The finding that LPS-TLR4 interactions govern in vivo virulence of A. baumannii and that an LpxC inhibitor antibiotic with no in vitro activity against A. baumannii protected mice from lethal infection are of considerable biological and translational importance. The protection despite a similar tissue bacterial burden but with reduced inflammatory cytokines in TLR4-mutant mice demonstrates that protection was driven by immunomodulation rather than by altering the bacterial density of infection. Interestingly, despite the lack of detectable in vitro killing of A. baumannii by LpxC-1 in standard susceptibility tests, treatment of mice with LpxC-1 markedly reduced tissue bacterial density, serum LPS levels, and serum inflammatory cytokine levels. As a result, LpxC-1 totally protected mice from lethal infection. Exposure of bacteria to the LpxC-1 inhibitor increased their susceptibility to opsonophagocytic killing by macrophages. The LpxC-1 inhibitor also reduced the LPS levels in serum relative to the bacterial density in blood, so the effect on reducing immunopathogenesis was greater than would be expected to be caused by another antibiotic that reduced CFU without reducing LPS density in the bacteria. Since LpxC inhibitors are already in advanced preclinical development, these results indicate that such inhibitors should be tested clinically in patients infected with A. baumannii irrespective of in vitro susceptibility results. Since there are few if any drugs in development with the potential to treat lethal XDR/PDR A. baumannii infections, the discovery that an entirely new class of compounds has therapeutic potential is of great potential clinical importance. Furthermore, the colistin-mediated in vitro neutralization of LPS activation of TLR4 suggests that adjunctive colistin therapy, potentially at lower and hence less-toxic doses than are typically used clinically, could reduce A. baumannii virulence in vivo irrespective of bactericidal activity. Thus, low-dose colistin merits study as an adjunctive, combination therapy even for A. baumannii strains that are susceptible to β-lactam antibiotics.
While colistin-resistant strains of A. baumannii
are reported to have reduced virulence in mice (46
), the current findings indicate that colistin resistance does not necessarily intrinsically affect virulence. Indeed, several publications have defined varying strain virulences unrelated to colistin resistance (47
). In the current study, two strains with regulatory mutations affecting polymyxin resistance through addition of phosphoethanolamine to LPS (43
), C14 and R2, were found to have highly divergent in vivo
virulences. The clinical isolate C14 was as virulent in vivo
as carbapenem-resistant, colistin-susceptible HUMC isolates. In contrast, R2 was avirulent. Extracted LPS from both strains led to enhanced TLR4 stimulation relative to that with LPS extracted from other, non-colistin-resistant strains. However, the pmrC
mutation in R2 did not increase its LPS shedding relative to that of its hypovirulent parent strain, 17978, and thus its virulence was not affected. In contrast, strain C14 had both increased TLR4 activation from extracted LPS and a very high level of LPS shed during growth, resulting in enhanced in vivo
virulence. The molecular genetics and structure of LPS that result in greater shedding by the more-virulent strains merits investigation, since elucidating these factors should result in novel targets for therapeutic intervention.
These results also provide direct experimental confirmation of the host-pathogen damage model of Casadevall and Pirofski (49
). Specifically, the A. baumannii
bacterial burdens were similar during lethal and nonlethal infection in wild-type versus mutant mice, and evaluation of bacterial burden or clearance did not describe virulence for this pathogen. Rather, virulence was related to induction of host hyperinflammation resulting in lethal sepsis. Thus, investigation of infections caused by A. baumannii
, whether preclinical or clinical, should focus as much on host response biomarkers as on microbiological eradication. Furthermore, caution must be exercised when evaluating the severity of infection in experimental models based solely on microbial burden. Microbial burden may not accurately reflect “damage” to the host, or actual outcome of infection, particularly in models that do not assess actual physiological consequences of infection (e.g., nonlethal models). For example, in a previous study, TLR4-KO mice on a C57BL/6 background were reported to be susceptible to A. baumannii
infection, which may appear to be discordant with our results. However, the previous study used a nonlethal model of infection and found slower early clearance of the organism from the lung (41
). By 48 h, the organism had been cleared similarly by wild-type and TLR4-KO mice, and there was no apparent clinical or physiological consequence for the mice of this slower initial bacterial clearance. Our data also showed a nonsignificant, modestly lower bacterial burden in tissue of C3H/HeJ TLR4-mutant mice than in wild-type mice and demonstrate that the clinical outcomes were not driven by the tissue bacterial burden but rather by the host response to the bacteria. Thus, our data are not discordant from those of the previous study and must be interpreted in the context of lethal versus nonlethal models.
How the LpxC-1 inhibitor enhances phagocytosis is not clear. The effect was not due to a direct impact of LpxC-1 on macrophages, because pretreatment of macrophages with the LpxC-1 inhibitor, followed by rinsing away the inhibitor, resulted in no substantive change in macrophage killing of the bacteria. Mutation of Lpx is known to result in upregulation of genes responsible for the biosynthesis of poly-β-1,6-N-acetylglucosamine (PNAG), which presumably replaces LPS as a predominant oligosaccharide in the outer membrane, enabling the bacteria to maintain cell viability (50
). It has long been known that the macrophage mannose receptor binds to N
), which may account for the enhanced phagocytosis of A. baumannii
in the setting of LpxC-1 exposure.
Antimicrobial discovery screens and development programs are typically built around lead compounds with low in vitro
MICs, preferably with microbicidal activity, against target bacteria. However, such screens fail to detect the potential for antimicrobial drugs to modulate pathogenesis aside from microbicidal activity against the organism. Most LpxC inhibitors, including LpxC-1, do not have in vitro
activity against A. baumannii
by standard susceptibility testing. However, A. baumannii
is known to express LpxC (52
), and the current study demonstrates that while the LpxC inhibitor tested did not inhibit A. baumannii
growth, it did markedly modulate the ability of the cells to activate TLR4 and induce septic shock in vivo
. These data underscore the importance of finding new, physiologically relevant ways to screen for small-molecule and biological agents to treat XDR/PDR GNB and other highly resistant microbes in order to discover novel therapeutic classes.
Bacteremia is one of the most common clinical syndromes caused by A. baumannii
and is often accompanied by sepsis syndrome (15
). Such infections typically occur in patients hospitalized in the ICU, most likely via bolus entry from catheters, which is similar to the mode of entry in the model studied. An advantage of the C3H model of infection is that relatively low inocula (e.g., 2 × 107
) induce fatal infection even without having to cause overt immunocompromise. This lethal inoculum is similar to that required to cause fatal infections by other virulent bacteria in noncompromised mice, such as Staphylococcus aureus
, and Pseudomonas aeruginosa
). In contrast, the same inoculum of A. baumannii
in other mouse models, such as BALB/c mice, is nonfatal unless accompanied by induction of diabetes mellitus or neutropenia (42
In summary, LPS-mediated activation of TLR4 was a primary pathogenic factor during systemic A. baumannii infection, and TLR4 was antiprotective against lethal infection. Of great translational importance is that inhibition of LpxC resulted in diminished LPS-mediated TLR4 activation and protected mice from lethal infection despite a lack of in vitro susceptibility of the bacteria to the inhibitor by traditional testing. These results underscore the urgent and pressing need to find in vitro screens that predict in vivo efficacy in a physiological way and the potential for small-molecule and biological therapies to be effective antibacterial agents even if they do not directly kill the target pathogen.