and particularly the genome variations contributed to the acquired polymyxin resistance which might have led to the fatal outcome of this patient.
The role of the hospital environment as a reservoir for
A. baumannii has been well defined [
32]. Hospital equipment, especially the medical equipment for invasive mechanical ventilation, is one of the important risk factors that predispose individuals to the acquisition of, and infection with,
A. baumannii [
6]. Other risk factors include prior antibiotic use and prior use of broad-spectrum drugs [
33]. In our study, H7N9 patients co-infected by
A. baumannii have a longer use of antibiotics, glucocorticoid, and the invasive mechanical ventilation, all of which may predispose to the occurrence of the
A. baumannii co-infection in these patients.
In fact, influenza virus infection itself also enhances the susceptibility of the patients to secondary bacterial infection by its impacts on the inflammatory signals and function of early innate immune defense [
34]. It is known that levels of inflammatory mediators such as IL-6 and IL-18 were higher in the patients with co-infection of influenza virus and bacteria than in patients with bacterial pneumonia or influenza virus infection alone [
35]. Concordantly, in our study, the IL-6 and IL-8 increased to a higher level in the H7N9-
A. baumannii co-infected patients compared to H7N9 patients without bacterial infection.
Influenza A infection could also increase susceptibility to secondary bacterial pneumonia by dysregulation of different innate immune cells. The influenza virus can inhibit Th17 immunity by the induction of type I IFNs [
36] and IL-27 [
37], and also can inhibit neutrophil attraction through the mediation of protein Setdb2 [
38] and the production of IL-10 [
39]. In our study, we found that the H7N9 patients with a persistently low level of CD8
+ and CD4
+ T-cell population, especially hyporesponsive antigen-specific T-cell responses, are susceptible to the
A. baumannii infection and the subsequent fatal outcome. This may indicate a T-cell anergy after the avian H7N9 influenza virus infection as previously defined in severe H1N1 infected patients [
40].
It is surprising that SMGC-AB1 quickly acquired resistance to polymyxin, an antibiotic that is considered to be the last hope to cope with MDR and or XDR gram-negative bacteria. Polymyxins are cyclic cationic peptides with a long hydrophobic tail that interact with the lipid A moiety of lipopolysaccharide (LPS) to disrupt the integrity of outer membranes of gram-negative bacteria. Three major resistance mechanisms to polymyxin in bacteria have been identified: modification of the bacterial outer membrane lipopolysaccharide, proteolytic cleavage of the drug and activation of broad-spectrum efflux pumps, among which modification of lipid A of LPS regulated by the two-component regulatory system PmrAB has been frequently reported in
A. baumannii [
41]. It is reported that a single mutation in
pmrB gene causing T235I amino acid substitution can lead to 16-fold increases in polymyxin B MIC in
A. baumannii [
29]. In this study, we found that the SMGC-AB2 harbors a mutation in
pmrB gene exactly resulting in T235I amino acid substitution; we believe that this mutation plays a decisive role for this isolate to resist polymyxin. As demonstrated, mutations in the
pmrA or
pmrB genes usually result in the constitutive expression of
pmrC, thus leading to LPS modification and reduction of the affinity of polymyxins [
42]. In clinical resistant isolates, similar mutations have also been found in
pmrC gene.
. However, in SMGC-AB2, the mutation located at the end of the C-terminus of pmrC protein (L533 T), beyond the functional sulfatase domain (aa 237–532), may not contribute to the resistance. It is reported that
A. baumannii can also develop resistance to polymyxin by mutating genes responsible for LPS production [
43]. Though no mutations were found in the biosynthesis pathway of LPS in SMGC-AB2, a mutation appeared in a pupative permease (Table
1, mutation No. 1) that probably functions in LPS transport. We speculate that this mutation may cause the decrease of LPS production, thus leading to the decrease of the drug target, which may also play a secondary role in the resistance phenotype of SMGC-AB2. Finally, we also found three mutations in a large repetitive protein harboring type I secretion C-terminal target domain leading to two amino acid substitutions (Val → Ile); whether and how this protein and the mutations are involved in the polymyxin resistance in SMGC-AB2 needs further investigations.
In conclusion, we described the occurrence of secondary infection of A. baumannii and its impacts on the disease severity in H7N9 patients. The dysfunctional immunity in the H7N9 patients correlated to the A. baumannii co-infection. Furthermore, the genome variations of A. baumannii contributed to the acquired polymyxin resistance in the patient with fatal outcome. We suggest that the enhancement of the anti-nosocomial infection measures for the prevention of A. baumannii in the H7N9 patients with risk factors for secondary infections, and the early administration of appropriate antibiotic regimen when such co-infection is detected by frequent microbiological testing.
Acknowledgements
We thank Dr. Jianfang Zhou and Dr. Hui Li for their helpful suggestions.